PI: Liu, Yingru Title: Experimental Gonococcal Vaccine

Received: 08/24/2016 FOA: PA16-302 Council: 01/2017

Competition ID: FORMS-D FOA Title: PHS 2016-02 OMNIBUS SOLICITATION OF THE NIH, CDC, FDA AND 

ACF FOR SMALL BUSINESS INNOVATION RESEARCH GRANT APPLICATIONS 

(PARENT SBIR [R43/R44])

2 R44 AI115877-02 Dual: DK,HD Accession Number: 3964434

IPF: 4529801 Organization: THERAPYX, INC.

Former Number: Department: TherapyX Inc.

IRG/SRG: ZRG1 IMM-R (12)B AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 2:

Year 3:

Animals: Y 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Yingru Liu 1450566070000 PD/PI 

Dominick Auci Ph.D TherapyXinc Co-PD/PI 

Stacia Furtado TherayXinc Other Professional-Senior Scientist

Edith Mathiowitz Brown University Other Professional-Collaborating 

Scientist

Nejat Egilmez University of Louisville Other Professional-Collaborating 

Scientist

Michael Russell TherapyXinc Consultant

Always follow your funding opportunity's instructions for application format. Although this application

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OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

3. DATE RECEIVED BY STATE State Application Identifier

1. TYPE OF SUBMISSION* 4.a. Federal Identifier

R43AI115877

❍ Pre-application ❍ Application ● Changed/Corrected

Application

b. Agency Routing Number

2. DATE SUBMITTED Application Identifier c. Previous Grants.gov Tracking Number

GRANT12235554

5. APPLICANT INFORMATION Organizational DUNS*: 1450566070000

Legal Name*: TherapyX Inc.

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*: -

Person to be contacted on matters involving this application

Prefix: Dr. First Name*: Dominick Middle Name: Last Name*: Auci Suffix: 

Position/Title: Vice President

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email: 

6. EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN)*

7. TYPE OF APPLICANT* R: Small Business

Other (Specify):

Small Business Organization Type ❍ Women Owned ❍ Socially and Economically Disadvantaged

8. TYPE OF APPLICATION* If Revision, mark appropriate box(es).

● New ❍ Resubmission ❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration

❍ Renewal ❍ Continuation ❍ Revision ❍ D. Decrease Duration ❍ E. Other (specify) :

Is this application being submitted to other agencies?* ❍Yes ●No What other Agencies?

9. NAME OF FEDERAL AGENCY*

National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

Experimental Gonococcal Vaccine

12. PROPOSED PROJECT

Start Date* Ending Date*

01/01/2017 12/31/2018

13. CONGRESSIONAL DISTRICTS OF APPLICANT

KY-003
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: Dr. First Name*: Yingru Middle Name: Last Name*: Liu Suffix: 

Position/Title: Senior Research Scientist

Organization Name*: 1450566070000

Department: TherapyX Inc.

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested* $

b. Total Non-Federal Funds* $0.00

c. Total Federal & Non-Federal Funds* $0.00

d. Estimated Program Income* $

16. IS APPLICATION SUBJECT TO REVIEW BY STATE

EXECUTIVE ORDER 12372 PROCESS?*

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

DATE:

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein

are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with

any resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to

criminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001)

● I agree*

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE

Prefix: Dr. First Name*: Nejat Middle Name: Last Name*: Egilmez Suffix: 

Position/Title*: Executive Vice President

Organization Name*: TherapyX Inc.

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

Signature of Authorized Representative*

Dominick Auci

Date Signed*

08/24/2016

20. PRE-APPLICATION File Name: Mime Type:

21. COVER LETTER ATTACHMENT File Name:1252-CoverLetterGCVaxPhaseII.pdf Mime Type: application/pdf
424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

 Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract(Description)----------------------------------------------------- 6

 Project Narrative------------------------------------------------------------------------- 7

 Facilities & Other Resources-------------------------------------------------------------- 8

 Equipment--------------------------------------------------------------------------------- 13

Research & Related Senior/Key Person-------------------------------------------------------------------------- 15

Research & Related Budget Year - 1---------------------------------------------------------------------------- 48

Research & Related Budget Year - 2---------------------------------------------------------------------------- 51

Budget Justification------------------------------------------------------------------------------------------ 54

Research & Related Cumulative Budget-------------------------------------------------------------------------- 59

SBIR STTR Information----------------------------------------------------------------------------------------- 60

 Commercialization Plan-------------------------------------------------------------------- 62

 Prior SBIR Phase II Awards---------------------------------------------------------------- 74

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 75

PHS 398 Research Plan----------------------------------------------------------------------------------------- 77

 Specific Aims----------------------------------------------------------------------------- 78

 Research Strategy------------------------------------------------------------------------- 79

 Vertebrate Animals------------------------------------------------------------------------ 91

 Bibliography & References Cited----------------------------------------------------------- 95

 Letters of Support------------------------------------------------------------------------ 97

Appendix

Number of Attachments in Appendix: 2

Contact PD/PI: Liu, Yingru

Table of Contents Page 3
Contact PD/PI: Liu, Yingru

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302. Received Date:

2016-08-24T11:01:24.000-04:00

Project/Performance Site Location(s)

Project/Performance Site Primary Location ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: TherapyXinc

Duns Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*: NY-027

Project/Performance Site Location 1 ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: TherapyXinc

DUNS Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*: KY-003

File Name Mime Type

Additional Location(s)

 Page 4
Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302. Received Date:

2016-08-24T11:01:24.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

 Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ● Yes ❍ No

IACUC Approval Date:

Animal Welfare Assurance Number A 3354-01

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an

environmental assessment (EA) or environmental impact statement (EIS) been performed?

❍ Yes ❍ No

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No

5.a. If yes, please explain:

6. Does this project involve activities outside the United States or partnership with international

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* 1247-ABSTRACTvaccinePhaseII

FINAL.pdf

Mime Type: application/pdf

8. Project Narrative* 1248-narativephaseIIvaccine.pdf Mime Type: application/pdf

9. Bibliography & References Cited 1249-REFERENCES GC Final.pdf Mime Type: application/pdf

10.Facilities & Other Resources 1250-Facilities and Other

Resources NKE.pdf

Mime Type: application/pdf

11.Equipment 1251-EQUIPMENT.pdf Mime Type: application/pdf

Contact PD/PI: Liu, Yingru

 Page 5
ABSTRACT 

Genital tract infection with Neisseria gonorrhoeae (gonorrhea) does not induce a state of specific protective 

immunity and it can be acquired repeatedly. Despite public health measures, the disease persists at an 

unacceptably high frequency; there is no vaccine against it, and resistance even to the latest generations of 

antibiotics continues to emerge. Recent findings have revealed that N. gonorrhoeae subverts the immune 

system for its own benefit by eliciting innate responses that it can survive and by suppressing specific adaptive 

responses that would eliminate it. However, this induced immunosuppression can be reversed by treatments 

that effectively redirect the immune response against N. gonorrhoeae. Proof-of-principle has been established 

for a novel strategy of prophylaxis against genital gonococcal infection using a mouse model that is accepted 

as the only currently available animal model. The current vaccine prototype, GvaX12® (a combinatorial 

formulation of our proprietary sustained-release nanoparticulate interleukin-12 and gonococcal outer 

membrane vesicles) induces anti-gonococcal T-cell and antibody responses, and generates protection against 

homologous and heterologous strains for up to 6 months. In this Phase II application, we will define, optimize, 

and validate a vaccination regimen including route, aiming for intranasal administration. We will determine the 

basis of cross-protection against diverse strains of naturally occurring N. gonorrhoeae, and begin preliminary 

pharmacokinetics and toxicology studies in preparation for subsequent toxicological testing in nonhuman 

primates. Along with the preclinical data, toxicology results and future plans will be incorporated into a briefing 

package that will be submitted to the FDA in a request for a Type C Meeting.

Contact PD/PI: Liu, Yingru

Project Summary/Abstract Page 6
NARRATIVE (PUBLIC HEALTH RELEVANCE STATEMENT) 

Gonorrhea is the second-most-frequent, notifiable infectious disease in the United States; the 

Centers for Disease Control report ~350,000 cases annually, and world-wide incidence is 

estimated at 78 million new infections per year. No vaccine is available and the emergence 

of multiple-drug-resistant strains now raises serious and urgent concerns over future treatment 

options. This proposal seeks to develop a novel strategy for prophylactic vaccination against 

gonorrhea by directing the immune response to generate lasting protective immunity. 

Contact PD/PI: Liu, Yingru

Project Narrative Page 7
Facilities

FACILITIES AND OTHER RESOURCES 

Office and Laboratory Space 

TherapyX

Inc recently moved its administrative offices to Louisville, KY in pursuit of potential new 

investment opportunities and state-supported programs for small-business development. Our research 

and development facilities remain in Buffalo, NY where we have an existing facilities use agreement with 

the State University of NY. All research work outlined in Aims 1 and 2 will be performed in our Buffalo, NY 

location (primary site). Dr. Yingru Liu and all research personnel work at the Buffalo facility. Dr. Dominick 

Auci, with the assistance of Ms. Samina Raza, works out of the Louisville office (site 1) but travels to the 

Buffalo facility once a month. 

Our administrative location in Louisville, KY consists of 600 square foot rental office space equipped 

with 2 Dell E520 desktop computers networked to the internet, a laser printer, photocopier, scanner, file 

cabinets, 3 desks and telephones. 

Our research office and laboratory space is located in Sherman Hall and our BSL2 and conventional 

animal facilities are located in the Biomedical Education Building at SUNY at Buffalo Medical School. 

These facilities include a total of 1200 sq. ft., of laboratory, 200 sq. ft. of office space and a 600 sq. ft. 

animal room. The office and laboratory in the Buffalo location are equipped with 3 Dell E520 computers, 

and 1 Dell Dimension computer, a networked laser printer and Ethernet connection to the internet with full 

access to the SUNY at Buffalo library and electronic journal subscriptions. The office is equipped with filing 

cabinets, office furniture, computers, FAX/Copier and telephones. An agreement entered into by 

TherapyX

Inc and SUNY at Buffalo also allows for employees access to common equipment rooms. 

Our research facility is adjacent to the Witebsky Center for Microbial Pathogenesis and Immunology, 

an interdepartmental center occupying two floors of the Biomedical Research Building on the State 

University of New York at Buffalo (UB) South Campus. The Center brings together a diverse group of 20 

active, extramurally funded faculty who share a common interest in various aspects of the constituent subdisciplines, including bacteriology, virology, parasitology, mycology, infectious diseases, and immunity to 

infection and to tumors. TherapyX

Inc has ongoing collaborations with a number of members of the 

Witebsky Center and the Department of Microbiology and Immunology at UB.

Animal Care Facilities 

Research animal facilities, including BSL-2 suites, are available through the UB Laboratory Animal 

Facility, which is adjacent to the TherapyX

Inc Laboratories in Sherman Hall. The company has a dedicated 

600 sq ft room in the animal facility that is maintained by the LAF staff for a fee. This facility is accredited 

by AAALAC, and complies with all applicable Federal and State laws and guidelines. The program 

provides procurement facilities, routine animal care, health surveillance, and veterinary support. 

Other Resources 

The Health Sciences Library is situated nearby on the UB South Campus. Inter-library loan service, 

photocopying, and both internal and external (Medline) computerized literature search and full-text 

retrieval services are available to TherapyX

Inc An inter-institutional agreement with SUNY at Buffalo allows 

full access to the Confocal Microscopy and Flow Cytometry core facility at the Witebsky Center at 

SUNY at Buffalo. Histology services for H & E staining of paraffin-embedded tissue sections as well as 

immuno- histochemical staining of frozen sections are available from the Histology services laboratory of 

the SUNY at Buffalo Medical School. 

The Confocal Microscopy and Flow Cytometry Facility is a University instrument laboratory operated 

by the School of Medicine and Biomedical Sciences for the benefit of the research community of the 

University at Buffalo. TherapyX

Inc has a facilities use agreement that allows for use of the equipment and 

the expertise and aid of the facility director Dr. Wade J. Sigurdson and Flow Cytometry administrator Dr. 

Ray Kelleher. 

Contact PD/PI: Liu, Yingru

Facilities & Other Resources Page 8
Facilities

Biohazard Protections 

This project includes work with Neissseria gonorrhoeae. The following section describes the special 

Standard Operating Procedures* that are followed while working with these biohazardous organisms. 

SUNY at Buffalo Occupational Health Monitoring Program and the Use of Hazardous Agents 

The Occupational Health Monitoring Program 

The Occupational Health Monitoring Program (OHMP) at SUNY at Buffalo is mandatory for all persons 

in contact with research animals or their unfixed tissues. The current program requires that all personnel 

working with animals must participate in, and must be trained in animal care and in safety. All personnel 

who are to handle animals, or to be in facilities where animals are kept, must complete a questionnaire 

describing their proposed activities, and must also provide information relevant to their health. These two 

components (activities and health) are reviewed by an occupational health physician, Dr. David Hughes, 

Occupational Physician at ECMC who then provides an assessment of the risk of the proposed activities 

(which is returned to IACUC administrator), and makes recommendations to the individual for vaccines 

etc. All costs for enrolling in the OHMP are covered by the University. 

Use of Hazardous Agents 

All researchers using potentially hazardous agents or materials such as infectious and cancerous 

agents, highly toxic chemicals or recombinant DNA molecules falling under NIH guidelines have an 

obligation to inform the University Biosafety Committee by registering their research program. Animal use 

is regulated by the Institutional Animal Care and Use Committee, in collaboration with these groups. It is 

the Principal Investigator's responsibility to provide appropriate information on these materials to all those 

who may be exposed to these materials and/or their hazardous degradation products and inform the CMLAF Director or Manager of when these projects will start. This information shall include but not be limited 

to (a) written hazard descriptions, such as material safety data sheets (MSDS); (b) hazard specific and 

documented training of personnel including both research workers and support personnel such as animal 

care workers; and (c) the posting of appropriate standardized signage. The CMLAF will arrange training of 

LAF personnel, posting of signage on the holding room doors, appropriate housing of animals and cage 

tags. 

The Office of Environmental Health and Safety Services can provide appropriate registration 

documents and NIH/CDC Guidelines, assist in obtaining appropriate MSDS information sheets, 

standardized signage and generic training and information on the establishment of a Chemical Hygiene 

Plan and/or a Blood Borne Pathogen Program (as required by OSHA). 

* The SUNY at Buffalo CM-LAF Standard Operating Procedure for research with Neisseria gonorrhoeae 

is included in the Biohazard attachment. 

Contact PD/PI: Liu, Yingru

Facilities & Other Resources Page 9
Facilities

BIOHAZARD PROTECTIONS 

SUNY at Buffalo Occupational Health Monitoring Program and the Use of Hazardous Agents 

The Occupational Health Monitoring Program 

The Occupational Health Monitoring Program (OHMP) at SUNY at Buffalo is mandatory for all persons in 

contact with research animals or their unfixed tissues. The current program requires that all personnel working 

with animals must participate in, and must be trained in animal care and in safety. All personnel who are to 

handle animals, or to be in facilities where animals are kept, must complete a questionnaire describing their 

proposed activities, and must also provide information relevant to their health. These two components 

(activities and health) are reviewed by an occupational health physician, Dr. David Hughes, Occupational 

Physician at ECMC who then provides an assessment of the risk of the proposed activities (which is returned 

to IACUC administrator), and makes recommendations to the individual for vaccines etc. All costs for enrolling 

in the OHMP are covered by the University. 

Use of Hazardous Agents 

All researchers using potentially hazardous agents or materials such as infectious and cancerous agents, 

highly toxic chemicals or recombinant DNA molecules falling under NIH guidelines have an obligation to inform 

the University Biosafety Committee by registering their research program. Animal use is regulated by the 

Institutional Animal Care and Use Committee, in collaboration with these groups. It is the Principal 

Investigator's responsibility to provide appropriate information on these materials to all those who may be 

exposed to these materials and/or their hazardous degradation products and inform the CM-LAF Director or 

Manager of when these projects will start. This information shall include but not be limited to (a) written hazard 

descriptions, such as material safety data sheets (MSDS); (b) hazard specific and documented training of 

personnel including both research workers and support personnel such as animal care workers; and (c) the 

posting of appropriate standardized signage. The CMLAF will arrange training of LAF personnel, posting of 

signage on the holding room doors, appropriate housing of animals and cage tags. 

The Office of Environmental Health and Safety Services can provide appropriate registration documents and 

NIH/CDC Guidelines, assist in obtaining appropriate MSDS information sheets, standardized signage and 

generic training and information on the establishment of a Chemical Hygiene Plan and/or a Blood Borne 

Pathogen Program (as required by OSHA). 

* The SUNY at Buffalo CM-LAF Standard Operating Procedure for research with Neisseria gonorrhoeae is 

included here for reference. 

Contact PD/PI: Liu, Yingru

Facilities & Other Resources Page 10
Facilities

COMPARATIVE MEDICINE 

LABORATORY ANIMAL FACILITIES 

STANDARD OPERATING PROCEDURE 

for 

NEISSERIA GONORRHOEAE SAFETY PRECAUTIONS 

1.0 PURPOSE: 

This SOP applies to all Researcher staff and LAF employees 

working in BSL 2 facility with this organism 

2.0 SCOPE: 

This SOP outlines the Occupational Health and Safety Guidelines 

when working with Neisseria gonorrhoeae. Gonorrheal organisms 

are infectious exclusively to humans. The greatest risk maybe the 

direct contact of excreta on bedding to mucous membranes 

(eyes, mouth, cuts) N. gonorrhoeae is not a life threatening 

infection and the strains used are fully susceptible to antibiotics. 

No vaccine is available. The organism is not transmissible from 

mouse to mouse. Organisms do not persist in the environment 

for long periods of time, and are susceptible to common 

disinfectants. However all material and mice that are in contact 

with gonorrhea must be treated as potentially infectious. 

3.1 PROCEDURE: 

3.2 Personnel must enroll in the Occupational Health Medical 

Program at UB. 

3.3 All work with this organism will be conducted in 

the BSL Level 2 Facility. 

3.4 Standard entry, gowning, and personal hygiene 

procedures of BSL2 will be followed 

3.5 NIOSH N95 face masks will be worn 

3.6 Safety glasses will be worn in the animal holding 

room and when cage changing as an additional 

precaution. 

3.7 Cages & bedding will be autoclaved prior to removal from 

BSL2. 

3.8 After autoclaving, bedding will be discarded into Red 

Biohazard bags, and disposed as Certified Medical 

Waste.

3.9 Autoclaved cages, water-bottles, lids etc will be 

taken to the cagewash area and sanitized in a routine 

manner 

3.10 All equipment and supplies must be autoclaved or 

disinfected with alcohol or 10% Bleach prior to removal 

from BSL2. 

Contact PD/PI: Liu, Yingru

Facilities & Other Resources Page 11
3.11 If a contaminated needle-stick or mucous membrane 

exposure occurs, follow SOP #3.F.1: Exposure to 

Blood and Other Body Fluids, Bites, Scratches and 

Needle Punctures 

A Notify your supervisor 

B Call ECMC 898-4153 and ask to see a doctor 

C Inform workers compensation at 645-5000 

X1025 and ask for Annette Lozo 

D After-hours, proceed to ECMC and explain your 

are enrolled in the OHMP at UB and you were 

hurt at work 

E Medical attention should be sought within 24 

hours of the exposure or injury 

3.12 Carcasses must be treated as infectious and incinerated. 

Contact PD/PI: Liu, Yingru

Facilities & Other Resources Page 12
EQUIPMENT

The TherapyX

Inc laboratories are equipped with standard biomedical laboratory apparatus, 

including: 

 Bio-Tek FLx800 multi-detection Microplate Reader 

 Tissue culture biosafety hood,

 Tissue culture incubator (CO2) 

 Microbiological incubator (CO2) 

 Stereo low-power microscope Inverted microscope 

 Mx3005P QPCR System 

 Image analysis system with digital camera for use with microscopes (Image Pro Plus ver. 

4)

 Conventional open column chromatography equipment with fraction collector, pump, UV 

monitor in a refrigerated cabinet, recorder, and selection of columns 

 FPLC system (Pharmacia) with 2 pumps, GP-250 controller, UV (280nm) monitor, 

fraction collectorMono Q 10/10, Mono S 10/10, various Superose, Sephacryl, Superdex, 

and HiTrap affinity columns 

 Polyacrylamide gel electrophoresis and electroblotting equipment 

 Stratagene Mx3005P QPCR System quantitative RT-PCR machine 

 Ultracold (-80°C) freezer, liquid nitrogen freezer 

 Refrigerators, freezers, water-baths, balances, pH meters, microcentrifuges, pipettors 

 Magnetic cell sorter apparatus (MACS) 

 High-speed centrifuge 

 Light microscope 

 Chemical fume hood

Microparticle, production, (as well as analysis and validation): 

 Silverson L4RT-A Shear Mixer and 4 overhead mixers 

 Waters 2695 Alliance System with 996 PDA detector 

 Virtis 4KBTZL-105 lyophilizer 

 Shimadzu GC-17A gas chromatograph with a flameionization detector and Tekmar 

7000HT autosampler 

 Coulter LS230 particle size analyzer 

 Shimadzu GPC system 

 Perkin Elmer Differential Scanning Calorimeter Puris 1 

 Perkin Elmer Differential Scanning Calorimeter Puris 7 

 Perkin Elmer FT-IR Spectrometer 1725 X,

 GBC Cintra 40 UV-Vis Spectrophotometer 

 Shimadzu UV-2501PC UV-Vis Recording Spectrophotometer 

Common equipment in the Witebsky Center available to Therapyx, Inc.: 

 Reagent-grade water system (Milli-Q)

 Walk-in cold room (4°C) 

 UV/visible spectrophotometer 

 Spectrofluorimeter 

 Luminometer 

 High-speed and ultra-centrifuges 

 Bacteriological shaker/incubators and autoclaves 

Contact PD/PI: Liu, Yingru

Equipment Page 13
 Li-Cor Odyssey CLx imager 

Confocal Microscopy and Flow Cytometry core facility equipment available to Therapyx, 

Inc. employees: 

 Epi-fluorescence and confocal microscopes fitted with digital cameras (Zeiss) 

 4-color flow cytometer (FACSCalibur) 

 7-color flow cytometer/cell sorter (FACSAria) 

 Leica AS/LMD laser capture microdissection system 

 ELISPOT plate-reader 

 Becton-Dickinson FACS Array Bioanalyzer multiplex bead and cellular assay system 

Contact PD/PI: Liu, Yingru

Equipment Page 14
Contact PD/PI: Liu, Yingru

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix: Dr. First Name*: Yingru Middle Name  Last Name*: Liu Suffix: 

Position/Title*: Senior Research Scientist

Organization Name*: 1450566070000

Department: TherapyX Inc.

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  2007

File Name Mime Type

Attach Biographical Sketch*: 1235-Bio-sk. Liu Y.pdf application/pdf

Attach Current & Pending Support: 1236-LIU Current Research

Support.pdf

application/pdf

 Page 15
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Dominick Middle Name  Last Name*: Auci Suffix: Ph.D

Position/Title*: Vice President, Research and Development

Organization Name*: TherapyXinc

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Co-PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  1990

File Name Mime Type

Attach Biographical Sketch*: 1237-Auci NEW NIH bio Phase II

vaccine.pdf

application/pdf

Attach Current & Pending Support: 1238-Auci current Funding.pdf application/pdf

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Stacia Middle Name  Last Name*: Furtado Suffix: 

Position/Title*: Senior Research Scientist

Organization Name*: TherayXinc

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Other Professional Other Project Role Category: Senior Scientist

Degree Type:  PhD Degree Year:  2008

File Name Mime Type

Attach Biographical Sketch*: 1239-Furtado biosketch

phaseIIvaccinePhase2.pdf

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Attach Current & Pending Support: 1240-FurtadoCurrentSupport.pdf application/pdf

 Page 16
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Edith Middle Name  Last Name*: Mathiowitz Suffix: 

Position/Title*: Professor of Medical Sciences and Engineeri

Organization Name*: Brown University

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Other Professional Other Project Role Category: Collaborating Scientist

Degree Type:  Ph.D. Degree Year:  1985

File Name Mime Type

Attach Biographical Sketch*: 1241-Mathiowitz_biosketch.pdf application/pdf

Attach Current & Pending Support: 1242-mathiowitzCurrent

Support.pdf

application/pdf

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Nejat Middle Name  Last Name*: Egilmez Suffix: 

Position/Title*: Professor and Chair

Organization Name*: University of Louisville

Department: Microbiology and Immunology

Division:

Street1*:

Street2:

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State*:

Province:

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Zip / Postal Code*:

Phone Number*:  Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Other Professional Other Project Role Category: Collaborating Scientist

Degree Type:  Ph.D. Degree Year:  1986

File Name Mime Type

Attach Biographical Sketch*: 1243-Egilmez bio vaccine Phase

II FINAL.pdf

application/pdf

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CurrentSupport.pdf

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 Page 17
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Michael Middle Name  Last Name*: Russell Suffix: 

Position/Title*: Advisor

Organization Name*: TherapyXinc

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone Number*:  Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Consultant Other Project Role Category: 

Degree Type:  Ph.D. Degree Year:  1973

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Attach Biographical Sketch*: 1245-Russell bio vaccine PhaseII

FINAL.pdf

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 Page 18
Contact PD/PI: Liu, Yingru

BIOGRAPHICAL SKETCH

NAME

Yingru Liu

POSITION TITLE

Senior Research Scientist, TherapyX, Inc. eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

Beijing Medical Univ., Beijing, China B.Med. (=M.D.) 08/90 Medicine

Univ. Brit. Columbia, Vancouver, BC, Canada M.S. 05/03 Neuroscience

Univ. Brit. Columbia, Vancouver, BC, Canada Ph.D. 11/07 Neuroimmunology

Harvard Medical School, Boston, MA, USA Postdoc 08/08 Neurology, Immunology

Univ. at Buffalo, Buffalo, NY, USA Postdoc 12/10 Immunology, Microbiology

A. Personal Statement 

Since completing my medical studies at Beijing Medical University, China, in 1990, I have been working on 

research in immunology and microbiology for more than 20 years, in both clinical and basic research institutes. 

My Ph.D. project at the University of British Columbia explored the immunomodulatory activities of bilirubin in 

rodent models of multiple sclerosis. I continued with immunological research at Brigham and Women’s 

Hospital, Boston MA (2007-8), and later at Roswell Park Cancer Institute, Buffalo NY (2008-9). Through all 

these years of study I solidified my medical knowledge and acquired a wide range of experimental techniques 

relating to cellular and molecular biology, immunology, and microbiology. In May 2009 I joined Dr. Michael 

Russell’s laboratory at the University at Buffalo, where I brought my expertise in T cell and cytokine biology to 

ongoing projects on immunity to Neisseria gonorrhoeae (Ng), resulting in novel understanding of the 

mechanism whereby this organism manipulates the immune system to its own advantage. 

In 2013 I joined TherapyX

Inc to lead their vaccine program. The current application focuses on the 

development of a vaccine against Ng. To this end, I have been the PI on 2 different SBIR projects focusing on 

novel Ng therapies at TherapyX

Inc. In the past 3 years we have shown how anti-Ng immunity can be countermanipulated to provide a potential new treatment for gonorrhea, and have developed a new approach to 

vaccine development. Based on my background and recent accomplishments I am uniquely qualified to 

continue directing this research and development program. 

B. Positions and Honors 

1990 – 1993 Neurological Resident, Department of Neurology, Neuroimmunology Lab, The Third 

Hospital of Beijing Medical University, Beijing, China 

1993 – 1996 Doctor in Charge, Department of Neurology, The Third Hospital of Beijing Medical 

University, Beijing, China 

1996 – 1999 Clinical Assistant Professor, Department of Neurology, Neuroimmunology Lab, The Third 

Hospital of Beijing Medical University, Beijing, China 

1999 – 2007 Graduate Research Assistant, Brain Research Center, University of British Columbia, 

Vancouver BC, Canada 

2007 – 2008 Postdoctoral Research Fellow, Center for Neurologic Diseases, Brigham and Women’s 

Hospital, Harvard Medical School, Boston, MA 

2008 – 2009 Postdoctoral Research Fellow, Department of Immunology, Roswell Park Cancer Institute, 

Buffalo, NY 

2009 – 2010 Postdoctoral Associate, Department of Microbiology & Immunology, University at Buffalo, 

Buffalo, NY 

2010 – present Research Assistant Professor, Department of Microbiology & Immunology, University at 

Buffalo, Buffalo, NY 

2013 – present Senior Research Scientist, TherapyX

 Inc., Buffalo, NY 

Biosketches Page 19
Awards/Credentials 

1987 – 1990 University Scholarship from Beijing Medical University, Beijing, China. 

1996 Distinguished Doctor, The Third Hospital of Beijing Medical University, Beijing, China. 

2005 – 2007 Senior Graduate Studentship Award from MICHAEL SMITH FOUNDATION FOR 

HEALTH RESEARCH, British Columbia, Canada. 

2010 NIH Travel Scholarship to attend the International Pathogenic Neisseria Conference, 

Banff, AB, Canada, 2010. 

C. Contributions to Science 

My contributions have been mainly in exploring new therapies for immunological and infectious diseases and 

novel approaches to the development of effective vaccines. 

1. During my MSc and PhD work at the University of British Columbia, I investigated the mechanisms of axon 

and neuron damage in multiple sclerosis, as this process contributes directly to disease progression and 

permanent neurologic damage. Using experimental autoimmune encephalomyelitis (EAE), an animal 

paradigm of multiple sclerosis, I investigated and exploited a new oxidative defense system that involves 

the conversion of heme to bilirubin. Although it had been shown that bilirubin was a powerful antioxidant 

substance both in vitro and in vivo, my study was the first to systematically explore its immunosuppressive 

activities, and demonstrated that bilirubin possesses various beneficial immunomodulatory effects, 

including its protective actions against autoimmunity, hemolysis, and antibody-dependent cell-mediated 

cytotoxicity. Although in traditional Oriental medicine animal biles have proved useful in the treatment of 

many human disorders, the potential utility of bilirubin in diseases is limited due to its insolubility and its 

toxicity at high concentrations. My PhD research was the first to identify biliverdin reductase, a watersoluble and nontoxic natural biological enzyme, as a novel strategy for treatment of stress- and immunerelated diseases. My results demonstrated that biliverdin reductase can regenerate bilirubin in a catalytic 

cycle and amplifies the action of bilirubin without significantly increasing its biological concentration. All 

these findings were published in highly respected journals, such as Journal of Immunology, and were 

highly cited in PubMed. 

a. Liu Y, Zhu B, Luo L, Li P, Paty DW, Cynader MS. Heme oxygenase-1 plays an important 

protective role in experimental autoimmune encephalomyelitis. Neuroreport 12: 1841-1845 (2001). 

b. Liu Y, Zhu B, Wang X, Luo L, Li P, Paty DW, Cynader MS. Bilirubin as a potent antioxidant suppresses 

experimental autoimmune encephalomyelitis: implications for the role of oxidative stress in the 

development of multiple sclerosis. J. Neuroimmunol. 139: 27-35 (2003). 

c. Liu Y, Liu J, Tetzlaff W, Paty DW, Cynader MS. Biliverdin reductase, a major physiologic 

cytoprotectant, suppresses experimental autoimmune encephalomyelitis. Free Radical Biol. Med. 40: 

960-967 (2006). 

d. Liu Y, Li P, Lu J, Xiong W, Oger J, Tetzlaff, and Cynader MS. Bilirubin possesses powerful 

immunomodulatory activity and suppresses experimental autoimmune encephalomyelitis. J. Immunol.

181: 1887-1897 (2008).

2. After I completed my graduate study in 2007, I accepted a postdoctoral research fellow position at Center 

for Neurologic Diseases, Harvard Medical School where I continued to look for new therapeutic options for 

a potentially disabling disease. To date, multiple sclerosis treatments have demonstrated effectiveness in 

reducing relapses but have had limited efficacy in slowing disease progression. At Harvard, I and Dr. 

Tanuja Chitnis identified a fusion protein, CD200, which could specifically protect neurons against 

inflammatory attack and halt symptom accumulation in chronic EAE. This finding has significant 

implications for the treatment of multiple sclerosis, as well as other neuroinflammatory diseases. The work 

was published in The Journal of Neuroscience. 

a. Liu Y, Bando Y, Vargas-Lowy D, Elyaman W, Khoury SJ, Huang T, Reif K, Chitnis T. CD200R1 agonist 

attenuates mechanisms of chronic disease in a murine model of multiple sclerosis. 

J. Neurosci. 30(6):2025-38 (2010). PMCID: 2837938 

Contact PD/PI: Liu, Yingru

Biosketches Page 20
3. After moving to Buffalo NY in 2009, I joined Dr. Michael Russell’s laboratory at the University at Buffalo and 

later, in 2013, began working at TherapyX

Inc in continuing collaboration with Dr. Russell. Dr. Russell is an 

internationally recognized expert in mucosal immunology, and is well known for his work on human 

immune responses to Neisseria gonorrhoeae. Gonorrhea is widespread globally and is a major cause of 

reproductive tract damage in women. In addition, it is well known that gonorrhea can be acquired 

repeatedly with apparently no development of protective immunity against reinfection, and no vaccine is 

available. Dr. Russell’s group had previously demonstrated that N. gonorrhoeae selectively induces Th17-

driven innate immune responses that it can survive, and concomitantly suppresses Th1/Th2-driven 

adaptive immune responses. My projects here address a very important, long-lasting question in 

gonococcal infection: how N. gonorrhoeae evades or inhibits host specific protective immunity that would 

eliminate it. My research at Buffalo has demonstrated that the induction of IL-10/Tr1 cells and TGF-β plays 

a critical role in the suppressive effect of N. gonorrhoeae on host adaptive immunity. Furthermore, my 

results showed for the first time that the inhibitory effect of this pathogen on Th1/Th2-mediated specific 

immune responses can be overcome by therapeutic intervention. Blockade of IL-10, TGF-β, or both, 

significantly enhanced specific protective immunity against N. gonorrhoeae, and resulted in the generation 

of immune memory and production of specific antibodies in a mouse model of vaginal gonococcal infection. 

In addition, we developed a novel technology of microencapsulated IL-12 or anti-IL-10 for local intravaginal 

treatment of N. gonorrhoeae infection, with minimal toxic potential. We have further demonstrated that 

microencapsulated IL-12 serves as an effective adjuvant for a prophylactic gonococcal vaccine 

administered intravaginally. The present studies are aimed at developing this finding further to provide the 

preclinical basis for a novel prophylactic vaccine against gonorrhea. 

a. Liu Y, Russell MW. Diversion of the immune response to Neisseria gonorrhoeae from Th17 to Th1/Th2 by 

treatment with anti-TGF-β antibody generates immunological memory and protective immunity. mBio 2: 

e00095-11 (2011). PMCID: 3101786. 

b. Liu Y, Islam E, Jarvis GA, Gray-Owen S, Russell MW. Neisseria gonorrhoeae selectively suppresses the 

development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-β-dependent 

mechanisms. Mucosal Immunol. 5: 320-31 (2012). PMCID:3328619 

c. Liu, Y, Liu, W, and Russell, MW. Suppression of host adaptive immune responses by Neisseria 

gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells. Mucosal Immunol. 7: 165-176 (2014). 

PMCID:3812424. 

d. Liu, Y, Egilmez, NK, and Russell, MW. Enhancement of adaptive immunity to Neisseria gonorrhoeae by 

local intravaginal administration of microencapsulated IL-12. J. Infect. Dis. 208: 1821-1829 (2013). 

PMCID:3814831. 

e. Liu, Y, Hammer, L, Hobbs, MM, Zielke, RA, Sikora, AE, Jerse, AE, Egilmez, NK, and Russell, MW. 

Experimental vaccine induces Th1-driven immune responses and protection against Neisseria 

gonorrhoeae in a murine model. MS submitted to Mucosal Immunol. (Aug. 2016)

Complete List of Published Work 

MyBibliography: http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/49301078/public

D. Research Support 

Current Research Support 

R44-AI-104067-02 (SBIR Phase II) Y. Liu (PI) 02/01/13 – 05/31/17 

NIH/NIAID 

Therapy and prophylaxis for genital tract infection 

The goal of phase II is to develop a novel approach to treatment of genital gonococcal infection by the local 

administration of microencapsulated IL-12. Role: PI. 

R43-AI-115877-01 (SBIR Phase I) Y. Liu (PI) 12/01/14 – 11/30/16 

Contact PD/PI: Liu, Yingru

Biosketches Page 21
NIH/NIAID 

Experimental gonococcal vaccine 

The goal of this project is to establish proof-of-principle for a novel vaccine against genital gonococcal 

infection. Role: PI. 

Completed Research Support 

R43-AI-104067-01 (SBIR Phase I) Y. Liu (PI) 02/01/13 – 01/14/14 

NIH/NIAID 

Therapy and prophylaxis for genital tract infection 

The goal of phase I is to establish proof-of-principle for a novel treatment of genital gonococcal infection by the 

local administration of microencapsulated IL-12. Role: PI. 

Contact PD/PI: Liu, Yingru

Biosketches Page 22
Contact PD/PI: Liu, Yingru

BIOGRAPHICAL SKETCH

Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Dominick L. Auci, MBA, Ph.D

POSITION TITLE

Vice President, Research and Development

eRA COMMONS USER NAME

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE

(if applicable) YEAR(s) FIELD OF STUDY

SUNY at Stony Brook, NY B.S. 1984 Biology and Philosophy

SUNY at Brooklyn, NY Ph.D. 1990 Pathology

Baruch College MBA 1999 Executive Management

A. Personal Statement 

I was recruited to TherapyX with over twenty years of pharmaceutical experience that includes leadership 

of research and development programs focused on infectious and immune mediated inflammatory 

diseases. As Director of Allergy, Autoimmunity and Inflammation at Hollis-Eden Pharmaceuticals, I led all 

aspects of the discovery and development of novel compounds through phase II clinical trials. At 

TherapyX, I have successfully lead the development of scale-up methods for TGFNanoCap, a 

nanoparticulate encapsulated form of TGF for oral delivery. I have coordinated efforts from multidisciplinary project teams that lead to several successful IND applications and phase I/II study protocols. 

My role in this project will be to oversee production and validation of drug product, animal studies (including 

toxicology studies at Comparative Biosciences), sample analysis, as well as data interpretation, evaluation 

and presentation. I will consult with Drs. Liu, Egilmez, Furtado and Mathiowitz to address any emergent 

difficulties. I will also continue to work closely with consultants at Regulatory Professionals to prepare a 

briefing package and request for a type C meeting with the FDA. 

B. Positions and Honors. 

2014- Present Vice President, R&D, TherapyXinc, Louisvillle, KY 

2011-2013 Cancer Vaccine Program Manager, University of Washington, Seattle, WA 

2001-2011 Director, Allergy, Autoimmunity, Inflammation, Hollis-Eden Pharmaceuticals, San 

Diego, CA 

2000-2001 Scientist/Manager Biomedical Laboratories, Pall Corporation, East Hills, N.Y.

1996-1998 Associate Director, Plasma and Blood Cell Repository, Women's Interagency AIDS 

Study, SUNY Health Science Center, Brooklyn, NY 

1992-2000 Assistant Professor, Department of Pathology, SUNY Health Science Center, 

Brooklyn, NY 

1992-2000 Adjunct Assistant Professor, Department of Natural Sciences, York College, The City 

University of New York, Jamaica, NY 

1992-1999 Associate Director, Clinical Immunology Laboratory/Flow Cytometry, SUNY Health 

Science Center, Brooklyn, NY 

1990-1991 Postdoctoral Fellow, Immunopharmacology, Joint appointments in the laboratories of 

Peter Dukor, M.D., Head, Sandoz Forschungsinstitut, Vienna, Austria, and Helen G. 

Durkin, Ph.D., Department of Pathology, SUNY at Brooklyn, NY 

Biosketches Page 23
C. Contributions to Science 

A full list of my published work can be found at: http://www.ncbi.nlm.nih.gov/pubmed/?term=Auci+Dominick

1. Control of IgE responses 

My early postdoctoral work focused on the ontogeny of IgE bearing B cells and IgE forming cells in

humans and mice. We found that unlike B cells bearing other isotypes, IgE bearing B cells never 

cloned in germinal centers, but appeared first in Peyer’s patches and Mesenteric Lymph nodes about a 

week after immunization. Later they were found in T cell dependent areas of spleen. These studies led 

to the development of in vivo and in vitro (IgE ELISPOT) assays to test pharmaceuticals aimed at 

turning off allergic responses at the source, at the level of the IgE producing B cell. I remained at 

Downstate Medical Center in Brooklyn as Assistant Professor of Pathology for the remainder of the 

decade exploring regulation of IgE responses and providing clinical immunology services during the 

HIV outbreak. 

I. Auci, D.L., Carucci, J.A., Chice S.M., Smith, M.C., Dukor, P., and Durkin, H.G., Control of IgE 

responses II. (1993). Isotype specific suppression of peak BPO specific IgE antibody forming cell 

responses in BPO-KLH sensitized mice after oral administration of muramyldipeptide or murabutide. J. 

Immunopharm. 26:157

II. Auci, D.L., Kleiner, G.I., Shaikh, A., Acosta, E., Athanassiades, T.J., and Durkin, H.G., (1993). 

Cytokine-induced suppression and potentiation of hapten specific immediate hypersensitivity 

responses. Immunol. Invest. 22:203

III. Auci, D.L., Chice, S.M., Heusser, C., Athanassiades, T.J., and Durkin, H.G., (1992). Origin and fate of 

IgE bearing lymphocytes II. Gut associated lymphoid tissue as sites of first appearance of IgE bearing 

B lymphocytes and hapten specific IgE antibody forming cells in mice immunized with benzylpenicilloylkeyhole limpet hemocyanin by various routes: relation to asialo Gm 1 ganglioside+ cells and IgE/CD23 

immune complexes. J. Immunol. 149:2241 

IV. Auci, D.L., Smith, T.N., Jagoo, A.D., Kleiner, G.I., Bluth, H.M., Chice, S.M., Bard, E., Anderson, V., 

Zevallos, E., and Durkin, H.D. (1997). IgE-Bearing Cells and Epsilon-Specific mRNA in Lymphoid 

Organs of Two Children with AIDS. Ped. AIDS HIV Infect. Fetus to Adolesc. 8:102-107

2. The Immunobiology and Therapeutic Potential of Novel Androstene Metabolites and Synthetic 

Derivatives. 

My subsequent work in industry focused on the discovery and development of anti-inflammatory and 

immune modulating steroid hormones derived from the dihydroepiandrosterone (DHEA) metabolome 

for use as immune therapy. Part of this work involved the discovery and development of natural, and 

novel synthetic DHEA derivatives for treatment of infectious diseases. Compounds were screened in 

vitro and in vivo for immune modulating activity and tested in rodent models. 

I. Ahlem, C., Auci, D.L., Nicoletti, F., Pieters R., Kennedy, M., Page, T., Reading C., Enioutina, E., and 

Frincke., J.M., (2011). Pharmacology and immune modulating properties of androst-5-ene-1 3,7 ,17 

-triol, a 2 DHEA metabolite in the human metabolome. J Steroid Biochem Mol Biol. 2011 Sep;126(3-

5):87-94

II. Conrad, D., Wang, A., Pieters, R., Nicoletti, F., Mangano, K., van Heeckeren, A.M., White, S.K., 

Frincke, J.M., Reading, C.L., Stickney, D., and Auci, D.L., (2010) HE3286, an oral synthetic steroid, 

treats lung inflammation in mice without immune suppression. Journal of Inflammation, 7:52.

Contact PD/PI: Liu, Yingru

Biosketches Page 24
III. Nicoletti, F., Conrad, D., Wang, A., Pieters, R., Mangano, K., van Heeckeren, A., White, S.K., Frincke, 

J., Reading, C.L., Auci, D.L., and Stickney, D. (2009). 16-Bromoepiandrosterone (HE2000) limits 

non-productive inflammation and stimulates immunity in lungs. Clin. Exp. Immunol. 158:308-316

3. Development of encapsulated cytokines as treatment for immune mediated inflammatory and infectious 

disease. 

I joined TherapyX in May of 2014 and have been focused on the development of nanoparticulate 

enpasulated cytokines and small molecules such as all trans retinoic acid, TGF, IL-12, and IL-10 as 

oral or mucosal treatments for various inflammatory and infectious diseases. I successfully lead the 

development of scale up spray drying methods for TGFNanoCap, a nanoparticulate encapsulated 

form of TGF for oral delivery in collaboration with the team at Bend Research, Bend, OR. 

I. Conway, T.F., Hammer, L., Furtado, S., Mathiowitz, E., Nicoletti, F., Mangano, K., Eglimez, N.K., and 

Auci, D.L. Oral Delivery of Particulate Transforming Growth Factor Beta 1 and All-Trans Retinoic Acid 

Reduces Gut Inflammation in Murine Models of Inflammatory Bowel Disease. J Crohns Colitis. (2015) 

Aug;9(8):647-58. 

D. Research projects ongoing or completed in the past 3 years 

Ongoing: 

“Therapy and Prophylaxis for Genital Tract Infection”. NIH 2R44-AI104067-02. 7/1/2014-6/30/2016. 

Role: Co-PI ( Liu, PD/PI), 5% effort. Total costs: $ 1,498,000. 

“Delivery of nanoencapsulated TGFbeta and ATRA for the Treatment of IBD”. NIH 2R44-AI080009- 

02A1. 4/1/2011-3/31/2016. 

Role: PI, 45% effort. Total costs: $ 2,999,994. 

Contact PD/PI: Liu, Yingru

Biosketches Page 25
Contact PD/PI: Liu, Yingru

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Stacia (Chmura) Furtado

eRA COMMONS USER NAME (credential, e.g., agency login):

POSITION TITLE: Senior Scientist

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Brown University, Providence, RI Sc.B. 1995 Psychology

Brown University, Providence, RI Sc.M. 2001 Biology

Brown University, Providence, RI Ph.D. 2008 Biology

A. Personal Statement 

My primary field of study is the development of novel polymeric drug delivery systems by utilizing various 

methods of microencapsulation as well as the in vitro and in vivo characterization of controlled drug delivery 

systems. For this grant, I will be responsible for the encapsulation of IL-12 as well as for performing the 

physico-chemical analytical methods in characterizing the drug product in vitro and in vivo as needed. 

B. Positions and Honors 

2008-present Senior Research Scientist, TherapyX

inc, Buffalo, New York. 

2008-2009 Visiting Scientist, Brown University, Providence, RI. 

2007-2008 Senior Scientist, Freedom2, Inc., Providence, RI. 

C. Contribution to Science 

My current research interests include the development and characterization of novel microsphere based 

delivery systems specifically for local delivery of proteins such as insulin and growth factors as well as in the 

development of nanotechnology approaches for therapeutic applications such as infectious diseases, cancer 

and autoimmune or immune mediated inflammatory diseases.

1. Conway, T.F., Hammer, L., Furtado, S., Mathiowitz, E., Nicoletti, F., Mangano, K., Eglimez, N.K., and 

Auci, D.L. Oral Delivery of Particulate Transforming Growth Factor Beta 1 and All-Trans Retinoic Acid 

Reduces Gut Inflammation in Murine Models of Inflammatory Bowel Disease. J Crohns Colitis. (2015) 

Aug;9(8):647-58. 

2. Chung AY, Li Q, Blair SJ, De Jesus M, Dennis KL, LeVea C, Yao J, Sun Y, Conway TF, Virtuoso LP, 

Battaglia NG, Furtado S, Mathiowitz E, Mantis NJ, Khazaie K, Egilmez NK. Oral interleukin-10 

alleviates polyposis via neutralization of pathogenic T-regulatory cells. Cancer Res. 2014 Oct 

1;74(19):5377-85.

3. Yuval Ramot, Raul Brauner, Kongbin Kang, John V. Heymach, Stacia Furtado and Abraham Nyska. 

Quantitative Evaluation of Drug-induced Microvascular Constriction in Mice Kidney Using a Novel Tool 

for 3D Geometrical Analysis of Ex vivo Organ Vasculature. Toxicol Pathol. 2014 Mar 26;42(4):774-783.

4. Jingxuan Shan, Shoba DSousza, Sasha Bakhru, Eman Al-Azwani, Maria Ascierto, Konduru, Sastry, 

Shahinaz Bedri, Dhanya Kizhakayil, Idil, Aigha, Joel Malek, Issam Al-Bozom, Salah, Gehani, Stacia 

Furtado, Edith Mathiowitz, EnaWang, Francesc Marincola, and Lotfi Chouchane. TNRC9 downregulates BRCA1 expression and promotes breast cancer aggressiveness. Cancer Research. 2013. 

May 1;73(9):2840-9 

Biosketches Page 26
5. Sasha H. Bakhru, Stacia Furtado, A. Peter Morello & Edith Mathiowitz. Oral delivery of proteins by 

biodegradable nanoparticles. Advanced Drug Delivery Reviews. 2013 Jun 15;65(6):811-21.

6. Bryan Laulicht, Alexis Mancini, Nathanael Geman, Daniel Cho, Kenneth Estrellas, Stacia Furtado, 

Russell Hopson, Anubhav Tripathi, Edith Mathiowitz. Bioinspired Bioadhesive Polymers: Dopa-Modified 

Poly(acrylic acid) Derivatives. Macromol Biosci. 2012 Nov 12 (11):1555-65. 

7. Patel RS, Cho DY, Tian C, Chang A, Estrellas KM, Lavin D, Furtado S, Mathiowitz E. Doxycycline 

delivery from PLGA microspheres prepared by a modified solvent removal method. J Microencapsul. 

2012;29(4):344-52. 

8. Lavin DM, Zhang L, Furtado S, Hopkins RA, Mathiowitz E. Effects of protein molecular weight on the 

intrinsic material properties and release kinetics of wet spun polymeric microfiber delivery systems. 

Acta Biomater. 2012 Aug 16. 

9. Lavin DM, Stefani RM, Zhang L, Furtado S, Hopkins RA, Mathiowitz E. Multifunctional polymeric 

microfibers with prolonged drug delivery and structural support capabilities. Acta Biomater. 2012 

May;8(5):1891-900. 

10. Stacia Furtado, Danielle Abramson, Roxanne Burrill, Gloria Olivier, Celinda Gourd, Emily Bubbers, and 

Edith Mathiowitz, Oral Delivery of Insulin Loaded Poly(Fumaric-co-sebacic) Anhydride Microspheres, 

International Journal of Pharmaceutics, Volume 347, Issue 1-2, January 2008, Pages 149-55. 

11. Stacia Furtado, Danielle Abramson, Liat Simhkay, Daniel Wobbekind and Edith Mathiowitz, 

Subcutaneous Delivery of Insulin Loaded Poly(fumaric-co-sebacic anhydride) Microspheres to Type 1 

Diabetic Rats, European Journal of Pharmaceutics and Biopharmaceutics, Volume 63, Issue 2, June 

2006, Pages 229-236. 

Contact PD/PI: Liu, Yingru

Biosketches Page 27
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OMB No. 0925-0001 and 0925-0002 (Rev. 10/15 Approved Through 10/31/2018) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Mathiowitz, Edith 

eRA COMMONS USER NAME (credential, e.g., agency login):

POSITION TITLE: Professor of Medical Science and Engineering 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION DEGREE (if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Tel Aviv University, Israel B.Sc. 1973 Chemistry

Weizmann Institute of Science, Israel M.Sc. 1979 Physical Chemistry

Weizmann Institute of Science, Israel Ph.D. 1985 Physical Chemistry

A. Personal Statement 

Dr. Edith Mathiowitz is a full Professor of Medical Science and Engineering at Brown University in the 

Department of Molecular Pharmacology, Physiology, and Biotechnology. She is also the Director of the 

Biotechnology Graduate Program. Her extensive experience includes development of polymers that are used 

in drug and gene delivery systems and tissue engineering applications, including stem cell recruitment. She 

holds an undergraduate degree in chemistry from Tel Aviv University and a master’s and PhD in physical 

chemistry from the Weizmann Institute of Science. Dr. Mathiowitz trained as a postdoctoral associate with 

Professor Robert Langer at the Massachusetts Institute of Technology. She is a Member of the National 

Academy of Inventors and an AIMBE fellow. Dr. Mathiowitz work resulted in major publications, but also 

translated into two start-up companies, Spherics, Inc. and Perosphere, Inc., which focus their efforts on 

developments to improve patient compliance. She has advised about 30 Graduate students about the same 

number of master and honor students over 100 undergraduate students and three postdoctoral fellows. 

Dr. Mathiowitz’ role will be to supervise in the encapsulation of cytokine as well advise and oversee the 

physico-chemical analytical methods used in the analysis of the encapsulated protein. She has a long-standing 

collaborative relationship with TherapyX

Inc and will continue to offer advice for the nanoparticle production, 

scale-up, stability and consistency portions of this project. 

B. Positions and Honors

Positions and Employment 

1975-1979 Research Chemist at the Israel Institute of Biological Research, Nes Ziona, Israel, Polymer 

 Department, in collaboration with the late Dr. A. Raziel, on applied polymer research. 

1984-1986 Postdoctoral Fellow at the Department of Applied Biological Sciences, with Prof. R. Langer, 

 Massachusetts Institute of Technology. 

 1987-1989 Research Associate at the Department of Surgery, Children's Hospital, Harvard Medical School. 

1989-1991 Senior Research Scientist in Drug Delivery. Enzytech, Inc. 

1989-1991 Visiting Scientist at the Department of Chemical Engineering, Massachusetts Institute of 

Technology 

1991-1994 Assistant Professor of Medical Science, Director of Graduate Students, Division of Biology and 

Medicine, Section of Artificial Organs, Biomaterials and Cellular Technology. 

1994-1999 Associate Professor of Medical Science and Engineering, Department of Molecular 

Pharmacology, Physiology & Biotechnology, Director of Graduate Program - Artificial Organs, 

Biomaterials and Cellular Technology 

2000- Professor of Medical Science and Engineering, Department of Molecular Pharmacology, 

 Physiology & Biotechnology, Director of Graduate Program - Biotechnology 

Biosketches Page 28
Other Experience and Professional Memberships 

1987-89 Research associate at the Department of Surgery, Children’s Hospital, Harvard Medical School 

Controlled Release Society 2008-2011 Founder and CEO of Perosphere Inc. 

1997-99 President of start up company, Spherics 

1999-2005 Chairwomen and consultant, Spherics 

American Chemical Society (Polymer Division) 

American Association for the Advancement of Science 

Material Research Society Biomaterials Society 

American Society for Artificial Internal Organs (ASAIO) 

Honors 

1973 Distinction Prize for B.Sc. students (Tel Aviv University, Israel). 

1979-84 Feinberg Fellowship, Weizmann Institute of Science, Israel. 

1982 Delek Prize for distinctive research work (Weizmann Institute of Science, Israel). 

1985-87 Bantrell Postdoctoral Fellowship (MIT).A competitive award from MIT in the field of surface 

science. 

1991-93 Whitaker Foundation Award. 

1994 Recognition Award for Excellence in Guiding Graduate Student Research. Controlled Release 

Society - Procter & Gamble. Awarded in Nice, France. 

1997 Fellow AIMBE

2000 The Eurand Award for Excellence in Research in the Area of Oral Drug Delivery Systems. 

2013 Fellow of the National Academy of Inventors 

2015 Fellow of the Controlled Release Society 

C. Contribution to Science 

My current research interests include development and characterization of novel bioadhesive delivery systems, 

oral delivery of proteins such as insulin and growth factors, development of nanotechnology approaches for 

therapeutic applications, such as cancer, development of bone repair delivery systems, development of drug 

eluting vascular grafts, use of progenitor cell to redirect healing of vascular grafts and treatment of obesity, 

development of novel liquid crystals as smart sensory devices, and gene/DNA delivery. I have published over 

120 research articles, acquired 75 patents and acted as a consultant to biotech companies. 

1. The work related to novel bioadhesive delivery systems ranged from basic engineering of fundamental 

science to the measurement of tensile forces between tissue/materials interactions, to designing smart 

delivery systems including the bioadhesive novel polymers. The overall aim was to develop an effective 

oral delivery system for peptide and proteins based on safe, biocompatible, biodegradable nanoparticles; 

specifically, to develop engineered bioerodible nanoparticles that, once delivered to the mucosal tissue, are 

capable to penetrate the barrier, reach the epithelium, penetrate the cell, and distribute to internal organs. 

We have achieved this by developing an encapsulation technique, which first stabilizes sensitive proteins 

for delivery after oral administration; the polymer then degrades, thereby releasing the encapsulated 

protein. Additionally, nanoparticles were designed using a very specific bioadhesive coating that is capable 

of delivering the nanoparticles to intestinal tissue in order to enhance penetration and control the uptake. 

Recently, two major publications (PNAS, 2013 and JCR, 2013) describe this work. My lab demonstrated 

that the relatively high degree of microsphere uptake in the absorptive and non-absorptive epithelium 

indicates that endocytosis as well as phagocytotic mechanisms are responsible for the uptake of 

microspheres in the small intestine. These findings may potentially guide research aimed at delivering 

specific molecules to target organs. In addition, the methods and in vivo model that were used may also be 

useful to toxicologists who are interested in determining the fate or tissue distribution of microparticles, 

including whether such particles can cross the blood-brain barrier. 

a. E. Mathiowitz, J. Jacob, Y. Jong, G. Carino, D. Chickering, P. Chaturvedi, C. Santos, K. 

Vijayaraghavan, S. Montgomery, M. Bassett and C. Morrell, “Biologically Erodable Microspheres as 

Potential Oral Drug Delivery Systems,” Nature 386, 410-414, 1997. 

Contact PD/PI: Liu, Yingru

Biosketches Page 29
b. Laulicht B, Gidmark NJ, Tripathi A, Mathiowitz E. Localization of magnetic pills. Proc Natl Acad Sci 

U S A. 2011 Feb 8;108(6):2252-7. doi: 10.1073/pnas.1016367108. Epub 2011 Jan 21. PubMed 

PMID: 21257903; PubMed Central PMCID: PMC3038721 

c. Laulicht B, Tripathi A, Schlageter V, Kucera P, Mathiowitz E. Understanding gastric forces calculated 

from high-resolution pill tracking. Proc Natl Acad Sci U S A. 2010 May 4;107(18):8201-6. doi: 

10.1073/pnas.1002292107. Epub 2010 Apr 19. PubMed PMID: 20404209; PubMed Central PMCID: 

PMC2889561 

d. Joshua J. Reineke, Daniel Y. Cho, Yu-Ting Dingle, A. Peter Morello III, Jules Jacob,, 

Christopher G. Thanos, Edith Mathiowitz a Unique insights into the intestinal absorption, transit, 

and subsequent biodistribution of polymer-derived microspheres. PNAS, vol. 110 no. 34, 13803–

13808, 20 PubMed PMID: 23922388; PubMed Central PMCID: PMC3752225 

2. In the area of microencapsulation, my work focuses on using basic concepts of polymer phase separation 

phenomenon to design self-assembled microspheres, vascular grafts, and fiber reinforced by solvent 

induced crystallization. One example is the development of multi-walled microspheres. (Nature 1994) 

Many approaches for the controlled release of drugs involve incorporation of the drug molecules into the 

matrix of microscopic polymer spheres or capsules. Those existing methods for preparing such 

microparticles do not, however, always guarantee a constant release rate. For example, drug molecules 

may be trapped preferentially at the surface; they have to diffuse through an increasing thickness of 

polymer when the particles are non-eroding, or the surface area changes for eroding particles. In other 

situations, pulsed release may be required—an application to which simple polymer microspheres do not 

readily lend themselves. Our work on how to engineer multi-walled microspheres might solve some of 

these problems. In addition we have developed a one-step process for preparing double-walled polymer 

microspheres based on phase separation between polymer mixtures. With an appropriate choice of 

interfacial tensions and evaporation rates, a spherical droplet of one polymer becomes coated with a highly 

uniform layer of the other. 

a. Pekarek, J. Jacob and E. Mathiowitz “Double-walled Microspheres for Controlled Drug Release,” 

Nature, 367, 258-260, January 20, 1994. 

b. K. Pekarek, J. Jacob and E. Mathiowitz “One-step preparation of double-walled microspheres,” 

Advanced Materials, 6, No. 9, 684-687, 1994. 

c. Morello AP 3rd, Burrill R, Mathiowitz E. Preparation and characterization of poly(methyl 

methacrylate) - iron (III) oxide microparticles using a modified solvent evaporation method. J 

Microencapsul. 2007 Aug;24(5):476-91. PubMed PMID: 17578736.. 

d. Laulicht B, Cheifetz P, Mathiowitz E, Tripathi A. Evaluation of continuous flow nanosphere formation 

by controlled microfluidic transport. Langmuir. 2008 Sep 2;24(17):9717-26. 2008 Aug 6 

3. Nanomedicine: Bioadhesive delivery systems ultimately led to the development of bioadhesive 

nanoparticles. Research in my laboratory started by developing novel bioadhesive polymers, which are 

different, then the traditional hydrogels. It was the understanding that polymers such as polyanhydrides 

are more adhesive in general and better delivery systems for certain drugs, since they retain hydrophilic 

and hydrophobic drugs more effectively than hydrogels such as poly acrylic acid. Specifically, we observed 

that bioadhesion with those new polymers enhanced the residence time in rats, dogs, and pigs as well as 

enhanced the bioavailability of hydrophobic drugs. The question my group then asked was what will now 

happen if we made nanoparticles and deliver insulin and DNA. The results were astonishing and were 

initially received with a lot of criticism, yet Nature did accept the evidence and publish the data. From here, 

my group has focused on studding the mechanism of action and more specifically what type of polymers 

will be both useful and non-toxic. By utilizing bioadhesive nanoparticles, we increase the residence time of 

these nanoparticle delivery systems in the GI tract, thereby producing a greater chance of uptake of these 

particles by both Peyer’s Patches as well as by the enterocytes of the intestine with subsequent release 

into the blood. 

a. Conway TF, Hammer L, Furtado S, Mathiowitz E, Nicoletti F, Mangano K, Egilmez NK, Auci DL. 

Oral Delivery of Particulate Transforming Growth Factor Beta 1 and All-Trans Retinoic Acid 

Reduces Gut Inflammation in Murine Models of Inflammatory Bowel Disease. J Crohns Colitis. 2015 

Aug;9(8):647-58. doi: 10.1093/ecco-jcc/jjv089. Epub 2015 May 18. PubMed PMID: 25987350; 

PubMed Central PMCID: PMC4817304 

b. Chung AY, Li Q, Blair SJ, De Jesus M, Dennis KL, LeVea C, Yao J, Sun Y, Conway TF, Virtuoso 

LP, Battaglia NG, Furtado S, Mathiowitz E, Mantis NJ, Khazaie K, Egilmez NK. Oral interleukin-10 

alleviates polyposis via neutralization of pathogenic T-regulatory cells. Cancer Res. 2014 Oct 

Contact PD/PI: Liu, Yingru

Biosketches Page 30
1;74(19):5377-85. doi: 10.1158/0008-5472.CAN-14-0918. Epub 2014 Sep 16. PubMed PMID: 

25228656; PubMed Central PMCID: PMC4322772. N.Egilmez, Y.Jong, J.Jacobs, C.Santos, 

E.Mathiowitz, Y.Iwanuma, R.Bankert, “Cytokine immunotherapy of cancer with controlled release 

biodegradable microspheres in a human tumor xenograft/SCID mouse model, Cancer Immunology 

Immunotherapy, 1998 Mar; 46(1):21-4.

c. M.Kuriakose, F-A.Chen, N.Egilmez, Y.Jong, E.Mathiowitz, M.Delacure, “W.Hicks, T.Loree, 

R.Bankert, Interleuckin-12 delivered by biodegradable microspheres promotes the antitumor activity 

of human peripheral blood lymphocytes in a human head and neck tumor xenograft/SCID mouse 

model”, Head and Neck. 2000 Jan;22(1):57-63. 

4. Tissue engineering: The idea behind drug-eluting vascular grafts was to develop a process for the inclusion of 

polymer microspheres in microporous polyurethane tubes and membranes. Our laboratory developed a very 

unique way to make porous vascular grafts. This work focused on the development of small diameter vascular 

grafts by optimizing the process based on phase separation phenomenon, one of my favorite topics. These 

composites were fabricated via a spray, phase-inversion technique using polyurethane, and either spray-dried 

poly (d,l-lactide-co-glycolide 50:50) microspheres or commercially available fluorescent polystyrene-latex 

microspheres. We intended to release heparin to prevent clotting as this is a major hindrance with the 

development of small diameter vascular grafts. This project reflected my desire to not only work on traditional 

dosage forms but to develop complex systems for tissue engineering. 

The last topic in this area relates to obesity. The first clinically-accepted, autologous cell-based therapy 

(CarticelTM) aimed at augmenting a cell population via harvest, expansion, and re-implantation of chondrocytes 

into cartilage defects. Applying a similar principle to increase the brown adipocyte population could be a highly 

effective therapeutic strategy to combat obesity; however, the invasiveness of extraction and difficulties facing 

expansion of brown adipocytes complicate a straightforward extraction and expansion approach. Instead, here 

we propose recruiting adipose stem cells using microencapsulated soluble factors to concentrate a more easily 

expandable population of cells in an easily accessible subcutaneous pouch. In the past, our group has 

successfully recruited hematopoietic stem cells into subcutaneous pouches by eluting soluble factors within a 

single week. The proposed study is using the same principle of polymer microencapsulation elution of soluble 

factors to recruit adipose stem cells for in vitro expansion. 

a. Shan J, Dsouza SP, Bakhru S, Al-Azwani EK, Ascierto ML, Sastry KS, Bedri S, Kizhakayil D, Aigha 

II, Malek J, Al-Bozom I, Gehani S, Furtado S, Mathiowitz E, Wang E, Marincola FM, Chouchane L. 

TNRC9 downregulates BRCA1 expression and promotes breast cancer aggressiveness. Cancer 

Res. 2013 May 1;73(9):2840-9. doi: 10.1158/0008-5472.CAN-12-4313. Epub 2013 Feb 27. PubMed 

PMID: 23447579N.

b. Egilmez, Y.Jong, M.Sabel, J.Jacob, E.Mathioiwitz, R.Bankert, “In Situ Tumor Vaccination with 

Interleukin-12-encapsulated Biodegradable Microspheres:Induction of Tunor Regression and Patent 

Antitumor Immunity”, Cancer Research 60, 3832-3837, July, 2000 

c. Lavin DM, Zhang L, Furtado S, Hopkins RA, Mathiowitz E. Effects of protein molecular weight on 

the intrinsic material properties and release kinetics of wet spun polymeric microfiber delivery 

systems. Acta Biomater. 2013 Jan;9(1):4569-78. doi: 10.1016/j.actbio.2012.08.005. Epub 2012 Aug 

16. PubMed PMID: 22902813 

d. Laulicht B, Cheifetz P, Tripathi A, Mathiowitz E. Are in vivo gastric bioadhesive forces accurately 

reflected by in vitro experiments? J Control Release. 2009 Mar 4;134(2):103-10. doi: 

10.1016/j.jconrel.2008.11.012. Epub 2008 Nov 27. PubMed PMID: 19087887. 

5. I am not only interested in publications, but also in translating my work into two start-up companies, Spherics, 

Inc. and Perosphere, Inc., which focus their efforts on developments to improve patient compliance. Spherics, 

Inc. was based on my patents in the area of bioadhesion and was focused on developing a long-acting drug for 

the central nervous system, specifically for Parkinson’s disease. Perosphere. Inc. is a specialty 

pharmaceutical company with a strong foundation in rescue molecules. The lead drug candidate is a reversal 

agent for new oral anticoagulants (NOACs). There is currently no approved reversal agent for these NOACs. I 

have submitted as co-inventor seven patent applications in the area of risk molecules, vaccines, and charge 

masking agents for oral delivery. 

I was lucky to have motivated outstanding students in the Biotechnology and BME programs. Each one of my 

graduate students took a unique course, Experimental Surgery, which is taught at Brown by a dedicated team. 

Contact PD/PI: Liu, Yingru

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Contact PD/PI: Liu, Yingru

Thus, almost each one of my graduate students was able to perform preclinical studies that depended on 

funding from institutions such as NIH and JDRF. Surgeries were performed on mice, rats, dogs and pigs. The 

transfer from the lab to a startup company went smoothly because of the experience my graduate students had 

in performing proficient animal studies. 

My publication list is available following the links:

http://www.ncbi.nlm.nih.gov/sites/myncbi/18KSYwaci_o5w/bibliography/48079688/public/?sort=date&direction=

ascending

A. Research Support

Ongoing Research Support 

Therapy X Mathiowitz (PI) 06/01/11-05/31/17 

Oral Delivery of Nanoencapsulated TGF1 and All-trans-Retinoic Acid for the Treatment of Inflammatory Bowel 

Disease 

The major goal of this project is to complete pre-clinical formulation and treatment optimization studies in the 

murine adoptive T-cell transfer model of established IBD 

 

 

 

 

Completed Research Support 

 

 

7R01CA100656-11 Mathiowitz (PI) 05/01/2014 – 04/30/2016 

NIH/University of Louisville (Subaward) 

Integrating Innate and Adaptive Immunity in Cancer Therapy 

The major goal of this project to encapsulate cytokes for eradicating cancer. 

 

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BIOGRAPHICAL SKETCH 

Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2. 

Follow this format for each person. DO NOT EXCEED FOUR PAGES. 

NAME

Egilmez, Nejat K.

POSITION TITLE

Professor and Chairman

eRA COMMONS USER NAME

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.) 

INSTITUTION AND LOCATION DEGREE

(if applicable) YEAR(s) FIELD OF STUDY

University of Minnesota, Minneapolis/St. Paul B.S. 1980 Biochemistry

State University of New York at Buffalo M.A. 1983 Cell & Molec. Biology

State University of New York at Buffalo Ph.D. 1986 Cell & Molec. Biology

Louisiana St. Univ. Med. Ctr., New Orleans, LA Postdoc 1986-88 Biochem. & Molec. Biol.

A. Personal Statement 

I currently hold the position of Chairman in the Department of Microbiology and Immunology, School of 

Medicine, University of Louisville. I have led an independent research program since 2001 focusing on the 

study of tumor immunity, immune therapy and immune regulation. During this period I authored/co-authored 

52 peer-reviewed original research and invited review articles, and trained 10 pre- and postdoctoral students. 

My laboratory originally pioneered the use of sustained-release biodegradable nanoparticulate cytokine 

formulations, particularly that of IL-12, in cancer therapy. More recently, in collaboration with TherapyX, Inc. 

and the Russell laboratory at SUNY Buffalo, we have extended the use of particulate IL-12 adjuvants to the 

treatment of genital tract infections. Therefore I am uniquely situated to assist TherapyX

Inc in advancing the 

studies proposed in this phase II SBIR application. My role in this project will be to provide scientific guidance 

and oversight of the planned studies. 

B. Positions and Honors. 

1988-1994 Assistant Professor, Robert College/Department of Biology, Istanbul, Turkey 

1994-2001 Cancer Research Scientist I, II, Assistant Member, Department of Immunology, Roswell Park 

Cancer Institute, Buffalo, NY

2001-2003 Research Assistant Professor, Department of Microbiology and Immunology, State University of 

New York, Buffalo, NY

2003-2006 Assistant Professor, J.G. Brown Cancer Center and Department of Microbiology and 

Immunology, University of Louisville, Louisville, KY.

2006-2012 Associate Professor, Department of Microbiology and Immunology, School of Medicine and 

Biomedical Sciences, State University of New York, Buffalo, NY.

2012-2013 Professor, Department of Microbiology and Immunology, School of Medicine and Biomedical 

Sciences, State University of New York, Buffalo, NY. 

2013-present Professor and Chairman, Department of Microbiology and Immunology, School of Medicine, 

University of Louisville, Louisville, KY. 

Ad-hoc member NIH/TTT study section, October 2005. 

Ad-hoc member NIH/TME study section, February 2008, October 2008, October 2009, June 2010. 

Ad-hoc member NIH/ZATI SM13 study section, March 2009. 

Ad-hoc member NIH/ZRG1 OTC-F study section, June 2010. 

Ad-hoc member NIH/ZRG1 BCMB-A 51R study section, March 2014. 

Ad-hoc member NIH/ZRG1 OTC-C(02)M study section, October 2014 

Ad-hoc member NIH/ZRG1 F09B-B920) study section, March 2015 

Ad-Hoc member NIH/ZAT1 SM (37) L study section June 2015 

Chartered member NIH/TME study section 7/2010 – 6/2014. 

Editor-in-Chief, International Journal of Immunology and Immunotherapy 

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Associate Editor: Frontiers in Immunology, Immunological Investigations, Journal of Immunology Research 

Member American Association of Immunology 

American Association for Cancer Research 

International Society for Biological Therapy of Cancer 

American Association for the Advancement of Science 

C. Contributions to Science 

A full list of my published work can be found at: http://www.ncbi.nlm.nih.gov/pubmed/?term=egilmez+n

1. Molecular factors that contribute to cellular aging. 

My early postdoctoral work focused on delineating the cellular factors that contributed to replicative 

lifespan in unicellular organisms. These studies led to the advancement of the notion that certain ageassociated factors are preferentially retained in the progenitor cells conferring the “senescent” 

phenotype. This notion was subsequently validated by numerous laboratories with the discovery of 

several such factors including extrachromosomal rDNA circles and sirtuins. 

Egilmez, N.K., J.B. Chen and S.M. Jazwinski. 1990. Preparation and Characterization of Old Yeast 

Cells. J. Gerontol., 45:B9-17.

Egilmez, N.K., J.B. Chen and S.M. Jazwinski. 1989. Specific Alterations in Transcript Prevalence 

During the Yeast Life-span. J. Biol. Chem. 264:14312-14317.

Egilmez, N.K. and S.M. Jazwinski. 1989. Evidence for the Involvement of a Cytoplasmic Factor in 

the Aging of the Yeast Saccharomyces cerevisiae. J. Bacteriol. 171:37-42. 

2. Human tumor/TIL-SCID mouse xenograft models for the study of tumor immunity and immune therapy. 

My subsequent postdoctoral training was in tumor immunology and immune therapy. Part of this work 

involved the development and optimization of intact human tumor/TIL xenograft models for evaluation 

of cancer immune therapy as a bridge model between purely mouse models and clinical studies. Our 

studies established and validated the use of autologous PBL/tumor and more importantly intact human 

tumor biopsy with autologous TIL xenografts in SCID mice as novel in vivo models of human cancer for 

study of human tumor immunity and immunotherapy. 

Hess, S.D., Egilmez, N.K., Bailey, N., Anderson, T.M., Mathiowitz, E., Bernstein, S. H. and Bankert, 

R.B. 2003. Human CD4+ T cells present within the microenvironment of human lung tumors are 

mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFNgamma. J Immunol. 170(1):400-12. 

Egilmez, N.K., Hess, S.D., Chen, F-A, Takita, H., Conway, T. and Bankert, R.B. 2002. Human 

CD4+ Effector T-Cells Mediate an Indirect IL-12 and IFN--Dependent Suppression of Autologous Lung 

Tumor Xenografts in SCID Mice. Cancer Res. 62:2611-2617. 

Egilmez, N.K., Jong, Y.S., Hess, S.D., Jacob, J.S., Mathiowitz, E. and Bankert, R.B., 2000. 

Cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by 

human peripheral blood lymphocytes in the SCID/Winn model. J. Immunother. 23(2):190-195.

Egilmez, N.K., R. Cuenca, S.J. Yokota, Sorgi, F. and R.B. Bankert. 1996. In vivo Cytokine Gene 

Therapy of Human Tumor Xenografts in SCID Mice. Gene Therapy 3:607-614.

3. Sustained-release Micro/nanoparticulate protein adjuvants for the treatment of inflammatory disease. 

In the past decade and a half my laboratory has pioneered the development and use of injectable 

micro/nanoparticulate sustained-release formulations of immune-modulatory macromolecules in 

disease therapy. Our earlier work focused primarily on the development of these novel formulations for 

cytokine-based therapy of cancer, utilizing numerous cytokines including IL-2, IL-12, GM-CSF and 

Contact PD/PI: Liu, Yingru

Biosketches Page 34
Contact PD/PI: Liu, Yingru

TNF as an alternative approach to gene therapy. We have published extensively on the utility of 

these reagents in numerous transplantable and spontaneous murine as well as human tumor/SCID 

mouse xenograft models. These reagents are currently under development for clinical trials in cancer 

patients. 

Recent work evaluated the potential of such formulations as oral therapeutics in the treatment of gutassociated inflammatory disorders including gastrointestinal cancers and IBD. These studies not only 

established the utility of microparticulate IL-10 and TGF in therapy of gut dysplasia, but provided 

unique insight into the immunological basis of their activity. 

Finally, we demonstrated that intragenital application of particulate cytokine or antibody can act as a 

therapeutic vaccine. Specifically, this strategy induced the eradication of established disease and 

provided long-term protection from subsequent challenge in a murine model of Neisseria gonorrheae 

infection, extending the clinical utility of these reagents to infectious disease. 

Chung AY, Li Q, Blair SJ, De Jesus M, Dennis KL, LeVea C, Yao J, Sun Y, Conway TF, Virtuoso 

LP, Battaglia NG, Furtado S, Mathiowitz E, Mantis NJ, Khazaie K, Egilmez NK. Oral Interleukin-10 

Alleviates Polyposis via Neutralization of Pathogenic T-Regulatory Cells. Cancer Res. 2014 Oct 

1;74(19):5377-85.

Liu Y, Egilmez NK, Russell MW. 2013. Enhancement of Adaptive Immunity to Neisseria 

gonorrhoeae by Local Intravaginal Administration of Microencapsulated Interleukin 12. J Infect Dis. 

208(11):1821-9.

Hill, H.C., Conway, T.F., Sabel, M.S., Jong, Y.S., Mathiowitz, E., Bankert, R.B. and Egilmez, N.K. 

2002. Cancer Immunotherapy with Interleukin-12 and Granulocyte-Macrophage Colony-Stimulating 

Factor-encapsulated microspheres: Coinduction of innate and adaptive immunity and cure of 

disseminated disease. Cancer Res. 62:7254-7263. 

4. Role of regulatory rebound in defining the long-term efficacy of immune therapy. 

In the past decade our mechanistic focus was on a central conundrum of cancer immune therapy, i.e. 

the general failure of immune-based therapies to achieve durable tumor regression in cancer patients 

despite the ability to induce potent antitumor T-cell activity. We approached this problem from the 

unique perspective of a specific therapeutic, i.e. IL-12, a potent antitumor cytokine with robust activity in 

murine models but only transient effects in patients. Our studies delineated the post-IL-12 cellular 

immune events that occur in the tumor microenvironment and the associated immune structures 

revealing the critical role of feedback regulation in short-circuiting IL-12-induced antitumor T-cell 

cytotoxicity. We found that IFN, the secondary effector cytokine downstream of IL-12, drives not only 

the cytotoxic but also the regulatory mechanisms that ultimately shut down the effector response. 

Specifically, we defined the role of IL-12-IFN-Indoleamine 2, 3 Dioxygenase axis in promoting the 

post-treatment T-regulatory cell rebound that ultimately restored suppressive homeostasis in tumors. 

We are currently dissecting the molecular mechanisms that drive the immunogenic (IL-12+ IDO-) to 

tolerogenic (IL-12- IDO+) switch in DC phenotype in post-IL-12 animals. Our findings are conceptually 

in line with the recent recognition of the critical role of T-cell co-inhibitory/regulatory molecules (now 

termed immune checkpoint molecules) in pre-mature termination of antitumor T-cell cytotoxicity, a 

paradigm that has revolutionized the field of cancer immunotherapy. 

Harden JL, Gu T, Kilinc MO, Rowswell-Turner RB, Virtuoso LP and Egilmez NK. 2011. 

Dichotomous effects of IFN on Dendritic cell function determine the extent of Interleukin-12-driven 

antitumor T-cell immunity. J Immunol. 187(1):126-32.

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Contact PD/PI: Liu, Yingru

Gu T, Rowswell-Turner RB, Kilinc MO and Egilmez NK. 2010. Central role of IFN-Indoleamine 

2,3-dioxygenase in regulation of Interleukin-12-mediated antitumor immunity. Cancer Res. 70(1):129- 

138.

Nair RE, Kilinc MO, Jones SA and Egilmez, NK. 2006. Chronic immune therapy induces a 

progressive increase in intra-tumoral T-suppressor activity and a concurrent loss of tumor-specific 

CD8+ T-effectors in her-2/neu transgenic mice bearing advanced spontaneous tumors. J. Immunol. 

176(12):7325-34. 

D. Research projects ongoing or completed in the past 3 years 

Ongoing: 

“Integrating Innate and Adaptive Immunity in Cancer Therapy”, NIH, 2R01-CA100656-01A1, 

7/1/10 – 4/30/16. NCE

Role: Principal Investigator, 10% effort. Total direct costs: $ 

“Delivery of nanoencapsulated TGFbeta and ATRA for the Treatment of IBD”. NIH 2R44-AI080009. 

4/1/2011-3/31/2016. 

Role: Co-I (Auci PD/PI), 5% effort. Total costs: $ 

Completed: 

“Oral Immune-modulatory Adjuvants for Treatment of Colorectal Carcinoma”, NIH 1-R21-AI092133-01A1. 

7/1/2011 - 6/30/2014. 

Role: Principal Investigator, 10% effort. Total direct costs $ 

Intratumoral T-suppressor cell Homeostasis in Breast Cancer, DOD Breast Cancer Program / Pre-doctoral 

fellowship. 

BC093325 (Rowswell-Turner, PI) 12/15/09-12/14/12 

Role: Mentor, 5% effort. Total direct costs $ 

Biosketches Page 36
Contact PD/PI: Liu, Yingru

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors 

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME

RUSSELL, Michael W.

eRA COMMONS USER NAME (credential, e.g., agency login)

POSITION TITLE

Professor Emeritus

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable)

Completion date

MM/YYYY FIELD OF STUDY

Cambridge University, England B.A. 06/1966 Biochemistry

Cambridge University, England M.A. 05/1970

Reading University, England Ph.D. 06/1973 Microbiology

Guy's Hospital Medical and Dental Schools, 

London, England

Post-doc 05/1979 Immunology

A. PERSONAL STATEMENT 

I have >40 years of research experience in mucosal immunology especially in relation to the generation and 

functions of IgA antibodies, and immunity to infections of the mouth and genital tract. I have published 140 

peer-reviewed papers and 88 book chapters or conference reports, which have garnered a total of >9200 

citations (ResearchGate; h-index = 54). My current research interests focus on immunity to Neisseria 

gonorrhoeae. Previous investigations on the human immune response to N. gonorrhoeae led to a novel 

approach in the study of immunity to gonorrhea, and to the hypothesis that this well-adapted pathogen 

suppresses adaptive immune responses that might eliminate it and instead elicits innate responses that it can 

resist. My lab has demonstrated that IL-17 and Th17-related immune responses are generated in a mouse 

model of vaginal gonococcal infection, and that clearance of the infection is dependent in part upon signaling 

through the IL-17 receptor, whereas Th1/Th2 immunity is suppressed by a mechanism dependent on TGF, 

IL-10, and the generation of type 1 regulatory T cells. More recently, in collaboration with TherapyX

inc, we 

found that this immunosuppression can be reversed by the intravaginal delivery of the company’s 

nanoparticulate IL-12 formulation, which permits the development of protective immune responses. We are 

now pursuing a prophylactic vaccine approach accomplishing the same objective that should be safer for 

human use. Although now formally retired, I continue to maintain a research laboratory at SUNY Buffalo, and 

in my role as consultant, will continue to provide scientific and technical advice and oversight for this Phase II 

application. Our accomplishments over the past few years lead us to believe that we are at the cutting edge of 

this endeavor. 

1. Liu, Y., Feinen, B., and Russell, M.W. New concepts in immunity to Neisseria gonorrhoeae: innate 

responses and suppression of adaptive immunity favor the pathogen, not the host. Front. Microbiol. 2: 52 

(2011). PMCID:3153028 

2. Woof, J.M., and Russell, M.W. Structure and function relationships in IgA. Mucosal Immunol. 4: 590-597 

(2011). PMID:21937984 

3. Jerse, A.E., Bash, M.C., and Russell, M.W. Vaccines against gonorrhea: current status and future 

challenges. Vaccine 32: 1579-1587 (2014). PMCID:4682887 

4. Russell, M.W. Thinking globally, acting locally: harnessing the immune system to deal with recalcitrant 

pathogens. mBio 6: e00382-15 (2015). PMCID:4436072

B. POSITIONS AND HONORS 

Employment 

Biosketches Page 37
1968-1972 Research Student, Department of Bacteriology, National Institute for Research in Dairying, and 

Department of Microbiology, University of Reading, UK. 

1972-1979 Postdoctoral Associate (1978-9, Lecturer), Department of Oral Immunology and Microbiology, 

Guy's Hospital Medical and Dental Schools, London, UK.

1979-1981 Visiting Investigator, Institute of Dental Research, University of Alabama at Birmingham (UAB). 

1982-1986 Research Assistant Professor of Microbiology; Investigator, Inst. of Dental Research, UAB. 

1986-1992 Research Associate Professor of Microbiology, UAB. 

1987-1988 Visiting Associate Professor, Dept. of Oral Biology, Royal Dental College, Aarhus, Denmark. 

1992-2000 Research Professor of Microbiology and Oral Biology, UAB.

2000-2016 Professor of Microbiology & Immunology, and Oral Biology, University at Buffalo (UB) 

2016- Professor Emeritus 

Other Experience and Professional Memberships 

1976- Member, British Society for Immunology 

1976-2014 Member, International Association for Dental Research (IADR) 

1980- Member, American Society for Microbiology (ASM) 

1982- Member, American Association of Immunologists 

1987- Member, Society for Mucosal Immunology 

2004-2006 Director, Witebsky Center for Microbial Pathogenesis and Immunology, UB 

2004- Member, American Association for the Advancement of Science 

2007- Member, American Society for Reproductive Immunology 

2010 Research Scholar, Department of Medical Microbiology and Immunology, University of 

Wisconsin, Madison (on sabbatical leave from UB, June – November). 

Honors and Awards (selected) 

1963-1965 Scholar of St. John's College, University of Cambridge, UK

1996-1998 WHO/IUIS Nomenclature Committee, Subcommittee on IgA nomenclature 

1999-2010 R37 MERIT award, NIH/NIDCR 

2000 Fellow, American Academy of Microbiology 

2007 Distinguished Scientist Award for Research in Oral Biology, IADR 

C. Contributions to Science 

My contributions, spanning >40 years, have been predominantly in the field of mucosal immunity, especially 

with respect to the biological functions of IgA antibodies, immunity to infections of the mouth and genital tract, 

and novel approaches to the development of mucosal vaccines, including the use of heat-labile enterotoxins as 

adjuvants and vaccine delivery systems. Most recently my work has focused on immunity to Neisseria 

gonorrhoeae, novel approaches to therapy and development of a prophylactic vaccine against gonorrhea. This 

has been pursued in collaboration with the early-stage research and development company, TherapyX, Inc. 

(Buffalo, NY), for which I serve on the Board of Scientific Advisors and as a Consultant. 

1. As a postdoctoral scientist at Guy’s Hospital, London, I was a member of a group working on a vaccine 

against dental caries using a rhesus monkey model. While there I discovered and characterized protein 

antigens (AgI/II and AgIII) of the oral bacterium, Streptococcus mutans. AgI/II came to be the prototype of 

a family of streptococcal surface proteins, and was the first such protein to be crystographically resolved. 

In addition, I was tasked with purifying monkey IgM, IgG, and IgA, and the production of specific antibody 

reagents against them. This allowed us also to demonstrate the transudation of circulating Igs into the oral 

cavity through the gingival crevices, and to determine the circulating half-lives of Igs in this species. After 

moving to UAB, I continued to work on immunity to dental caries and my lab showed that oral or intranasal 

immunization with AgI/II was effective in generating protective mucosal antibody responses against S. 

mutans in rodent and monkey models. We also demonstrated the ability of S-IgA antibodies to inhibit S. 

mutans adherence to tooth-like sufaces. 

a. Russell, M.W., Bergmeier, L.A., Zanders, E.D., and Lehner, T. Protein antigens of Streptococcus 

mutans: Purification and properties of a double antigen and its protease-resistant component. Infect. 

Immun. 28: 486-493 (1980). 

b. Lehner, T., Russell, M.W., Caldwell, J., and Smith, R. Immunization with purified protein antigens from 

Streptococcus mutans against dental caries in rhesus monkeys. Infect. Immun. 34: 407-415 (1981). 

Contact PD/PI: Liu, Yingru

Biosketches Page 38
c. Hajishengallis, G., Nikolova, E., and Russell, M.W. Inhibition of Streptococcus mutans adherence to 

saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface 

protein antigen I/II: reversal by IgA1 protease cleavage. Infect. Immun. 60: 5057-5064 (1992). 

d. Katz, J., Harmon, C.C., Buckner, G.P., Richardson, G.J., Russell, M.W., and Michalek, S.M. Protective 

salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal 

immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin. Infect. Immun. 61: 

1964-1971 (1993). 

2. In contrast to the role of secretory IgA antibodies at mucosal surfaces, the biological significance of 

circulating IgA antibodies has remained incompletely understood. Intrigued by this on moving to UAB, I 

first demonstrated the ability of serum polymeric IgA to transport bound antigen through the liver into bile, 

thereby revealing a novel function of IgA antibodies in the non-inflammatory elimination of non-degradable 

antigens. Later, while on sabbatical in Denmark, I showed that complement activation by human IgA was 

essentially artifactual, being dependent on the interfacial denaturation of IgA, whereas IgA antibody-antigen 

complexes failed to activate complement. In contrast, human IgA antibodies, or their Fab fragments, 

inhibited complement activation by IgG antibodies, thereby affirming the essentially anti-inflammatory 

nature of IgA. Further elaboration of these studies on my return to UAB indicated that the glycan chains of 

interfacially aggregated IgA contributed to activation of the alternative complement pathway, and that the 

ability of IgA antibodies to promote phagocytosis by neutrophils depended on their activation status and 

expression of FcR.

a. Russell, M.W., Brown, T.A., and Mestecky, J. Role of serum IgA: Hepatobiliary transport of circulating 

antigen. J. Exp. Med. 153: 968-976 (1981). 

b. Russell, M.W., and Mansa, B. Complement-fixing properties of human IgA antibodies: alternative 

pathway complement activation by plastic-bound, but not by specific antigen-bound IgA. Scand. J. 

Immunol. 30: 175-183 (1989). 

c. Russell, M.W., Reinholdt, J., and Kilian, M. Anti-inflammatory activity of human IgA antibodies and their 

Fab fragments: inhibition of IgG-mediated complement activation. Eur. J. Immunol. 19: 2243-2249 

(1989). 

d. Nikolova, E.B., and Russell, M.W. Dual function of human IgA antibodies: inhibition of phagocytosis in 

circulating neutrophils and enhancement of responses in IL-8-stimulated cells. J. Leukocyte Biol. 57: 

875-882 (1995). 

3. Holmgren’s group at the University of Göteborg, Sweden, developed the first generation oral cholera 

vaccine (Dukoral) which consists of killed cholera vibrios plus the nontoxic B subunit of cholera toxin (CT). 

To determine whether other vaccine antigens could be “piggybacked” on to CTB, I embarked in 

collaboration with this group on studies of chemically coupling S. mutans AgI/II to CTB to generate a novel 

mucosal vaccine, exploiting the model of dental caries in rodents. We subsequently applied this to 

intranasal immunization and investigated the cellular immune responses in nasal lymphoid tissues, and 

further demonstrated the effectiveness of intranasal immunization for inducing disseminated mucosal 

immune responses in primates. We also developed a novel recombinant genetic approach to create 

nontoxic chimeric mucosal immunogens from AgI/II and CT. 

a. Czerkinsky, C., Russell, M.W., Lycke, N., Lindblad, M., and Holmgren, J. Oral administration of a 

streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary 

glands and extramucosal tissues. Infect. Immun. 57: 1072-1077 (1989). 

b. Wu, H.-Y. and Russell, M.W. Induction of mucosal immunity by intranasal application of a 

streptococcal surface protein antigen with the cholera toxin B subunit. Infect. Immun. 61: 314-322 

(1993). 

c. Hajishengallis, G., Hollingshead, S.K., Koga, T., and Russell, M.W. Mucosal immunization with a 

bacterial protein antigen genetically coupled to cholera toxin A2/B subunits J. Immunol. 154: 4322- 

4332 (1995). 

d. Russell, M.W., Moldoveanu, Z., White, P.L., Sibert, G.J., Mestecky, J., and Michalek, S.M. Salivary, 

nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial 

protein antigen and the cholera toxin B subunit. Infect. Immun. 64: 1272-1283 (1996). 

Contact PD/PI: Liu, Yingru

Biosketches Page 39
4. The development of mucosally administered vaccines, which are needed especially for infections of the 

gastrointestinal, respiratory, and genital tracts, is limited by the lack of suitable adjuvants for use by these 

routes. In collaboration with Connell’s group at UB, we first demonstrated that type II heat labile 

enterotoxins possessed similar adjuvant properties to CT, but with significant differences that were 

amenable to exploitation for vaccine development. Further development of these studies after I had moved 

to UB revealed the basis of the adjuvanticity of these type II enterotoxins and their mutants, and led to the 

discovery that their B subunits activated Toll-like receptor 2.

a. Martin, M.H., Metzger, D.J., Michalek, S.M., Connell, T.D., and Russell, M.W. Distinct cytokine 

regulation by cholera toxin and the type II heat-labile enterotoxins involves differential regulation of 

CD40 ligand on CD4+ T cells. Infect. Immun. 69: 4486-4492 (2001). 

b. Hajishengallis, G., Tapping, R.I., Martin, M.H., Nawar, H., Lyle, E.A., Russell, M.W., Connell, T.D. Tolllike receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins. Infect. 

Immun. 73: 1343-1349 (2005). PMCID:1064972 

c. Nawar, H., Arce, S., Russell, M.W., Connell, T.D. Mucosal adjuvant properties of mutant LT-IIa and LTIIb enterotoxins that exhibit altered ganglioside-binding activities. Infect. Immun. 73: 1330-1342 (2005). 

PMCID:1064923 

d. Arce, S., Nawar, H.F., Muehlinghaus, G., Russell, M.W., Connell, T.D. In vitro induction of IgA- and 

IgM-secreting plasma blasts by cholera toxin depends upon T cell help and is mediated by CD154 upregulation and inhibition of IFN- synthesis. Infect. Immun. 75: 1413-1423 (2007). PMCID:1828582 

5. For the past 20 years I have investigated immunity to Neisseria gonorrhoeae. This infection is widespread 

globally and is a major cause of reproductive tract damage in women. Moreover it can be contracted 

repeatedly as there is no induction of protective immunity against reinfection, and no vaccine is available. 

The continuing emergence of antibiotic resistance is raising concerns that gonorrhea could become 

untreatable. Initially at UAB we found that antibody and cytokine responses to uncomplicated genital tract 

infection in both men and women were minimal, suggesting that N. gonorrhoeae had the ability to interfere 

with the normal development of immune responses. Subsequent studies at UB have sustained this 

hypothesis, and we have demonstrated in a mouse model of genital tract infection that N. gonorrhoeae 

selectively induces Th17-driven innate immune responses that it can survive, and concomitantly 

suppresses Th1/Th2-driven adaptive immune responses by mechanisms dependent on the cytokines TGF- 

 and IL-10, and involving type 1 regulatory T cells. Our further studies have identified novel approaches 

to the treatment of gonococcal infection by the local administration of agents that reverse the induced 

immunosuppression, thereby affording a new approach to the treatment of infectious disease. These 

findings have also informed a novel approach to the development of a vaccine against gonorrhea, utilizing 

microencapsulated IL-12 as a Th1-driving adjuvant for a gonococcal outer membrane vesicle vaccine, 

which we are pursuing in this project. 

a. Liu, Y., and Russell, M.W. Diversion of the immune response to Neisseria gonorrhoeae from Th17 to 

Th1/Th2 by treatment with anti-TGF- antibody generates immunological memory and protective 

immunity. mBio 2: e00095-11 (2011). PMCID:3101786

b. Liu, Y., Islam, E., Jarvis, G.A., Gray-Owen, S., and Russell, M.W. Neisseria gonorrhoeae selectively 

suppresses the development of Th1 and Th2 cells, and enhances Th17 cell responses, through 

TGF--dependent mechanisms. Mucosal Immunol. 5: 320-331 (2012). PMCID:3328619 

c. Liu, Y., Egilmez, N.K., and Russell, M.W. Enhancement of adaptive immunity to Neisseria 

gonorrhoeae by local intravaginal administration of microencapsulated IL-12. J. Infect. Dis. 208: 

1821-1829 (2013). PMCID:3814831. 

d. Liu, Y., Liu, W., and Russell, M.W. Suppression of host adaptive immune responses by Neisseria 

gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells. Mucosal Immunol. 7: 165-176 

(2014). PMCID:3812424 

e. Liu, Y., Hammer, L., Hobbs, M.M., Zielke, R.A., Sikora, A.E., Jerse, A.E., Egilmez, N.K., Russell, 

M.W. (2016) Experimental vaccine induces Th1-driven immune responses and protection against 

Neisseria gonorrhoeae in a murine model. MS submitted to Mucosal Immunol. (August 2016). 

Complete List of Published Work (140 peer-reviewed publications and 88 book chapters) 

MyBibliography: http://www.ncbi.nlm.nih.gov/myncbi/collections/bibliography/41133423/public/

Contact PD/PI: Liu, Yingru

Biosketches Page 40
D. RESEARCH SUPPORT 

Current Research Support

R44-AI-104067-02 (SBIR Phase II) 

NIH/NIAID

Therapy and prophylaxis for genital tract infection

Y. Liu (PI) 02/01/13 – 05/31/17

(No-cost extension)

The goal of phase II is to develop a novel approach to treatment of genital gonococcal infection by the local 

administration of microencapsulated IL-12. 

Role: collaborating scientist. 

R43-AI115877-01 (SBIR Phase I) Y. Liu (PI) 12/01/14 – 11/30/16 

NIH/NIAID (No-cost extension) 

Experimental gonococcal vaccine 

The goal of this project is to establish proof-of-principle for a novel vaccine against genital gonococcal 

infection. 

Role: collaborating scientist. 

Completed Research Support 

R21-AI092348 M.W. Russell (PI) 12/03/10 – 11/30/13 

NIH/NIAID 

Imaging antigen and adjuvant uptake for enhancing response to mucosal vaccines 

The major goals of this project were to characterize the cells and tissues involved in the uptake and processing 

of enterotoxin adjuvants and recombinant chimeric immunogens based on enterotoxins when administered by 

mucosal routes, utilizing imaging flow cytometry. 

Role: PI 

Contact PD/PI: Liu, Yingru

Biosketches Page 41
Liu Current Support 

Current Research Support 

R44-AI-104067-02 (SBIR Phase II) Y. Liu (PI) 02/01/13 – 05/31/16 

NIH/NIAID 

Therapy and prophylaxis for genital tract infection 

The goal of phase II is to develop a novel approach to treatment of genital gonococcal infection 

by the local administration of microencapsulated IL-12.

Role: PI. 

R43-AI-115877-01 (SBIR Phase I) Y. Liu (PI) 12/01/14 – 11/30/16 

NIH/NIAID 

Experimental gonococcal vaccine 

The goal of this project is to establish proof-of-principle for a novel vaccine against genital 

gonococcal infection. 

Role: PI. 

Contact PD/PI: Liu, Yingru

Current and Pending Support Page 42
Contact PD/PI: Liu, Yingru

D. Research projects ongoing or completed in the past 3 years 

Ongoing: 

“Therapy and Prophylaxis for Genital Tract Infection”. NIH 2R44-AI104067-02. 7/1/2014-

6/30/2016. 

Role: Co-PI ( Liu, PD/PI), 5% effort. Total costs: $ 

“Delivery of nanoencapsulated TGFbeta and ATRA for the Treatment of IBD”. NIH 2R44-

AI080009- 

02A1. 4/1/2011-3/31/2016. 

Role: PI, 45% effort. Total costs: $ 

Current and Pending Support Page 43
Furtado, Current Support 

ACTIVE 

 

Therapy X NIH 2R44-AI104067-02 06/01/2011 – 05/31/2017 

Oral Delivery of Nanoencapsulated TGF1 and All-trans-Retinoic Acid for the Treatment of Inflammatory Bowel 

Disease 

The major goal of this project is to complete pre-clinical formulation and treatment optimization studies in the 

murine adoptive T-cell transfer model of established IBD Role: Senior Scientist. 

NIH/University of Louisville (Subaward 7R01CA100656-11 NCE) 05/01/2014 – 04/30/16 

Integrating Innate and Adaptive Immunity in Cancer Therapy 

The major goal of this project to encapsulate cytokes for eradicating cancer. Role:Senior Scientist 

Contact PD/PI: Liu, Yingru

Current and Pending Support Page 44
ACTIVE

(Mathiowitz) 06/01/2011 – 05/31/2017

Therapy X

Oral Delivery of Nanoencapsulated TGF1 and All-trans-Retinoic Acid for the Treatment of Inflammatory

Bowel Disease

The major goal of this project is to complete pre-clinical formulation and treatment optimization studies in the

murine adoptive T-cell transfer model of established IBD

NPRP 4-748-2-277 (Mathiowitz) 03/15/2012 – 03/14/2015

Qatar National Research Foundation

Nanotechnologies and Treatment of Obesity: From Polymeric Nanoparticle-Based Recruitment of Brown

Adipose Stem cells Toward Autologous Cell-Based Therapy.

The major goal of this project is to use the principle of polymeric nanoencapsulation and elution of soluble

factors to recruit adipose stem cells for in vitro expansion.

(Mathiowitz) 03/25/2014 – 03/24/2016

Takeda Pharmaceuticals, Inc.

Oral Formulation of Antibody for Gut Disease

The major goal of this project is developing oral delivery for large proteins...

(Mathiowitz) 07/21/2014 – 7/20/15

TADGEP, LLC

Characterization of Algae Delivery Systems Developed by TADGEP

The major goal of this project to elucidate the mechanism of action of proprietary technology developed by the

company.

7R01CA100656-11 (Mathiowitz) 05/01/2014 – 04/30/16 (NCE)

NIH/University of Louisville (Subaward)

Integrating Innate and Adaptive Immunity in Cancer Therapy

The major goal of this project to encapsulate cytokes for eradicating cancer.

(Mathiowitz) 12/01/2014 – 11/30/2017

Bio-Tree Systems, Inc.

Characterization of Vascular Casts Created using Polymeric Materials

The major goal of this project is to develop scaffold for vascular imaging....

Contact PD/PI: Liu, Yingru

Current and Pending Support Page 45
Contact PD/PI: Liu, Yingru

D. Research projects ongoing or completed in the past 3 years 

Ongoing: 

“Integrating Innate and Adaptive Immunity in Cancer Therapy”, NIH, 2R01-CA100656-01A1, 

7/1/10 – 4/30/16. NCE

Role: Principal Investigator, 10% effort. Total direct costs: $ 

“Delivery of nanoencapsulated TGFbeta and ATRA for the Treatment of IBD”. NIH 2R44-AI080009. 

4/1/2011-3/31/2016. 

Role: Co-I (Auci PD/PI), 5% effort. Total costs: $ 

Completed: 

“Oral Immune-modulatory Adjuvants for Treatment of Colorectal Carcinoma”, NIH 1-R21-AI092133-01A1. 

7/1/2011 - 6/30/2014. 

Role: Principal Investigator, 10% effort. Total direct costs $ 

Intratumoral T-suppressor cell Homeostasis in Breast Cancer, DOD Breast Cancer Program / Pre-doctoral 

fellowship. 

BC093325 (Rowswell-Turner, PI) 12/15/09-12/14/12 

Role: Mentor, 5% effort. Total direct costs $ 

Current and Pending Support Page 46
CURRENT SUPPORT 

Michael W. Russell 

Current Research Support 

R44-AI-104067-02 (SBIR Phase II) Y. Liu (PI) 02/01/13 – 05/31/16 

NIH/NIAID 

Therapy and prophylaxis for genital tract infection 

The goal of phase II is to develop a novel approach to treatment of genital gonococcal infection by the local 

administration of microencapsulated IL-12. 

Role: collaborating scientist. 

R43-AI115877-01 (SBIR Phase I) Y. Liu (PI) 12/01/14 – 11/30/16 

NIH/NIAID (No-cost extension) 

Experimental gonococcal vaccine 

The goal of this project is to establish proof-of-principle for a novel vaccine against genital gonococcal 

infection. 

Role: collaborating scientist. 

Completed Research Support 

R21-AI074791 M.W. Russell (PI) 02/01/09 – 01/31/13 

NIH/NIAID 

Gonococcal inflammatory immune responses 

The major goals of this project were to determine the significance of IL-17 in host responses to Neisseria 

gonorrhoeae in a mouse vaginal infection model. 

Role: PI 

R21-AI092348 M.W. Russell (PI) 12/03/10 – 11/30/13 

NIH/NIAID 

Imaging antigen and adjuvant uptake for enhancing response to mucosal vaccines 

The major goals of this project were to characterize the cells and tissues involved in the uptake and processing 

of enterotoxin adjuvants and recombinant chimeric immunogens based on enterotoxins when administered by 

mucosal routes, utilizing imaging flow cytometry. 

Role: PI 

Contact PD/PI: Liu, Yingru

Current and Pending Support Page 47
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date: 2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: TherapyX Inc.

Start Date*: 01-01-2017 End Date*: 12-31-2017 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Dr. Yingru Liu PD/PI 6.00

2 . Dominick Auci Co-PI 3.00

3 . Stacia Furtado Senior Scientist 9.00

4 . Michael Russell Consultant 2.40

5 . Nejat Eglimez Collaborating

Scienist

0.60 0.00 0.00 0.00

6 . Edith Mathiowitz Collaborating

Scientist

0.60 0.00 0.00 0.00

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Laura Hammer (Senior Research

Technician)

6.00

1 Julianny Perez (Research

Technician)

12.00

1 Samina Raza 1.20

3 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Liu, Yingru

 Page 48
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: TherapyX Inc.

Start Date*: 01-01-2017 End Date*: 12-31-2017 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 49
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: TherapyX Inc.

Start Date*: 01-01-2017 End Date*: 12-31-2017 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Overhead 40.00

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: 1234-

BudgetJustificationVaccinePhase II.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 50
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date: 2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  2

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: TherapyX Inc.

Start Date*: 01-01-2018 End Date*: 12-31-2018 Budget Period: 2

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Dr. Yingru Liu PD/PI 6.00

2 . Dominick Auci Co-PI 3.00

3 . Stacia Furtado Senior Scientist 6.00

4 . Michael Russell Consultant 2.40

5 . Nejat Eglimez Collaborating

Scienist

0.60 0.00 0.00 0.00

6 . Edith Mathiowitz Collaborating

Scientist

0.60 0.00 0.00 0.00

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Laura Hammer (Senior Research

Technician)

6.00

1 Julianny Perez (Research

Technician)

12.00

1 Samina Raza 1.20

3 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Liu, Yingru

 Page 51
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 2

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: TherapyX Inc.

Start Date*: 01-01-2018 End Date*: 12-31-2018 Budget Period: 2

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 52
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 2

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: TherapyX Inc.

Start Date*: 01-01-2018 End Date*: 12-31-2018 Budget Period: 2

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . overhead 40.00

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: 1234-

BudgetJustificationVaccinePhase II.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 53
THERAPYX, INC. BUDGET JUSTIFICATION

We feel that the number of personnel and the proposed effort are needed to complete the 

anticipated studies in the allotted 2 year period. The justification for each individual and their 

proposed percent effort is shown below. 

Personnel

Yingru Liu, M.D., Ph.D., P.I. (Effort: 50% Year 1, 50% Year 2) Dr. Liu was the PI on the original 

Phase I award which produced the results presented in this Phase II application. He is an 

experienced scientist with expertise in a wide range of experimental techniques relating to 

cellular and molecular biology, immunology, and microbiology. His training in Dr. Michael 

Russell’s laboratory at the University at Buffalo, which combines microbiological and 

immunological aspects, makes him uniquely qualified to continue the development of our novel 

prophylactic vaccine against gonorrhea, GvaX12®. Dr. Liu will work closely with Drs Furtado and 

Auci to coordinate microsphere production and validation. He will supervise Ms. Hammer and 

Mr Perez, and along with them, perform the in vivo mouse inoculations, microparticle therapy, 

tissue collection and the in vitro studies proposed here. He will also be responsible for data 

interpretation/evaluation and will address any emergent issues. He will also contribute to the 

production of regulatory documentation for submission to the FDA, participate in the type C 

meeting, and contribute to continued development plans. 

Dominick L. Auci MBA, Ph.D., Co-P.I. (Effort: 25% Year 1, 25% Year 2) Dr. Auci is an 

experienced immunologist with expertise in immune therapy of infectious disease, cancer, and 

immune mediated inflammatory diseases. He has extensive industrial and academic experience 

with drug development, IND creation and working with fee for service contract regulatory 

organizations. He has effectively directed the development of small molecules and biologicals, 

including phase I/II clinical trials. While at TherapyX

Inc, he has directed the successful 

completion of the SBIR phase I and phase II studies employing microparticles to treat cancer 

and Inflammatory Bowel Disease (IBD). He has overseen the proof of principle studies, 

optimization, toxicology and scale up of the company’s TGFβ and all-trans retinoic acid microparticle formulations for the treatment of IBD. He has led the company in its past dealings with 

the FDA, working with consultants from Regulatory Professionals and experts from Charles 

River Laboratories and Comparative Biosciences to plan and execute toxicology studies and 

produce regulatory documentation and meeting requests for the FDA. Dr. Auci has decades of 

experience managing and coordinating international research and development efforts at 

multiple locations. He will provide administrative direction and will assist Dr. Liu to ensure that 

the programmatic and scientific goals of this project are met as described in the application. 

Along with consultants, he will monitor toxicology studies, lead the production and submission of 

a type C meeting request to the FDA, the associated briefing package, interrogatives, and other 

regulatory documentation. He will lead the company’s team at the meeting and the subsequent 

formulation of development plans in response to agency recommendations. 

Stacia M Furtado, Ph.D. Senior Scientist (Effort: 75% Year 1, 50% Year 2) Dr. Furtado joined 

TherapyX

Inc in June 2008 and has contributed to several SBIR projects involving the 

development of oral microparticulate peptide/protein formulations for the treatment of diabetes 

and inflammatory bowel disease. She has extensive experience in microparticle design, 

production, and testing. She has produced the IL-12 microparticles used in the associated 

phase I studies. Dr. Furtado will perform all microparticle production and analytical testing (i.e., 

HPLC, gas chromatography, DSC, particle sizing, and FTIR) for drug product validation.

Contact PD/PI: Liu, Yingru

Budget Justification Attachment Page 54
Dr. Michael W. Russell, Ph.D., Consulting Scientist (Effort: 20% Year 1, 20% Year 2) Dr. 

Russell has over 40 years of research experience in mucosal immunology especially in relation 

to infections of the mouth and genital tract. While working with Dr. Russell, Dr. Liu has 

elucidated the mechanisms of suppression of Th1/Th2 immunity in gonococcal infection. These 

studies have led to the proposal of the novel approach to the treatment of gonococcal infection 

by redirecting host immunity using IL-12 microparticles. Dr. Russell will spend one day a week 

at TherapyX

Inc. and will serve as a consulting scientist on the both the murine studies (in vivo 

and in vitro) and toxicology plans proposed in this application, as well as in the preparation of 

regulatory documents and meetings with regulatory agencies. 

Nejat K. Egilmez, Ph.D., Executive Vice-President. (Effort: 5% Year 1, 5% Year 2) Dr. 

Egilmez has pioneered the development of the cytokine-encapsulated proteins for use in 

immunotherapy and has published in this area extensively with his collaborator Dr. Mathiowitz. 

Dr. Egilmez’s laboratory, which collaborates with TherapyX

Inc uses many of the 

cellular/molecular immune assays that will be used in the proposed studies and he will be 

available for consultation and technical advice when needed. 

Edith Mathiowitz, Ph.D., Collaborating Scientist. (Effort: 5% Year 1, 5% Year 2). Dr. 

Mathiowitz is an internationally recognized leader in the field of nano-encapsulation and drug 

delivery. She has led many projects involving the encapsulation of small molecule drugs, and 

proteins and is a leader and innovator in nano/microparticle formulation development. The 

formulation development studies detailed in the preliminary results section were designed and 

performed under her direction and supervision. TherapyX

Inc and Dr. Mathiowitz have a long 

standing relationship with past collaborations on nanoparticle-based delivery of cytokines for the 

therapy of cancer, nano-particulate vaccine adjuvant development and nano-particulate 

therapeutics. Dr. Mathiowitz will continue to offer guidance and support, particularly for drug 

product manufacturing and validation portions of this project as well as in the preparation of 

regulatory documents and meetings with regulatory agencies. 

Laura Hammer, B.S., Senior Research Technician. (Effort: 50% Year 1, 50% Year 2) Ms. 

Hammer has over 25 years’ of experience in molecular biology, pharmacology and immunology. 

Ms. Hammer has been with TherapyX

Inc for 9 years and along with developing in vitro bioassays 

for numerous cytokines including IL-12, she has worked extensively in FACS analysis, the in 

vitro testing of proteins and microparticle release assays. Ms. Hammer has contributed to 

previous TGFβ microparticle studies by conducting in vitro release bio-assays as well as FACS 

analysis and ELISA. She assisted Dr. Liu with the phase I in vivo and in vitro work and will 

continue to work closely with him and Mr. Perez to execute the proposed studies. 

Julianny Perez, B.S., Research Technician. (Effort: 100% Year 1, 100% Year 2) is a recent 

graduate with laboratory experience in Western Blotting, PCR, RNA isolation and several cellbased assays. He will assist Dr. Liu with the in vivo mouse inoculations, microparticle therapy 

and tissue collection. He will also assist Ms. Hammer with the in vitro microparticle studies 

including, bio-assays and ELISA. 

Samina Raza, Ph.D. Director of Operations (Effort: 10% Year 1, 10% Year 2) has been with 

the company since its inception in 2000. She will continue to oversee operations, administration 

and management efforts for the period of the award. She will work closely with Dr. Auci in 

budgeting, planning, accounting, as well as documentation and compliance with all state, 

federal and local regulatory agencies. 

Contact PD/PI: Liu, Yingru

Budget Justification Attachment Page 55
Contact PD/PI: Liu, Yingru

Animals, Materials and Supplies

The materials and supplies for the project consist of recombinant IL-12, microparticle polymer, 

HPLC Columns, β-Estradiol, Ceftriaxone, bacterial and cell culture materials, antibodies for 

FACS analysis and immunofluorescence, intracellular staining kits, ELISA kits and mice to 

conduct the studies proposed in the research design section. A detailed breakdown of expenses 

can be found below. 

*As per agreement with the Division of Comparative Medicine Laboratory Animal Facilities (CMLAF) at the State University of New York at Buffalo per diem charges for BSL2 mice housed in 

Micro-isolator cages are $0.58 per mouse per day. Please see the vertebrate animal section for 

the justification for the numbers of mice used. 

Equipment or Facility Rental/User Fees

Fluorescence Microscopy and flow cytometry

As per agreement with the Confocal Microscopy and Flow Cytometry core facility at the 

Witebsky Center at SUNY at Buffalo charges are $80.00 per hour for use of the Becton 

Dickinson FACSCalibur 4-color flow cytometer and the Zeiss LSM 510 Meta NLO Confocal 

Microscope ($ x 40 hours = $ 

Histology Fees

Histology services to processing and serial sectioning of samples for the studies described in 

aim 1A-II cost $32 per sample for outside concerns (20 mice x 4 tissues each = 80 x $ = 

$

$ + + = $146, 

Equipment Purchase 

We are requesting funds to purchase a replacement for an aging cell culture hood. We propose to 

purchase a CLASS II A2 4 115V package from Fisher Scientific at a cost of $6,872.62.

Animal Care and Use: Balb/c mice (792 x $30.00) $

Animal Care (14,256 days x $0.58/day)* $

$

Materials and Reagents: Recombinant mIL-12 (10 mg)

Microparticle Polymers and Solvents

HPLC Columns, vials, inserts etc.

Antibodies (Fluorochrome Labeled)

Intracellular Staining Kits

ELISA kits

Antibiotics, Estradiol

Agar, Culture Media, buffers etc.

Plastic ware, pipettes, syringes etc.

Gonococcal Ag, Prep. and ELISA

$

Total for animals, materials and reagents $

Budget Justification Attachment Page 56
Contact PD/PI: Liu, Yingru

YEAR 2

The materials and supplies needed for the second year of the project consist of recombinant IL12, microparticle polymer, β-Estradiol, Ceftriaxone, bacterial and cell culture materials, 

antibodies for FACS analysis and immunofluorescence, intracellular staining kits, ELISA kits 

and mice to conduct the studies proposed in the research design section. A detailed breakdown 

of expenses can be found below. 

Animals, Materials and Supplies

Animal Care and Use: Balb/c mice (604 x $30.00) $

Animal Care (21,744 days x $0.58/day)*

$

Materials and Reagents: Recombinant mIL-12 (5mg)

Microparticle Polymers and Solvents

Intracellular Staining Kits

ELISA kits

Antibiotics, Estradiol

Agar, Culture Media, buffers etc.

Plastic ware, pipettes, syringes etc.

Gonococcal Ag, Prep. and ELISA

$

Total for animals, materials and reagents $

*As per agreement with the Division of Comparative Medicine Laboratory Animal Facilities (CM- LAF) at 

the State University of New York at Buffalo per diem charges for BSL2 mice housed in Micro-isolator 

cages are $0.58 per mouse per day. Please see the vertebrate animal section for the justification for the 

numbers of mice used. 

Aim 2B Standard teratogenicity toxicity in rats (see Appendix 1)

Subcontracted to Comparative Biosciences..................................................$ 

Aim 2 C Standard Reproductive Toxicology 

Subcontracted to Comparative Biosciences...................................................$

TOTAL Subcontract costs.................................................................................

Consultant Fees ...............................................................................$

A pre-pre-IND meeting will be requested during last half of the second year of the project. The 

type C meeting will pose questions to the FDA concerning, the design of primate toxicology 

studies, and to confirm FDA expectations for data to support the Phase I clinical study, primarily 

with respect to safety. The consultants at Regulatory Professionals have extensive expertise in 

the preparation of clinical (IND) and marketing applications for various drug formulations, 

including biologics and polymeric drug delivery systems, and are especially well qualified to 

guide us through this process (see Appendix 2).

Budget Justification Attachment Page 57
Contact PD/PI: Liu, Yingru

Budget Justification Attachment Page 58
Contact PD/PI: Liu, Yingru

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 6

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment

Section D, Travel

1. Domestic

2. Foreign

Section E, Participant/Trainee Support

Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

6. Number of Participants/Trainees

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

Fees

7. Alterations and Renovations

8. Other 1

9. Other 2

10. Other 3

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

 Page 59
Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302. Received Date:

2016-08-24T11:01:24.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

Program Type (select only one)*

● SBIR ❍ STTR ❍ Both  (See agency-specific instructions to determine whether a particular agency allows a single submission for

both SBIR and STTR)

SBIR/STTR Type (select only one)*

❍ Phase I ● Phase II ❍ Fast-Track (See agency-specific instructions to determine whether a particular agency participates in FastTrack)

Questions 1-7 must be completed by all SBIR and STTR Applicants:

1a. Do you certify that at the time of award your organization will meet the eligibility criteria for a

small business as defined in the funding opportunity announcement?*

● Yes ❍ No

1b. Anticipated Number of personnel to be employed at your organization at the time of award.* 10

2. Does this application include subcontracts with Federal laboratories or any other Federal

Government agencies?*

❍ Yes ● No

If yes, insert the names of the Federal laboratories/agencies:*

3. Are you located in a HUBZone? To find out if your business is in a HUBZone, use the mapping

utility provided by the Small Business Administration at its web site: http://www.sba.gov *

❍ Yes ● No

4. Will all research and development on the project be performed in its entirety in the United

States?*

● Yes ❍ No

If no, provide an explanation in an attached file. Explanation:*

5. Has the applicant and/or Program Director/Principal Investigator submitted proposals for

essentially equivalent work under other Federal program solicitations or received other Federal

awards for essentially equivalent work?*

❍ Yes ● No

If yes, insert the names of the other Federal agencies:*

6. Disclosure Permission Statement: If this application does not result in an award, is the

Government permitted to disclose the title of your proposed project, and the name, address,

telephone number and e-mail address of the official signing for the applicant organization, to

organizations that may be interested in contacting you for further information (e.g., possible

collaborations, investment)?*

● Yes ❍ No

7. Commercialization Plan: If you are submitting a Phase II or Phase I/Phase II Fast-Track Application, include a Commercialization

Plan in accordance with the agency announcement and/or agency-specific instructions.*

Attach File:* 1259-Commercalization Plan GC FINAL.pdf

Contact PD/PI: Liu, Yingru

 Page 60
Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302. Received Date:

2016-08-24T11:01:24.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

SBIR-Specific Questions:

Questions 8 and 9 apply only to SBIR applications. If you are submitting ONLY an STTR application, leave questions 8 and 9 blank and

proceed to question 10.

8. Have you received SBIR Phase II awards from the Federal Government? If yes, provide a

company commercialization history in accordance with agency-specific instructions using this

attachment.*

● Yes ❍ No

Attach File:* 1260-COMMERCIALIZATION STATEMENT.pdf

9. Will the Project Director/Principal Investigator have his/her primary employment with the small

business at the time of award?*

● Yes ❍ No

STTR-Specific Questions:

Questions 10 and 11 apply only to STTR applications. If you are submitting ONLY an SBIR application, leave questions 10 and 11

blank.

10. Please indicate whether the answer to BOTH of the following questions is TRUE:*

(1) Does the Project Director/Principal Investigator have a formal appointment or commitment either with the small business directly

(as an employee or a contractor) OR as an employee of the Research Institution, which in turn has made a commitment to the small

business through the STTR application process; AND

(2) Will the Project Director/Principal Investigator devote at least 10% effort to the proposed project?

❍ Yes ❍ No

11. In the joint research and development proposed in this project, does the small business

perform at least 40% of the work and the research institution named in the application perform at

least 30% of the work?*

❍ Yes ❍ No

Contact PD/PI: Liu, Yingru

 Page 61
1 | P a g e

COMMERCIALIZATION PLAN 

 

The rapid spread of antimicrobial resistance throughout the U.S. and the world is considered a major threat to 

public health and national security. The President has emphasized that tackling this issue is a priority; in 

September 2014, the White House introduced the President’s National Strategy to Combat Antibiotic Resistant 

Bacteria (CARB), released concurrently with the President’s Council of Advisors on Science and Technology 

(PCAST) report and recommendations to the President on combating antibiotic resistance.1

 Together, these 

reports identify priorities and guide coordination across U.S. government agencies for 1) better prevention and 

response to the spread of antibiotic resistance; 2) increased surveillance of emerging antibiotic resistance in 

humans, animals, and the environment; 3) improved capability for detection and diagnostics; 4) accelerated 

development of new products, including new classes of antibiotics, therapeutics, and vaccines; and 5) 

enhanced international collaboration.39 The federal commitment to addressing antimicrobial resistance was 

further emphasized by a Presidential budget request to Congress for $1.2 billion to support these efforts.2

 

One disease that is particularly resistant, Neisseria gonorrhoeae (i.e., gonorrhea) is known to be acquired 

repeatedly with apparently no development of protective immunity resulting from previous infection. The 

reasons for this are poorly understood, but the generally accepted view is that this pathogen has an 

extraordinary capacity of for antigenic variation, involving most of its surface antigens. Coupled with multiple 

mechanisms for resisting complement, N. gonorrhoeae can evade whatever adaptive immune responses the 

host develops against it. Although these considerations imply that the development of a successful vaccine 

may be improbable, studies using a mouse model of vaginal gonococcal infection have led to a different view: 

that N. gonorrhoeae (Ngo) selectively elicits innate host defenses that it can survive and concomitantly 

suppresses adaptive immune responses that would eliminate it. These findings put comprehension of immunity 

to N. gonorrhoeae in a completely new light, and moreover reveal novel approaches to treatment and 

prevention based on the manipulation of cytokines that control Th1, Th2, and regulatory immune responses. 

TherapyX

inc’s goal is to generate safe and effective sustained-release nano-particulate immunotherapeutics for

the treatment of inflammation and infectious disease. Our lead product, GvaX12®, is a nanoencapsulated 

interleukin 12 (IL-12) plus outer membrane vesicles (OMV) formulated for mucosal delivery. This vaccine is 

intended to enhance specific adaptive immune responses that provide lasting, protective immunity against 

genital tract infection with heterologous strains of N. gonorrhoeae. The goals of this commercialization plan are 

to provide information regarding US gonorrhea infections, identify the target market for vaccine 

commercialization, address market strategy, discuss industry outreach efforts, and measure the value such a 

vaccine would have for both health organizations and TherapyX

Inc

. 

1. Gonorrhea Overview 

Sexually transmitted diseases (STDs) affect people in all walks of life. More than half of all Americans will be 

infected with an STD at some point in their lifetime.

3 STDs are a substantial health challenge facing the United 

States, as an estimated 110 million US men and women have contacted STDs.4 Because the majority of

infections may go undiagnosed and unreported, the true burden of STD’s in America is likely far greater than 

reporting would indicate. Reported cases of the three nationally notifiable STDs – chlamydia, gonorrhea, and 

syphilis – have increased for the first time since 2006, according to data published by the Center for Disease 

Control and Prevention (CDC) in the 2014 STD Surveillance Report. Each of these infections is a threat to an 

individual’s immediate and long-term health and well-being. America’s worsening STD epidemic is a clear sign 

that better diagnosis, treatment, and prevention techniques are urgently needed.

Young people aged 15-24 are particularly at risk for STDs, accounting for half of the nearly 20 million new

cases that occur each year, according to the CDC. In this age segment, STDs account for nearly $16 billion in 

health care costs.3 Thus, preventing STDs among youth is a key priority.

 

1 The White House. “National Action Plan for Combating Antibiotic Resistant Bacteria.” 2015

2 President’s Council of Advsiors on Science and Technology. Report to the President on Combating Antibiotic Resistance. 2014. 

3 Koutsky L. (1997). Epidemiology of genital human papillomavirus infection. American Journal of Medicine, 102(5A), 3-8.

4 Satterwhite CL, et al. Sexually transmitted infections among U.S. women and men: Prevalence and incidence estimates, 2008. Sex Transm Dis 2013; 

40(3): pp. 187-193

Contact PD/PI: Liu, Yingru

Commercialization Plan Page 62
2 | P a g e

Gonorrhea, an STD caused by the N. gonorrhoeae bacterium, infects the mucous membranes of the 

reproductive tract, including the cervix, uterus, and fallopian tubes in women, and the urethra in women and 

men.5 It can also infect the mucous membranes of the mouth, throat, eyes, and rectum. Complications from 

gonorrhea infections are frequent and debilitating, and disproportionately affect women.

The surge in antimicrobial resistant strains of the gonorrhea organism is threatening the effectiveness of

treatment both in the United States and internationally. While medication for gonorrhea has been available for 

decades, the bacteria have grown resistant to every drug ever used to treat it. To ensure that available 

antibiotics remain effective for as long as possible, a multi-faceted approach will be required. However, the role 

of vaccines in preventing bacterial infections (as well as the potential role of vaccines (direct and indirect) in 

reducing the overall use of antibiotics) is surprisingly under-represented in both discussion and action to date. 

Vaccines not only prevent infectious diseases before they occur, thereby eliminating the need for antibiotic 

treatments and unnecessary contacts with the healthcare system, but also decrease circulation of the 

pathogen, which protects the community under the well-established principle of “herd immunity”.

Complications: The first symptoms are usually discrete and occur over a period of two to ten days after 

sexual intercourse with an infected partner. Often, no symptoms develop in people already infected with 

gonorrhea: 10 to 15% of men and up to 80% of women do not show any symptoms at all with new 

infections.

6

In sexually active individuals, gonorrhea can cause clinically unapparent mucosal infections, 

symptomatic urethritis and cervicitis, and upper urogenital tract infections. Systemic or disseminated 

gonococcal infections (DGI) are infrequent (0.5–3%), occur mainly in women, and include a characteristic 

gonococcal arthritis-dermatitis syndrome, suppurative arthritis, and (rarely) endocarditis, meningitis or other 

localized infections.

7

In men ~15% of untreated urethritis cases progress to epididymitis, a common cause of

male infertility.8In women, untreated cervical infections commonly progress to the upper reproductive tract, 

which contributes to pelvic inflammatory disease (PID) and HIV:5

 Pelvic Inflammatory Disease (PID) is the most common complication associated with gonorrhea, 

resulting in an estimated 26% of cases; ~4.2% of U.S. women report being treated for PID in their 

lifetime.

9 PID results from the ascension of microorganisms from the cervix and vagina to the upper 

genital tract. The wide variation in symptoms and signs associated with PID can make diagnosis 

challenging; when symptoms are mild, PID can go unnoticed by women and their health care providers.

No single historical, physical, or laboratory finding is both sensitive and specific to the diagnosis of 

PID.

7 Direct medical expenditures for PID and its sequelae have been estimated at $2.7 billion.7

 Infertility: PID can lead to infertility and permanent damage of a woman’s reproductive organs. While 

recurrent episodes of PID are associated with a greater risk of infertility,10 even subclinical PID has 

been associated with infertility.11 Perhaps the most significant burden attributed to PID comes from the 

long-term reproductive sequelae of tubal infection: tubal factor infertility, ectopic pregnancy, and pelvic 

adhesions, which can lead to chronic pelvic pain. Prompt antibiotic treatment can prevent severe 

damage to the reproductive organs, but is not guaranteed. Women diagnosed with PID are six times 

more likely to have ectopic pregnancy, are 18% more likely to experience chronic pelvic pain after a 

single episode, and are increasingly likely to suffer from tubal factor infertility, with incidence ranging 

from 8% after the first episode to as high as 40% after three episodes.7 At least 15 percent of all 

American women who are infertile can attribute it to tubal damage caused by PID.12

 

 

5 Center for Disease Control. “Gonorrhea – CDC Fact Sheet” (2016)

6 Center for Disease Control. “ Pelvic Inflammatory Disease (PID) – CDC Fact Sheet” (2016)

7 Jerse, Ann, Margaret C. Bash, Michael W. Russell. “Vaccines against Gonorrhea: Current status and Future Challenges” Vaccine. 2014 Mar 20; 

32(14): 1579–1587

8 Chesson, Harrell et al. “The estimated Direct medical cost of sexually transmitted diseases among American youth, 2000” Perspectives on Sexual and 

Reproduction Health Volume 36, Issue 1 January/February 2004.

9 Satterwhite CL, et al. Sexually transmitted infections among U.S. women and men: Prevalence and incidence estimates, 2008. Sex Transm Dis 2013; 

40(3): pp. 187-193

10 Westrom L, Incidence, prevalence, and trends of acute pelvic inflammatory disease and its consequences in industrialized countries. Am J Obstet 

Gynecol 1980; 138:880-892.

11 Wiesenfeld HC, Hillier SL, Meyn LA, Amortegui AJ, and Sweet RL. Subclinical pelvic inflammatory disease and infertility. Obstet & Gyn, 2012; 120:37-

43.

12 Ness RB et al. (2004). Condom use and the risk of recurrent pelvic inflammatory disease, chronic pelvic pain, or infertility following an episode of 

pelvic inflammatory disease. American Journal of Public Health, 2004, 94:1327-1329.

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 Gonorrhea & HIV. Untreated gonorrhea can increase a person’s risk of acquiring or transmitting HIV, 

the virus that causes Acquired Immune Deficiency Syndrome (AIDS). Untreated gonorrhea can make a 

person with HIV more infectious, because untreated STD’s can increase the HIV viral load in genital 

fluids.13 Having gonorrhea also can make it more likely that an HIV-negative person will be infected with 

HIV if they are exposed to the virus.11

In summary, it is extremely important to prevent or treat gonorrhea in the early stages of the disease, 

especially in high-risk populations.

Occurrence: In 2014, 350,062 cases of gonorrhea were reported; however, the CDC estimates that 

approximately 820,000 new gonorrheal infections occur in the United States each year. The discrepancy

between the two numbers is due to infections being undetected (asymptomatic) and/or unreported.

14 This 

makes gonorrhea the second most commonly reported STD behind only chlamydia (~1.4M). The overall US 

gonorrhea infection rate has increased 5.1% since 2013, and increased 10.5% since 2010.

15 Furthermore, the 

World Health Organization (WHO) estimates that globally 106 million new gonorrhea cases occur among 

adults each year, representing a 21% increase in just three years.

16 Gonorrhea disproportionately affects

certain groups of individuals more than others when factors of age, sex, location, race and socioeconomic 

status are considered.:

2

 Age: 2014 CDC data (Figure 1) shows that youths are at the highest risk of acquiring an STD, 

accounting for half of the estimated 20 million new STDs diagnosed each year. This is especially true 

for gonorrhea:

17 despite being a relatively small portion of the sexually active population, young people 

between 15 and 24 accounted for nearly two-thirds of gonorrhea cases, estimated at ~570,000.

15

Young individuals are at a greater risk for several reasons: young women’s bodies are biologically more 

susceptible to STDs because of increased cervical ectopy; some young people do not get the 

recommended STD tests; many young people are hesitant to talk openly and honestly with a doctor or 

nurse about their sex lives; the absence of insurance or transportation can make it more difficult for 

young people to access STD testing; and some young people have more than one sex partner.

18

 Sex: In 2014 (Figure 2) the rate of reported gonorrhea cases among men (120.1 cases per 100,000 

males) was higher than the rate among women (101.3 cases per 100,000 females).15 Between 2013 

and 2014, the gonorrhea rate among men increased 10.5%, while the rate among women decreased 

0.4%. During 2010–2014, the rate among men increased 27.9%, while the rate among women 

decreased 4.1%.15 “The magnitude of the increase among men compared with a decrease among 

women suggests either increased transmission or increased case ascertainment among gay, bisexual, 

and other men who have sex with men (collectively MSM). However, most jurisdictions do not routinely 

report the sex of the partner or the site of infection for gonorrhea cases; hence, trends in gonorrhea 

rates among MSM over time cannot be assessed.”15

 

13 Fleming D, Wasserheit J. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to 

sexual transmission of HIV infection. Sex Transm DIs, 75(1), 3–17 (1999).

14 Centers for Disease Control and Prevention. “Sexually Transmitted Disease Surveillance 2014”

15 Satterwhite CL, et al. Sexually transmitted infections among U.S. women and men: Prevalence and incidence estimates, 2008. Sex Transm Dis 2013; 

40(3): pp. 187-193

16 Magnus Unemo and Robert A Nicholas. “Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea.” Future Microbiol. 

2012 Dec; 7(12): 1401–1422

17 Center for Disease Control. “Reported STDs in the United States: 2014 National Data for Chlamydia, Gonorrhea, and Syphilis” (2014)

18 Center for Disease Control. “Information for Teens & Young Adults: Staying Healthy and Preventing STDs.”

Figure 1: Rates of Reported Cases by 

Age and Sex, United States, 2014

Figure 2: Rates of Reported Cases by 

Sex, United States, 1994–2014

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 Location: In 2014, rates of reported gonorrhea cases 

per 100,000 in population ranged by state (Figure 3), 

from a low of 13.4 in Vermont to a high 194.6 in 

Louisiana. In the District of Columbia the gonorrhea rate 

was 291.3 per 100,000 population.

15 Between 2013 and 

2014, gonorrhea rates increased in 70% (35/50) of the 

states and decreased in 30% (15/50) of the states and 

the District of Columbia.

15 In 2014, as in previous years, 

the South had the highest rate of reported gonorrhea 

cases (averaging 131.4 cases per 100,000 population) 

among the four US regions, followed by the Midwest 

(106.6 cases per 100,000 population), West (101.1

cases per 100,000 population), and Northeast (84.7 cases per 100,000 population).

15 Between 2013 

and 2014, the gonorrhea rate increased 22.2% in the West and 3.1% in the South, but decreased 1.5% 

in the Midwest and 0.6% in the Northeast.19

The overall rate of reported gonorrhea cases in the 50 most populous metropolitan statistical areas 

(MSAs) was 122.8 cases per 100,000 population in 2014, representing a 5.0% increase compared with 

2013 (117.0 cases per 100,000 population).

17 In 2014, 60.6% of reported gonorrhea cases were 

reported by these MSAs.

17 Since 2010, the gonorrhea rate among women in the 50 most populous 

MSAs has been lower than the rate among men. In 2014, the rate among women in these MSAs was 

102.0 cases per 100,000 females, while the rate among men was 144.1 cases per 100,000 males.

17

 Race: In 2014, among the 48 states that submitted data on 

race and ethnicity (Figure 4), the rate of reported gonorrhea 

cases remained highest among blacks (405.4 cases per 

100,000 population), 10.6 times the rate among whites (38.3 

cases per 100,000 population). The gonorrhea rate among 

American Indians/Alaska Natives (159.4 cases per 100,000 

population) was 4.2 times that of whites, the rate among 

Native Hawaiians/Other Pacific Islanders (102.1 cases per 

100,000 population) was 2.7 times that of whites, the rate 

among Hispanics (73.3 cases per 100,000 population) was 

1.9 times that of whites, and the rate among Asians (19.3 

cases per 100,000 population) was 0.5 times that of whites.17

During 2010–2014, among the 43 states that submitted race 

and ethnicity, the gonorrhea rate increased among American Indians/Alaska Natives (100.3%), whites 

(59.3%), Hispanics (51.2%), Asians (45.3%), and Native Hawaiians/Other Pacific Islanders (44.2%). 

During this same time period, the gonorrhea rate decreased 8.2% among blacks.17

 Socioeconomic Status: Gonorrhea infections tend to cluster in geographically definable risk spaces, 

often located in low socioeconomic status (SES) urban neighborhoods, suggesting that socio-cultural 

determinants may influence the clustered spatial pattern observed for the infection.20 Prevalence of the 

infection has a direct impact on the incidence of infection. Low SES can impair timely access to 

services in risk spaces, thereby increasing the duration of infection and, ultimately, the prevalence of 

infection within a sexual network. Neighborhoods representing the lowest SES quartile accounted for 

58% of reported gonorrhea cases, and individuals from the lowest SES neighborhoods were more than 

four times more likely to acquire infection than those from the highest SES neighborhoods21

 

19 Center for Disease Control. “Reported STDs in the United States: 2014 National Data for Chlamydia, Gonorrhea, and Syphilis”

20 Ashleigh B Sullivan et al “Are neighborhood socio-cultural factors influencing the spatial pattern of gonorrhea in North Carolina.” Ann Epidemiol. 2011 

Apr; 21(4): 245–252.

21 Lacey CJ, Merrick DW, Bensley DC, Fairley I. “Analysis of the sociodemography of gonorrhoea in Leeds, 1989-93” (1997) BMJ. 1997 Jun 

14;314(7096):1715-8.

Figure 4: Rates of Reported Cases by 

Race/Ethnicity, United States, 2010–

2014 Socioeconomic Status 

Figure 3: Rates of Reported Cases by State, 

United States and Outlying Areas, 2014

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Diagnosis: Physicians and other health care professionals typically use three methods of diagnosing 

gonorrhea: biological samples from the infected area with staining (gram staining), detection of bacterial genes 

in urine, and bacteria growth in laboratory cultures.

22 Many doctors prefer to use dual testing to increase the 

accuracy of the diagnosis. Although gram staining is a very accurate test for diagnosing men, it is not effective 

in women: only one woman out of two infected with gonorrhea will show an identifiable trace. Most often, 

doctors use urine and cervical secretions to detect bacterial genes. These tests give even better results than 

culturing the bacteria and therefore are widespread. Culture tests involve placing a sample of the secretion in a 

culture medium and incubating it for up to two days, to allow the bacteria to grow and multiply.

23 The sensitivity 

of this test depends on the area from which the sample is taken: cultures of cervical samples detect infection in 

about 90 percent of the cases. It should be noted that cultures also are used to determine the drug resistance 

of the bacteria, which increases the overall usage of this method.

21

Treatment: Gonorrhea can currently be cured with the right treatment. However, in order to eliminate 

reinfection, both sex partners must be treated, because typically both are infected. The CDC now recommends 

dual therapy (using two drugs) for the treatment of gonorrhea. On the basis of experience with other microbes 

that rapidly develop antimicrobial resistance, a theoretical basis exists for combination therapy, in which two 

antimicrobials with different mechanisms of action are used to improve treatment efficacy and potentially slow 

the emergence and spread of antimicrobial resistance.

24 The current treatment recommendation is Ceftriaxone 

(250 mg intramuscular dose) plus Azithromycin (1g orally in single dose).

22 Cefixime (400 mg in a single dose) 

can be used if Ceftriaxone is not available.22 Several other antimicrobials are active against N. gonorrhoeae, 

but none have substantial advantages over the recommended regimen, and efficacy data (especially for 

pharyngeal infection) is limited.22 In up to one-half of infected patients, chlamydia occurs simultaneously with 

gonorrhea; for such patients, oral tetracycline, doxycycline, minocycline, or erythromycin is prescribed for a 

period of 7 days, in addition to the Ceftriaxone and Azithromycin.

22

Resistance to Treatment: Unfortunately, gonorrhea treatment is complicated by the ability of N. 

gonorrhoeae to develop resistance to antimicrobials:

 Antimicrobial resistance: In 1986, the Gonococcal Isolate Surveillance Project (GISP), a national 

sentinel surveillance system, was established to monitor trends in antimicrobial susceptibilities of 

urethral N. gonorrhoeae strains in the United States.

25 This system annually collects approximately 5-

6,000 N. gonorrhoeae samples from men with urethral gonorrhea at STD clinics in approximately 25-30 

U.S. cities and measures the concentration of various antimicrobials needed to stop the bacteria’s 

growth in the laboratory. A minimum inhibitory concentration (MIC) is the lowest concentration of drug 

needed to stop growth and is an indication of how susceptible the bacteria is to treatment with an 

antibiotic.

24 The higher the MIC, the greater the dose required for effective treatment. If MICs become 

too high, the antibiotic will not work at all. 

 Historical Trends in Drug Resistance: In response to the ongoing threat of drug resistance, the CDC 

has repeatedly revised its gonorrhea treatment guidelines to phase out the use of antibiotics that have 

become less effective in treating the infection, due to shifts in antimicrobial resistance patterns. N. 

gonorrhoeae has progressively developed resistance to all of the antimicrobials used for treatment:26

- 1930s: Introduction of sulfanomide antimicrobials to treat gonorrhea

- 1940s: Due to increasing resistance, sulfanomides no longer recommended for treatment;

 penicillin becomes treatment of choice

- 1980s: Due to increasing resistance, penicillin and tetracycline no longer recommended

- 1990s: Fluoroquinolones become predominant treatment 

- 2007: Fluoroquinolones no longer recommended; cephalosporins (including injectable

 ceftriaxone and oral cefiximine) become backbone of treatment 

 

22 Jerse, Ann, Margaret C. Bash, Michael W. Russell. “Vaccines against Gonorrhea: Current status and Future Challenges” Vaccine. 2014 Mar 20; 

32(14): 1579–1587

23 Jerse, Ann, Margaret C. Bash, Michael W. Russell. “Vaccines against Gonorrhea: Current status and Future Challenges” Vaccine. 2014 Mar 20; 

32(14): 1579–1587

24 Lacey CJ, Merrick DW, Bensley DC, Fairley I. “Analysis of the sociodemography of gonorrhoea in Leeds, 1989-93” (1997) BMJ. 1997 Jun 

14;314(7096):1715-8.

25 Center for Disease Control. “2015 STD Treatment Guidelines: Gonococcal Infections”

26 Center for Disease Control. “Addressing the Threat of Drug-Resistant Gonorrhea- CDC Fact Sheet.” (2016)

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- 2012: Cefixime no longer recommended as a first-line regiment, leaving ceftriaxone-based

 dual treatment as recommended treatment 

- 2015: Cetraixone plus azithromycin is the only recommended treatment 

Recently labeled a “superbug,” N. gonorrhoeae has retained resistance to antimicrobials previously 

recommended for first-line treatment and has now demonstrated its capacity to develop resistance to 

the extended-spectrum cephalosporin (ceftriaxone), the last remaining option for first-line empiric 

treatment of gonorrhea.

27 Alarmingly, society may have reached the point where untreatable infections 

can be expected. Such fears have become a reality in Australia, France, Norway, Sweden and the UK, 

as some infections can no longer be treated with current antibiotic classes.

26 Unfortunately, few 

antibiotic options remain that are simple, well-studied, well-tolerated, and highly effective. The 

combination of persistently high gonorrhea morbidity in some populations, along with cephalosporinresistant gonorrhea, reinforces the need for more effective methods of prevention, particularly vaccines.

Treatment Centers: The number of gonorrhea cases reported by 

STD clinics declined between 2005 and 2014.28 In 2014 (Figure 5), 

15.1% of gonorrhea cases were reported by STD clinics, a decrease 

from 2013 when 16.3% of gonorrhea cases were reported by STD 

clinics.27 In 2014, among women, private physicians or health 

maintenance organizations (HMOs) were the most common reporting 

source (25.1%), followed by family planning clinics (8.5%), STD 

clinics (8.2%), other health department clinics (6.6%), and 

emergency rooms (5.5%). Among men, private physicians/HMOs 

(20.5%) and STD clinics (17.2%) were the most common reporting 

sources. Other reporting sources for men included other health 

department clinics (8.7%), emergency rooms (5.9%), and family 

planning clinics (4.7%)29 Research aimed at characterizing the client demographics of STD clinics found that 

most clinics see young (51% under 25 years of age), minority (64% nonwhite), and poor (43% make less than 

$10,000 per year) individuals with high STD rates (66% diagnosed with 1 or more STDs). Roughly half had 

previously visited the STD clinic, and STD symptoms were cited as the reason for the visit in 63% of cases.30

This research supports the continuing need for publicly funded, categorical STD clinics in urban areas with 

high STD morbidity and the importance of easily accessible, confidential, expert STD services.29

Economic Burden of Treatment: In 2014, 163,208 women and 86,943 men were treated for gonorrhea in 

the US. These treatments resulted in $43.4M and $9.9M in direct medical care payments for men and women, 

respectively, a total of $53.3 million. When considering that the CDC estimates that 820,000 new cases of 

gonorrhea occur annually, total medical care expenditures have the potential to nearly triple to $124.8M, with 

women totaling $101.6M and men totaling $23.2M. These costs can be broken down between the costs of 

screening, the costs of diagnosis and treatment, and the costs of sequelae resulting from untreated acute 

infections or from delayed or improper treatment: 

 Screening Costs: Screening tests usually are administered during visits for non-gonorrhea related 

issues in which gonorrhea screening is provided.31 The cost for cases detected through screening –

covering expenses for office visits (including treatment visits, where appropriate), diagnostic testing and 

treatment – range from $36 to $69.30 The lower estimate is for screening in a correctional setting, and 

the higher estimate reflects a private setting.30

 

27 Magnus Unemo and Robert A Nicholas. “Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea.” Future Microbiol. 

2012 Dec; 7(12): 1401–1422

28 Centers for Disease Control and Prevention. “Sexually Transmitted Disease Surveillance 2014”

29 Center for Disease Control. “Reported STDs in the United States: 2014 National Data for Chlamydia, Gonorrhea, and Syphilis” 

30 CL Celum, et al. “Patients attending STD clinics in an evolving health care environment. Demographics, insurance coverage, preferences for STD

services, and STD morbidity.” Sex Transm Dis. 1997 Nov;24(10):599-605.

31 Marrazzo JM et al., Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women, Sexually 

Transmitted Diseases, 1997, 24(3):131-141.

Figure 5: Percentage of Reported 

Cases by Sex & Reporting Sources, 

United States, 2014

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 Diagnostic and Treatment Costs: Other cases are detected through a diagnostic visit initiated by 

patients with symptoms. For these cases, costs range from $69 to $125.32 The cost per case would be 

lower if most symptomatic men were treated on the basis of symptoms or a clinic-performed gram stain 

(without diagnostic laboratory-based testing), but research has found high rates (83-92%) of expensive 

diagnostic testing among private providers.33 The $69 estimate was determined to be the average cost 

per acute asymptomatic case or symptomatic case treated.34 Data on the proportion of gonorrheal 

infections that are asymptomatic or that do not have recognized symptoms vary. Women are generally 

more likely than men to have asymptomatic infections, but even among men, asymptomatic infections 

and infections without recognized symptoms are estimated at approximately 34%.

35

 (It is noted that the 

medication to treat gonorrhea is relatively inexpensive: the price of Ceftriaxone is between $9.50 and 

$23.00, while Azithromycin is priced between $25.00 and $30.00, depending on the pharmacy. 

However, the public sector typically receives price breaks for use in low-cost clinics; for example, 

Azithromycin costs only $11.50 for public providers.36) 

 Costs of Sequelae: Although untreated acute infections for which patients never seek care entail no 

direct costs, treating their sequelae can be costly. Data for chlamydia (due to higher data availability) 

can be used to determine the gonorrhea-related costs of epididymitis in men and PID in women 

(because no evidence exists that costs vary significantly according to the organism involved). 37

Estimated rates, less than 15%, of progression from chlamydial infection to epididymitis were used to 

estimate those for acute gonorrhea. PID develops approximately 26% of the time following cervical 

Gonococcal infection. 38 As PID develops in an estimated 20% of women with untreated acute 

Gonococcal infections and in 6% of those with successfully treated infections.36

Taking all of these costs into account, the average cost per case for men was $53 and $266 for women. 

These costs were calculated by assuming that acute infections were untreated in 29% of men and 72% of 

women.39 In women, 81% of the cost per case is attributable to sequelae, whereas in men, 92% of the cost per 

case is attributable to acute infection treatment.37

2. Market Analysis 

Current gonorrhea control measures are inadequate and are seriously threatened by the rapid emergence of 

pathogens with antimicrobial resistance. Unfortunately, virtually no promising candidates are in sight for either 

therapeutic or prophylactic vaccines. In fact, the role of vaccines in preventing bacterial infections, as well as 

their potential role in reducing the overall use of antibiotics, has been under-represented in current 

approaches. Nonetheless, an appreciation for the role vaccines, as opposed to focusing solely on the 

development of novel antibiotics, is growing. Although vaccines will not be a practical or feasible solution for all 

antibiotic resistant bacteria, supporting vaccine development against some of these pathogens, particularly 

gonorrhea, can have a major impact in decreasing the burden of resistant infections. For some infections, 

vaccines represent the most logical strategy for protecting high-risk patients who are repeatedly exposed to 

resistant bacterial pathogens, due to frequent interactions with the healthcare system, demographic risk 

factors, and non-modifiable host factors.41 If there is a bright side, it is that U.S. and world policymakers have 

focused efforts on gonorrhea treatment, prevention, and the clear need for a vaccine like never before. 

 

32 Begley CE, McGill L and Smith PB, The incremental cost of screening, diagnosis, and treatment of gonorrhea and chlamydia in a family planning 

clinic, Sexually Transmitted Diseases, 1989, 16(2):63-67

33 Ratelle S et al., Management of urethritis in health maintenance organization members receiving care at a multispecialty group practice in 

Massachusetts, Sexually Transmitted Diseases, 2001, 28(4):232-235.

34 Marrazzo JM et al., Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women, Sexually 

Transmitted Diseases, 1997, 24(3):131-141.

35 Turner CF et al., Untreated gonococcal and chlamydial infection in a probability sample of adults, Journal of the American Medical Association, 2002, 

287(6):726-733.

36 Blandford JM1, Gift TL.” The cost-effectiveness of single-dose azithromycin for treatment of incubating syphilis.” Sex Transm Dis. 2003 Jun;30(6):502-

8.

37 Washington AE, Johnson RE and Sanders LL, Jr., Chlamydia trachomatis infections in the United States: what are they costing us? Journal of the 

American Medical Association, 1987, 257(15):2070-2072

38 Westrom L and Eschenbach D, Pelvic inflammatory disease, in: Holmes KK et al., eds., Sexually Transmitted Diseases, New York: McGraw-Hill, 1999, 

pp. 783-809

39 St. Lawrence JS et al., STD screening, testing, case reporting, and clinical and partner notification practices: a national survey of US physicians, 

American Journal of Public Health, 2002, 92(11):1784-1788.

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Commercialization Plan Page 68
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Corporate Investment in Vaccines: In recent years, major pharmaceutical companies have poured most of 

their research dollars into highly profitable medicines to fight cancer, metabolic and immune mediated 

diseases, and prevalent infectious disease such as HIV and hepatitis C. These drugs not only command high 

prices but also are typically used for far longer periods of time than vaccines. 

Many factors confound the production of vaccines, which are more risky and less lucrative for larger 

pharmaceutical companies: 

 The cost of developing a vaccine, from research and discovery to product registration, is estimated to 

be between $200 million and $500 million.40 It costs roughly the same amount of money to bring new 

cancer drugs to market, which can command $100,000 or more per year per patient. 

 Low-cost existing vaccines can serve as a disincentive for developing improved vaccines that will be 

considerably more costly to advance than those already on the market. Pharmaceutical companies 

often cannot make an economic case for investing in superbugs, as they have a fiduciary duty to 

maximize shareholder profit. 

 Live vaccines are troublesome to manufacture and are closely regulated by the Food and Drug 

Administration (FDA) for quality control. 

 For decades, vaccines have been a neglected corner of the drugs business, relying on old technology, 

little investment and abysmal profit margins for both the firm producing them and the practices 

deploying them, at times doing so at a loss.41 

 The anti-vaccine movement has grown. This movement typically relies on an old, discredited study 

linking vaccination to autism and the theory that unscrupulous doctors and pharmaceutical companies 

stand to make huge profits on vaccinations. Yet, after review, that argument is historically unfounded. 

Altogether, a combination of high production costs, low market prices, and heavy regulation contribute to the 

shortage of novel vaccines. In the absence of a new ways of compensation, the production of vaccines does 

not make economic sense. Even though the vaccine industry is likely more profitable now than in previous 

years, the result of global market forces, the economic truth is that pharmaceutical companies need financial 

support or incentives to keep producing vaccines. Yet, regardless of company profits, the economic and social 

benefits of vaccination are unsurpassed; wide spread vaccination is the only strategy that has ever come close 

to eradicating a pathogen. Thus, it is up to governments and foundations to spearhead efforts to combat the 

problem, which includes supporting innovative small businesses with promising approaches to tackling 

resistant superbugs. 

Market Size: Estimates put the vaccine market value at $24 billion; while huge, it is a mere 2 to 3 percent of a 

trillion-dollar worldwide pharmaceutical industry.43 The most recently marketed vaccine for an STD was for the 

Human Papillomavirus (HPV). The first HPV vaccine (Gardasil) was approved by the FDA in June of 2006 and 

became part of the free Vaccines for Children (VFC) program within in year of approval.42 The private sector 

cost of treatment is ~$540 as 3 doses must be given, each costing $180. However, the CDC purchases this 

same vaccine for $120 per dose, resulting in a $360 cost of treatment, a 33% discount.43 

Given that antimicrobial-resistant gonorrhea is a top priority at the CDC a premium can be expected for a 

single dose vaccine. It is estimated the market would be willing to pay $350 (similar to two doses of the private 

sector HPV vaccine cost), with a conservative estimate of $250 (similar to two doses of public sector HPV 

vaccine cost) and an aggressive estimate of $450 (less than the cost of HPV treatment in the private sector, 

but more than the public sector). With this information in mind, it can be estimated that the revenue potential of 

the gonorrhea vaccine will be somewhere between $250 and $350. If the vaccine is selling to the private sector 

for $350, it will be sold to the public sector at a 33% discount (~$235). In addition, the CDC has indicated that 

gonorrhea is a high priority target; therefore, it is likely that the CDC would purchase a substantial percentage 

of the vaccines produced, perhaps more than 50%. 

When estimating the market size of the gonorrhea vaccine, it is probable that the estimate will fall between the 

CDC reported (350,000) and estimated total rate of infection (over 800,000). It is expected that once the 

 

40 André FE. “How the research-based industry approaches vaccine development and establishes priorities.” Developmental Biology (Basel). 2002

41 Bourree, Lam. “Vaccines are profitable, so what?” The Atlantic; February 10, 2015 

42 Center for Disease Control. “Vaccines for Children Program: Price List” 

43 Center for Disease Control. “CDC Vaccine Price List”

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vaccine becomes more known to the public, women will be more likely to seek treatment. Thus, the revenue 

potential might be estimated at $4.1M in year 1, $16.4M in year 5 and $17.9 in year 10. In our estimates of 

market value, we used the more conservative reported (known) rates (Table 1). Note that these estimates do 

not account for international sales. 

Table 1. Market Penetration & Value 

Assumptions. 1. Price will be determined based on a best-estimate derived from similar vaccines 

along with “higher and lower” cost considerations to better understand the sensitivity of the price 

point; 2. The treatment is for the female population only, equating to 46.6% of infections; 3. After 

year 1 of market penetration, public agencies should begin to purchase the product at a volume 

discount; 4. The overall rate of infection will decline overtime due to immunization; 5. Patient 

population differences between CDC reported and estimated; 6. General population growth; 7. 

Rate of vaccination; 8. Increase in vaccination rate due to public visibility; 9. Cost and sales to 

the private sector; 10. Cost and acceptance into public sector programs. 

3. Company Overview 

TherapyX

Inc is a privately held biopharmaceutical company, headquartered in Louisville Kentucky, with 

research facilities in Buffalo, NY. The company currently has 8 employees and remains focused on the 

development and commercialization of nanoparticulate protein-based immunotherapeutics for the treatment of 

infectious disease, inflammation and cancer. 

TherapyX

inc and collaborators have developed a patented phase-inversion nano-encapsulation (PIN®

) 

technology to create clinically targeted slow-release cytokine adjuvants for use in autoimmune and cancer 

therapies, and now bacterial infectious diseases. The process affords a mild encapsulation methodology with 

minimal loss of structural integrity or biological activity of macromolecules. Various cytokines – including IL-2, 

GM-CSF, TNFα, TGFβ-1, and IL-12 – have been successfully encapsulated and tested in murine systems, and 

encapsulation efficiency, release kinetics, and post-encapsulation specific activity of selected cytokines have 

been evaluated. Formulation studies have resulted in IL-12 encapsulated in poly-lactic acid microspheres, with 

90% efficiency, fully-preserved cytokine bioactivity, and full stability for 2 years. 

Recently, we developed proprietary scale up spray drying methods that can commercially produce kilogram 

sized batches suitable for non-clinical studies in primates and for clinical studies in humans. Batch-to-batch 

consistency and stability was demonstrated. The polymers used to create these formulations already have 

been approved for use in a number of applications by the FDA. The commercial production of bacterial outer 

membrane vesicles (OMVs) or specific bacterial peptides eventually will be outsourced to specialty 

microbiology companies.

Research at TherapyX

Inc has been funded primarily through the SBIR program of the NIH in the past 8 years 

(Table 2). In the Phase I SBIR project (R43-AI115877), we, proved the concept that our lead product GvaX12®

– a combination of an interleukin-12 (IL-12) formulation plus gonococcal outer membrane vesicles (OMV) –

could serve as prototype prophylactic vaccine. Our present proposal to exploit local application of GvaX12® is 

based on several factual considerations: (i) soluble IL12 is ineffective; (ii) systemic manipulation of cytokines in 

US Penetration & Value Year 1 Year 5 Year 10

US Patient Population (known) 350,000 280,000 154,000

Decline in Patient Population (YoY) 0 20% 45%

Percentage Women 46.6% 46.6% 46.6%

Women Patient Population 163,100 130,480 71,764 

Vaccination Rate 5% 25% 50%

Women Vaccinated 8,155.00 32,620.00 35,882.00 

Sales: Price per vaccine @ $250 $2,038,750 $8,155,000 $8,970,500 

Sales: Price per vaccine @ $350 $2,854,250 $11,417,000 $12,558,700 

Sales: Price per vaccine @ $450 $3,669,750 $14,679,000 $16,146,900 

Average: Price Per Vaccine @$300 $2,446,500 $9,786,000 $10,764,600 

Contact PD/PI: Liu, Yingru

Commercialization Plan Page 70
10 | P a g e

humans can have adverse consequences; (iii) IL-12 counteracts IL-10 and other suppressive cytokines; (iv) 

local application of GvaX12® should avoid major systemic complications and direct favorable responses to the 

site of infection; (v) the combination of OMV and particulate IL-12 (GvaX12®) serves as a prototype 

prophylactic vaccine. 

Table 2. Selected Awards. 

SBIR Description 

R43-AI080009 Delivery of Nanoencapsulated TGF and ATRA for the Treatment of IBD. 7/20/2008-

12/31/2009, Thomas Conway, P.I

R44-AI080009 Nanoencapsulated TGF and ATRA for the Treatment of IBD. 4/01/2012-3/31/2016, Dominick 

Auci, P.I. 

R43-AI104067 Therapy and Prophylaxis for Genital Tract Infection. 02/01/2013 – 01/31/2015, Yingru Liu, P.I. 

R44-AI104067 Therapy and Prophylaxis for Genital Tract Infection. 06/15/2014 – 05/31/2016, Yingru Liu, P.I.

R43-AI115877 “Experimental Gonococcal Vaccine” 01/01/2015 – 12/31/2015, Yingru Liu, P.I. 

4. Intellectual Property Protection 

Patents and other proprietary rights are critical to TherapyX's business. We file patent applications to protect 

our technology, inventions, and improvements to our inventions that we consider important to our product 

development. TherapyX

inc has exclusive worldwide licenses from Brown University for the protein micronization 

process and the PIN® technology used to create GvaX12®, its other lead product, TGFNanoCap® for 

treatment of IBD, and other formulations targeting immune mediated inflammatory diseases and cancer. The 

method for the polymeric micro/nanoencapsulation of proteins by phase inversion nanoencapsulation (PIN®) 

process is covered under a number of patents. PIN®is unique in that, because the process does not require 

emulsification, it is gentler and less likely to denature sensitive proteins. Table 3 lists the PIN® technology 

patents included in the agreement with Brown: 

Table 3. Patents 

Title Number Country Issue 

Date

Process for preparing microparticles through phase inversion 

phenomena

6,143,211 USA 2000

Process for preparing microparticles through phase inversion 

phenomena

6,235,224 USA 2001

Process for preparing microparticles through phase inversion 

phenomena

6,616,869 USA 2003

Process for preparing microparticles through phase inversion 

phenomena

12/171,275 USA Pending

Process for preparing microparticles through phase inversion 

phenomena

718482 Canada 2009

Process for preparing microparticles through phase inversion 

phenomena

0844871 Europe 2004

Micronized freeze-dried particles 7,029,700 USA 2006

Micronized freeze-dried particles 2,397,404 Canada 2008

Micronized freeze-dried particles 1246609 Europe 2010

Compositions for stabilizing and delivering proteins Provisional filed USA Pending

 

5. Commercialization Strategy 

The CDC is urging the acceleration of research on new vaccines or drug combinations now, as it takes years 

to bring them to market. This puts TherapyX

Inc in a prime position to leverage these efforts in the development 

of its vaccine. The company’s PIN®nano-encapsulation platform represents a general strategy for the 

immunotherapy of disease through the application of slow-release cytokines and drugs. The overall timing for 

development of a GvaX12®-based prophylactic vaccine to prevent gonorrhea is described below, based on the 

Contact PD/PI: Liu, Yingru

Commercialization Plan Page 71
11 | P a g e

successful completion by Q1/Q2 2019 of the proposed PK/PD preclinical studies and GLP nonclinical 

toxicology studies respectively: 

 File an IND in late 2019; start Phase I/IIa clinical studies, complete the trial in late 2020, and assess the 

drug’s safety in gonorrhea patients. 

 In 2021, initiate Phase IIb/III clinical studies to demonstrate the safety and efficacy of the optimized 

vaccine to prevent infection with gonorrhea. The trial design will enable a Go/No-Go decision to convert 

the initial Phase IIb trial to a Phase III pivotal trial. The projected study completion date is late 2022. 

 File the biologics license application (BLA) in the 2nd half of 2023 and receive FDA approval in the 2nd 

half of 2024. 

 Immediately launch after approval in 2025. 

The target market for launch of the vaccine will be to STD clinics as they serve young, minority, and poor 

individuals with high STD rates. The clinic demographic data syncs perfectly with the demographic of potential 

target patients who are at the highest risk of infection. There are currently 1,036 of the clinics across all 50 

states with the vast majority of them being in California (92), Texas (52), New York (49), Florida (48), and 

Pennsylvania (41).44

 In addition, the company’s efforts can be easily fast-tracked with bulk purchasing from 

the CDC, quickly putting vaccines into STD clinics across the US and allowing for rapid market penetration. 

The company's current strategic direction is to complete the IND filing and the Phase II/IIb SBIR work, and 

then partner with major pharmaceutical and biotechnology companies to complete development to market 

registration. The ideal partner will have biologic drug expertise as well as an effective global sales and 

marketing organization. It is possible that the partner would conduct some, if not all of the production and 

marketing activities, which would serve to reduce capital investment requirements. 

Table 4. SWOT Analysis

Such collaboration will produce key benefits for 

TherapyX

Inc, including obvious financial aspects, 

and aid the company with key expertise needed 

to develop its other pipeline products on its own. 

The company plans to make extensive use of 

outsourced manufacturing activities, coupled with 

strong internal quality management and 

assessment systems to ensure an adequate 

supply of high quality clinical and commercial 

products while minimizing capital investment. 

Overall the company’s strategy has the potential 

to rapidly commercialize an innovative approach 

that will benefit patients worldwide. Strengths, 

weaknesses, opportunities and threats are 

summarized in Table 4. 

6. Key Operations Analysis 

Finance Plan: In 2017, TherapyX

inc intends to raise an estimated $2.625 million through the sale of Series A 

Preferred Shares. These funds will be in addition to the SBIR award and will be used primarily to (i) initiate 

phase I/II studies of GvaX12® with a view to out-licensing the product to a partner, and (ii) for general corporate 

purposes. The first investor group targeted will be the Buffalo Angel Investor Network. While this group votes 

on investment opportunities, the investors do not pool their funds; rather, they participate as individual 

investors. To limit the number of new shareholders, we will limit the sale to 16 individuals at $100,000 each; 

the remaining $1.025 million will be sold to a seed funding venture capital firm (25 potential seed venture funds 

with life science investments in Western New York and the northeast U.S. have been targeted) or to any 

combination of the two investments that achieves the capital requirement for items (i) and (ii) . 

 

44 A list of contact information for all the publicly funded free and low-cost STD clinics can be found in support document “Free and Low Cost Providers 

Contact List.”

Contact PD/PI: Liu, Yingru

Commercialization Plan Page 72
12 | P a g e

Production and Marketing: TherapyX

inc plans to focus its operations on R&D for the foreseeable future. The 

company intends to outsource the manufacturing, distribution and sales of its products. In previous capacities 

the company has partnered with Bend Research, Inc. for all of its drug manufacturing. Bend Research, Inc. is 

now part of Capsugel’s Dosage Form Solutions business unit headquartered in Morristown, NJ. This 

partnership has provided access to additional technologies, R&D capabilities, and a global manufacturing 

infrastructure. TherapyX

inc also has partnered with Neumedicines, Inc., a private biotechnology firm developing 

protein therapeutics that address unmet clinical and societal needs in oncology, hematology, and immunology. 

Our agreement with Neumedicines insures unique and continued access to clinical grade rhIL-12. The 

company operates from its headquarters in Pasadena, CA. Recently, we have begun discussions with 

researches at Allergan inc, regarding possible co-development of encapsulated peptides for oral delivery. We 

expect that any GvaX12® prophylactic vaccine partner would conduct some, if not all of the production and 

marketing activities, which serves to reduce development-associated capital requirements. 

Revenue Stream: Based on our expected development timelines and other assumptions, we project a profit of 

~$5 million for GvaX12® in 2030. License and milestone payments will primarily drive revenues through 2022. 

After 2022 (projected launch) royalties (projected at ~5%) on sales will drive top-line revenue growth. 

Projections are based upon (1) the known female patient populations indicated above; (2) vaccination rates of 

5% in year 1, growing steadily to 25% by year 5 (2030); and an average cost of $300 per vaccine. The financial 

forecasts do not include the commercialization of additional pre-clinical stage projects that could impact the 

revenue growth of the company as early as 2020. Operating expenses include anticipated development costs. 

 

($000s) 2020 2021 2022 2023 2025 2026 2027 2028 2029 2030

Licensing Fees &

Royalties $0 $0 $500 $500 $1000 $1,800 $3,500 $5,500 $7,500 $10,000

Grants $500 $1,000 $1,000 $1,000 $400 $400 $850 $850 $850 $850

Investments $0 $2,625 $0 $0 $0 $0 $0 $0 $0 $0

Revenue $0 $3,625 $1,500 $1,500 $1,400 $2,200 $4,350 $6,350 $8,350 $10,850

Operating Expenses $400 $1,000 $1,000 $1,000 $1,000 $1,500 $2,000 $3,000 $4,000 $5,000

EBIT $100 $2,625 $500 $500 $400 $700 $2,350 $3,350 $4,350 $5,850

Contact PD/PI: Liu, Yingru

Commercialization Plan Page 73
COMPANY COMMERCIALIZATION STATEMENT 

Therapyx, Inc. has not received more than 15 SBIR Phase II awards from the 

Federal Government during the preceding five fiscal years. 

Contact PD/PI: Liu, Yingru

Prior SBIR Phase II Awards Page 74
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ● Yes ❍ No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

● Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Liu, Yingru

 Page 75
PHS 398 Cover Page Supplement

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302 . Received Date:

2016-08-24T11:01:24.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Liu, Yingru

 Page 76
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12235571 Funding Opportunity Number: PA-16-302. Received Date:

2016-08-24T11:01:24.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

Research Plan Section

2. Specific Aims 1253-SPECIFIC AIMS.pdf

3. Research Strategy* 1254-Res plan NKE FINAL.pdf

4. Progress Report Publication List

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals 1255-VERTEBRATE ANIMALS VACCINE phase II FINAL.pdf

10. Select Agent Research

11. Multiple PD/PI Leadership Plan

12. Consortium/Contractual Arrangements  

13. Letters of Support 1256-VaccinePhaseII LOS.pdf

14. Resource Sharing Plan(s)

15. Authentication of Key Biological and/or

Chemical Resources

Appendix

16. Appendix 1257-Appendix 1 Repo study designs and quotes.pdf

1258-Appendix 2 IND Budget Proposal Master-TherapyX.pdf

Contact PD/PI: Liu, Yingru

 Page 77
SPECIFIC AIMS 

The overall objective of this SBIR project is to develop a prophylactic vaccine against Neisseria 

gonorrhoeae (i.e., gonorrhea). In Phase I, using a murine model of genital gonococcal infection, we have 

established Proof-of-Principle that i.vag. immunization with a proprietary sustained-release particulate 

interleukin-12 (IL-12) formulation plus gonococcal outer membrane vesicles (OMV) - the combination we refer 

to as GvaX12® - induces durable Th1-driven adaptive immune responses that protect against genital tract 

infection with heterologous strains of N. gonorrhoeae [progress report]. In fulfilling the Phase I milestones we 

demonstrated (i) induction of gonococcus-specific Th1 cells, (ii) generation of anti-gonococcal antibodies in 

serum and genital tract secretions, (iii) protection against diverse strains of N. gonorrhoeae, (iv) recall of 

specific antibodies and reactivation of T cells after challenge infection, and (v) duration of protection against 

infection for 6 months. In addition we have shown that protection depends on both antibodies and IFN. 

 

In Phase II, we will use the same murine model to identify alternative routes of immunization that may be more 

applicable to humans (potentially including males), and determine the basis of protective immunity against 

antigenically diverse clinical isolates of N. gonorrhoeae. In addition, we will perform preliminary 

pharmacokinetics and toxicity studies and prepare materials for submission to the FDA for a type C meeting. 

The specific aims for Phase II are as follows: 

Aim 1: Define, optimize, and validate a vaccination regimen. This aim will encompass three tasks: 

A. Determine a route of administration, intranasal vs. intravaginal, with respect to: 1) protection 

against challenge with N. gonorrhoeae ; and 2) immune response correlates of protection (i.e., 

antibody titers, antibody specificity and T cell cytokine responses). 

B. Determine the basis of cross-protective immunity against antigenically diverse clinical isolates of 

N. gonorrhoeae 

C. Determine whether immunization with detergent-extracted OMV-containing GvaX12® induces 

protective immune responses against N. gonorrhoeae

Aim 2: Rodent pharmacokinetics and reproductive toxicology. This aim involves two tasks: 

A. Bioavailability and pharmacokinetics of IL-12 following intravaginal and intranasal administration 

of GvaX12® in mice. 

B. Conduct reproductive toxicology studies in rats (i.e., embryonic-fetal development and effects on 

pre- post-natal development ) 

Aim 3: Request a type C pre-pre-IND meeting with the FDA and prepare a briefing package with written 

questions. Upon successful completion of Aims 1 and 2, TherapyX

inc will request a type C pre-pre-IND 

meeting with the FDA and prepare a briefing package with written questions. At this meeting, we will seek 

agency guidance to shape future toxicological and clinical study designs 

Milestones to be accomplished: 

Year 1: 

1. Demonstrate efficacy of intra-nasal versus intra-vaginal routes of vaccine delivery; 

2. Elucidate the basis of cross-protection against antigenically diverse strains; 

Year 2: 

3. Determine whether detergent-extracted OMV are effective as a vaccine immunogen; 

4. Define systemic exposure and the potential for reproductive toxicity; 

5. Receive agency guidance that will shape toxicological and clinical study designs. 

Upon successful completion of these aims TherapyX

inc will submit an SBIR Phase IIb grant application, which 

will seek to evaluate the pharmacokinetics and potential toxicity of this vaccine in non-human primates, and 

prepare for an IND submission. 

Contact PD/PI: Liu, Yingru

Specific Aims Page 78
RESEARCH STRATEGY 

1. Significance 

Despite public health control measures, gonorrhea remains an all-too-common disease, and is the second 

most frequent reportable infectious disease in the US. Although the reported incidence of this disease 

exceeds 350,000 cases per annum (1), the real incidence is estimated to be 820,000 cases per annum (2). 

World-wide incidence is now estimated at 78 million new cases per annum (3). Women bear the greater 

morbidity because untreated gonorrhea can lead to upper tract infection and pelvic inflammatory disease, with 

tubal scarring, infertility, and risk for ectopic pregnancy which can be life-threatening. Gonorrhea also 

increases the risk of acquisition and transmission of HIV up to 5-fold in both sexes (4). Because no vaccine is 

available (5), both its control and treatment depend upon antibiotics. However, N. gonorrhoeae has steadily 

developed resistance to each class of antibiotics deployed against it, including fluoroquinolones and most 

recently even extended-spectrum cephalosporins, raising serious concerns that gonorrhea could become 

untreatable (6). The CDC has listed antibiotic-resistant N. gonorrhoeae as one of the top three pathogens 

presenting “an immediate public health threat that requires urgent and aggressive action” (7). A recent WHO 

technical consultation on vaccines against sexually transmitted infections (to which our collaborator Dr. Russell 

contributed) called for renewed efforts to develop a vaccine against gonorrhea (8, 9). In June 2015, the NIAID 

convened a workshop to discuss the status of gonococcal vaccine development that both Dr. Russell and the 

PI attended (see report published in ref. 10). 

Several previous attempts have been made to develop vaccines against gonorrhea, three in recent times 

although only two went into clinical trial (reviewed in 11, 12). The first, based on killed whole gonococci, 

proved ineffective as did a later and substantially larger effort based on gonococcal pili; the third based on 

porin protein was abandoned. These disappointments discouraged further efforts and engendered pessimism 

that vaccination against gonorrhea might not be feasible. Contributing to this situation is the well-known 

observation that uncomplicated gonorrhea can be acquired repeatedly with apparently no development of 

protective immunity resulting from previous infection. The reasons for this are poorly understood, but it is 

generally thought that extensive antigenic variation coupled with multiple mechanisms for resisting complement 

enable N. gonorrhoeae to evade whatever adaptive immune responses the host develops against it. 

Consequently, in the absence of a reproducible state of immunity, the determinants of human immune 

protection have not been defined. 

Recent studies in our collaborator’s laboratory using a mouse model of genital gonococcal infection have cast 

new light on this problem. It was shown that N. gonorrhoeae selectively elicits innate host defenses that it 

can survive and concomitantly suppresses adaptive immune responses that would eliminate it, by

mechanisms involving IL-10, TGF, and type 1 regulatory T cells (13-17). These studies further showed 

that: 1) immunosuppression can be reversed by neutralizing TGF and IL-10 (14, 17); and 2) local intravaginal 

(i.vag.) treatment with TherapyX

inc’s sustained-release nanoparticulate IL-12 formulation (but not soluble IL-12) 

during gonococcal infection accelerates clearance of the infection and induces resistance to secondary 

infection several weeks later (18). This latter finding implies the development and recall of immune memory 

(18). We therefore inferred that i.vag. nanoparticulate IL-12 effectively turned the infection into a live vaccine, 

and consequently proposed that it could serve as an adjuvant for a non-viable prophylactic gonococcal vaccine. 

This notion was directly tested in our Phase I work. The data demonstrated that prophylactic vaccination with 

a formulation containing particulate IL-12 plus gonococcal outer membrane vesicles (GvaX12®) administered 

i.vag resulted in long-term protection from subsequent challenge (see Phase I data below, 19). Thus we now 

have a candidate prophylactic vaccine for clinical development.

2. Innovation: 

The innovative aspects of our proposal are two-fold. First, we now have a novel approach for developing a 

vaccine against N. gonorrhoeae based on a small animal model in which we can reliably induce a state of 

protective immunity against genital gonococcal infection. This situation was never attained in the previous 

unsuccessful efforts to create a gonococcal vaccine and represents a major conceptual advance. We have 

found that mice can be immunized i.vag. with GvaX12® to generate protection against subsequent challenge 

Contact PD/PI: Liu, Yingru

Research Strategy Page 79
with live N. gonorrhoeae (progress report). Having established Proof-of-Principle, we now hypothesize that 

protective immunity against genital gonococcal infection depends upon: 1) Th1-driven production of antigonococcal antibodies (Abs) in serum and genital secretions; and 2) immune memory that can be recalled on 

subsequent exposure to N. gonorrhoeae. 

Second, the sustained-release nanoparticulate IL-12 formulation, a critical component of GvaX12®, represents 

an important technological innovation. Our patented phase inversion nanoencapsulation (PIN®) process 

provides a mild encapsulation methodology without loss of structural integrity or biological activity of 

macromolecules (20). Studies of encapsulation efficiency, release kinetics, and post-encapsulation specific 

activity studies established that excipient-stabilized macromolecules can be incorporated in poly-lactic acid 

(PLA) particles in a two-step process with 90% efficiency, fully-preserved bioactivity and storage stability for up 

to one year (21). Finally, recent scale-up work at TherapyX

inc has achieved commercial methods for the 

production of kilogram size batches of the formulation removing a major hurdle that has thwarted the 

development of similar products. Thus GvaX12® is now ideally situated for development as a vaccine that 

induces Th1-driven protective immunity against N. gonorrhoeae. 

3. Approach 

3.1 Phase I Results 

In Phase I of this SBIR grant we established proof-of-principle that i.vag. immunization with GvaX12®

induces adaptive Th1-driven immune responses that protect against genital gonococcal infection. 

Gonococcal outer membrane vesicles (OMV) were selected as the immunogen because they contain most 

gonococcal surface antigens (Ags) in native conformation, whereas in heat- or formaldehyde-killed cells some 

Ags may be denatured. Moreover, gonococcal OMV have been used to immunize mice intranasally, though 

with variable results (11, 12, 22), and meningococcal OMV vaccines have been developed against N. 

meningitidis and successfully used in humans (23). 

We have attained the Milestones stated in the Phase I application as follows: 

Milestone 1: Intra-vaginal immunization with GvaX12® protects from genital gonococcal infection by inducing 

IFN-secreting T cells specific for gonococcal antigen in the draining iliac lymph nodes (Fig. 1). 

Figure 1: GvaX12® protects from Ngo challenge and induces Ngo-specific T-cell immunity in a murine model of 

Ngo infection. In this model (19) , young female mice in diestrus are treated with estradiol, placed on antibiotics 

to suppress the overgrowth of commensals, and then vaginally infected by instilling a suspension of freshly grown, 

virulent N. gonorrhoeae. Infection, monitored by vaginal swabbing and plating, typically clears in 2-3 weeks, and is 

accompanied by an influx of neutrophils. Vaccine efficacy is determined by the change in the rate of Ngo clearance after 

challenge. Mice (N=8) were immunized twice with gonococcal (Ngo; FA1090) OMV (50μg protein) plus blank or IL-12 

particles (1μg IL-12); controls were sham-immunized with blank particles or with NTHI OMV plus IL-12 particles 

(GvaX12®). Two weeks later, all mice were challenged with Ngo strain FA1090. A: recovery of Ngo (mean ±SEM, CFU), 

P <0.01 (ANOVA) gonococcal OMV + IL-12 particles vs. blank particles; B: % of animals remaining infected at each time 

point, P <0.0001 (Kaplan-Meier, log-rank test) Ngo OMV + IL-12 particles vs. blank particles. C: T cell cytokine responses 

A B C

Contact PD/PI: Liu, Yingru

Research Strategy Page 80
induced by immunization with gonococcal OMV plus IL-12/ms, prior to gonococcal challenge. ILN cells were isolated 2 

weeks after the last immunization with 1, 2, or 3 doses of gonococcal OMV (50μg protein) plus IL-12/ms (1μg IL-12). 

Control ILN were obtained from mice sham-immunized with blank ms (3 doses) and additional mice were immunized 3x 

with gonococcal OMV plus blank ms. Data shown as mean ±SEM % of CD4+ or CD8+ cells staining for each cytokine. * 

P < 0.01 (ANOVA) comparing immunization with IL-12/ms vs. blank ms, N=3 samples per group. 

The above data establish that GvaX12® enabled mice to reject a bacterial challenge more effectively than 

vaccination with OMV/blank particles and that the clearance of bacteria was associated with a Th1 response. 

Milestone 2: Anti-gonococcal Ab were generated in serum and vaginal secretions by i.vag. immunization with 

GvaX12® (Fig. 2).

Fig. 2: T-cell cytokine responses induced by 

immunization with gonococcal OMV plus IL-12, 

prior to gonococcal challenge. ILN cells were 

isolated 2 weeks after the last immunization with 1, 2, 

or 3 doses of gonococcal OMV (50μg protein) plus IL12 particles (1μg IL-12). Control ILN were obtained 

from mice sham-immunized with blank controls (3 

doses) and additional mice were immunized 3x with 

gonococcal OMV plus blank particles. Data shown as 

mean ±SEM % of CD4+ or CD8+ cells staining for each 

cytokine. * P < 0.01 (ANOVA) comparing 

immunization with IL-12 vs. blank, N=3 samples per 

group.

These results demonstrate that GvaX12® induced potent Ng-specific IgG and IgA responses both in the serum 

and in the mucosa and that maximal response required 2 immunizations. 

Milestone 3: i.vag. immunization with GvaX12® induced protection against antigenically different strains of N. 

gonorrhoeae (Fig. 3).

Fig. 3 . Protection against heterologous gonococcal challenge. A. Challenge with strain MS11. One month after 

immunization with FA1090 GvaX12® or control, mice were challenged with N. gonorrhoeae strains FA1090 or MS11. Left 

panel: recovery of N. gonorrhoeae (mean ±SEM, CFU), * P <0.001 (ANOVA) N=8; right panel: % of animal remaining 

infected at each time point), P <0.02 for FA1090 challenge, IL-12 vs. blank ; P <0.001 IL-12 vs. blank for MS11 challenge 

(Kaplan-Meier analysis, log-rank test) N=8. B: Challenge with strain FA19. One month after immunization with FA1090 

GvaX12® or blank control, mice were challenged with N. gonorrhoeae strains FA1090 or FA19. Left panel: recovery of N. 

gonorrhoeae (mean ±SEM, CFU), * P <0.001 (ANOVA) N=8; right panel: % of animal remaining infected at each time 

point, P <0.02 for FA1090 challenge, IL-12 vs. blank; P <0.001 for FA19 challenge, IL-12 vs. blank (Kaplan-Meier analysis, 

log-rank test), N=8. 

The above data reveal that GvaX12®-induced immunity to strain FA1090 provided cross-protection from strains 

MS11 and FA19. 

Milestone 4: Immune responses (T cells and Ab) induced by i.vag. immunization with GvaX12® were recalled 

after challenge infection (Fig. 4). Note that IL-17-secreting T cells are induced by gonococcal infection alone, 

regardless of prior immunization or treatment (13, 16, 18). A B

Contact PD/PI: Liu, Yingru

Research Strategy Page 81
Figure 4: Recall of responses in mice after challenge with N. gonorrhoeae FA1090 6 months after immunization 

with FA1090 OMV plus IL-12. A: Vaginal wash antibodies (N=5 samples), B: serum antibodies (N=5 samples), C:

cytokine production by CD4+ ILN cells (N=3 samples). # P <0.05, * P <0.01 vs control samples from sham-infected mice 

(ANOVA). 

These results establish that GvaX12® induced long-term Ng-specific Th1 and antibody responses. 

Milestone 5: Protective immunity against genital infection with N. gonorrhoeae persisted for at least 6 months 

after i.vag. immunization with GvaX12® (Fig. 5) and that long-term protection required both Th1 and B-cell 

responses (Fig 6). 

Fig. 5: Protection against gonococcal (FA1090) challenge 

persisted for at least 6 months after immunization with 2 doses 

of gonococcal (FA1090) OMV plus IL-12. Data shown as mean 

±SEM CFU of N. gonorrhoeae recovered at each time point; * P <0.01, 

N=8 (ANOVA). 

The enhanced clearance of a bacterial challenge after 6 

months establishes that GvaX12® induced long-term memory. 

In addition we demonstrated that long-term protection against 

genital gonococcal infection depended on both B cells 

(presumably to produce Ab) and IFN (Fig. 6).

Fig. 6: Protection induced by immunization with GvaX12® depends on IFN and B-cells. A: Course of infection 

(FA1090) in IFN-ko vs wild-type mice immunized with FA1090 OMV GvaX12®. 

B: Course of infection (FA1090) in μMT (B-cell ko) vs wild-type mice immunized with FA1090 OMV GvaX12®. Left panels, 

recovery of N. gonorrhoeae (mean ±SEM, CFU) • P <0.01 (ANOVA); right panels, % of animal remaining infected at each 

time point, P <0.0001 for wild-type mice, IL-12 vs. blank (Kaplan-Meier analysis, log-rank test). 

Publication: A manuscript reporting these and additional findings has been submitted to Mucosal 

Immunology. If accepted before this application is reviewed we will inform the SRO: 

Liu, Y., Hammer, L., Liu, W., Hobbs, M.M., Zielke, R.A., Sikora, A.E., Jerse, A.E., Egilmez, N.K., Russell, M.W. 

(2016) Experimental vaccine induces Th1-driven immune responses and protection against Neisseria 

gonorrhoeae in a murine model. 

A B

Contact PD/PI: Liu, Yingru

Research Strategy Page 82
3.2 The Phase II Project 

The goal of this proposed Phase II SBIR project is to advance GvaX12® towards clinical trials by: 1) developing 

a more practicable vaccination regimen in terms of the route of administration (intranasal vs. intravaginal); 2) 

determining immune response correlates of protection: 3) determining the basis for protection against naturally 

occurring, antigenically diverse, clinical isolates of N. gonorrhoeae; 4) perform bioavailability, pharmacokinetic, 

and initial safety studies on GvaX12® in rats; and 5) prepare a briefing package with written questions for the 

FDA, requesting a pre-pre-IND meeting. The FDA’s response will be used to guide continued development 

including OMV scale up, and immune response and toxicological studies in non-human primates. The results 

should also enable (i) defining the specificity and type of immune responses that need to be generated to elicit 

protective immunity, and (ii) framing hypotheses for testing in clinical trials.

The proposed studies will be limited to female mice. The female mouse is the only animal model currently 

available for experimental in vivo studies of genital gonococcal infections and immunity to them (24); there is 

no experimental animal model for male tract gonococcal infection. Moreover, women suffer the greater burden 

of morbidity from gonorrhea, because of the potential for damage to the reproductive tract that arises from 

ascending infection. This infection can lead to tubal scarring, hence infertility; increased risk of ectopic 

pregnancy; and painful and debilitating pelvic inflammatory disease. A developing fetus can be infected in 

utero, leading to pre-term birth or septic abortion, and an infant delivered through an infected birth canal risks 

ocular infection that can lead to blindness. Therefore, limiting our studies to female mice is not only 

unavoidable but also justifiable in addressing the greater problem in females. 

3.2.1 Specific Aim 1: Define, optimize and validate a vaccine regimen 

Task 1A: To determine an optimal route of immunization: intravaginal vs. intranasal 

The current i.vag. immunization regimen, although shown to be effective in our Phase I studies, is thought to 

be impracticable for human prophylactic application, because i.vag. immunization likely will be unacceptable 

for adolescent girls and many women, and inapplicable for males. However, substantial literature, including 

studies in our collaborator’s laboratory, shows that intranasal (i.n.) immunization elicits responses in the genital 

tract, in primates and in males as well as females (25-27), and may be even more effective than i.vag. 

immunization for generating genital antibody responses (28). 

Compare protection against challenge with N. gonorrhoeae 

Based on our previous gonococcal immunization and infection studies (13, 14, 16-18), including the phase I 

results shown above, we will use groups of 8-12 female mice, BALB/c or C57BL/6 strains, at 2-4 months of 

age. The mice will be prepared for gonococcal challenge (24) by injecting s.c. with 0.5mg estradiol (Premarin) 

on days –2, 0, and +2, placed on an antibiotic regime, and challenged by i.vag. instillation of 5x106 CFU of 

freshly grown N. gonorrhoeae. The course of infection will be monitored by taking daily vaginal swabs for 

viable cell counting on selective agar (a GC agar base supplemented with IsoVitalex, hemin, and antibiotics: 

streptomycin, vancomycin, nystatin, colistin, and trimethoprim). Statistical analysis by 2-way ANOVA of CFU 

recovered at each time point or by Kaplan-Meier analysis of the number of mice remaining infected each day 

(clearance is defined as at least 3 successive days of negative gonococcal recovery) has revealed significant 

differences (P <0.05 or lower) between control and experimental groups, each with at least 8 animals 

completing the experiments. To guard against inadvertent loss of mice through accident (a rare event) or 

overgrowth of commensal microbiota under estrogen treatment, experiments will be commenced with groups 

of at least 10 animals. Experiments requiring challenge after a prolonged interval, e.g., up to 6 months will 

start with 12 animals. Experiments will be performed and gonococcal recovery will be enumerated by persons 

who are “blinded” to the group designations. All experiments will be repeated 2-3 times. 

I.vag. immunization has been successfully achieved by instilling GvaX12® containing OMV prepared as 

described (19, 29) from N. gonorrhoeae strains FA1090, MS11, or FA19 (containing 40μg protein) plus IL-12 

particles (1μg murine IL-12) and repeating the dose 2 weeks later (see above). We will compare this regimen 

with administering the same dose of GvaX12® intranasally (i.n.) to a group of 10-12 female mice, and giving a 

second similar dose 2-3 weeks later. Control groups will include sham-immunized mice given vehicle only by 

the same routes. Additional controls for the i.n. route will include mice immunized with OMV plus blank 

particles, or with IL-12 particles alone. As these latter controls have previously been performed repeatedly by 

Contact PD/PI: Liu, Yingru

Research Strategy Page 83
the i.vag. route and shown to be no different from sham-immunization, they will not be routinely repeated. Two 

weeks after the second immunization, mice will be challenged with N. gonorrhoeae of the same strain as used 

to prepare the OMV, and infection monitored by vaginal swabbing and plating. The results will be evaluated 

statistically using 2-way ANOVA of gonococcal CFU recovered, and Kaplan-Meier analysis of the number of 

mice remaining infected, to determine (a) if protection against gonococcal challenge is achieved by i.n. 

immunization with GvaX12® (compared to controls), and (b) whether i.n. immunization is as effective as i.vag. 

immunization. 

 

Compare immune responses with i.n. versus i.vag. immunization 

As in our previous studies, serum, saliva, and vaginal wash samples will be collected from mice prior to 

immunization, ~2 weeks after immunization and prior to challenge, and at termination (after clearance of 

infection) for assay of anti-gonococcal Abs (IgG and IgA; IgM Ab are low, variable, and uninformative) by 

ELISA. Western blot analysis of the Abs also will be performed using OMV subjected to SDS-PAGE to reveal 

the predominant Ag bands detected by these Abs. 

 For the i.vag.-immunized mice, the iliac lymph nodes (ILN) will be excised from i.vag.-immunized mice 

to assess T cell responses in the genital tract-draining lymph nodes. In some instances, the mice will 

be euthanized after immunization and prior to challenge 

 For the i.n.-immunized mice, we will instead use the superficial and deep cervical lymph nodes (CLN) 

which drain the nasal lymphoid tissues (NALT) (30-32). T cell responses will be assessed by: (a) 

intracellular staining and flow cytometry of ILN or CLN cells (co-stained for surface markers including 

CD4 and CD8) for the production of cytokines ex vivo (IFN, IL-4, and IL-17); (b) culture of ILN or CLN 

cells preloaded with carboxyfluorescein succinimide ester (CFSE) with or without gonococcal Ag (OMV) 

for 4 days followed by staining for surface markers (CD4 and CD8) and flow cytometry to assess 

proliferation by attenuation of CFSE staining (secreted cytokines can also be assayed by ELISA); and 

(c) RT-PCR assay of mRNA extracted from ILN or CLN cells for the key Th1, Th2, and Th17 

transcription factors Tbet, GATA3, and RORt, respectively. 

Differences in the quantitative responses shown by different groups of mice will be analyzed statistically using 

Student’s t tests (for 2 groups) or ANOVA (for multiple groups). All of these experimental and statistical 

methods have been used in our previous studies (14, 16-18). 

Outcomes, potential difficulties, further experiments/alternatives, and significance/interpretation for 

Task 1A: 

The primary objectives are to determine: 1) whether i.n. immunization with GvaX12® is as effective as i.vag. 

immunization in protection against i.vag. challenge with N. gonorrhoeae; and 2) whether similar immune 

responses are induced by these two routes. If i.n. immunization is effective, we would expect it to be 

associated with similar immune responses (i.e., anti-gonococcal Abs in serum and vaginal wash, and IFN-

secreting CD4+ T cells in the draining lymph nodes). Evaluation of salivary IgA Ab responses would reveal the 

induction of a common mucosal immune response, which would be expected for i.n. immunization, but not for 

i.vag. immunization, because the genital tract does not contain the mucosal inductive site tissues found in the 

nasal passages of mice (28, 30). If protection is weak or ineffective, we expect it to be associated with 

inadequate immune responses, and in that case we will adjust the dose of vaccine (GvaX12®) given i.n., and/or 

the number of doses (e.g., 3 or more doses at 2-week intervals) to increase the immune responses and 

generate protection. 

An important consideration will be the generation of Ab (IgG and IgA) in vaginal wash after i.n. immunization as 

presumably this will be critical for protection. Based on previous experience we expect i.n. immunization to be 

at least as effective in generating genital tract Ab responses as i.vag. immunization (25, 28). Discordance 

between protection and immune response induction by the two routes would require further investigation, and 

possibly provide insight into the mechanisms of protective immunity. If i.n. immunization effectively generates 

both protection and immune responses equivalent to i.vag. immunization, we will immunize male mice i.n. with 

the same GvaX12® regime to determine if they generate the same serum and salivary Ab responses as female 

mice. Although it is not possible to evaluate protective immunity in male mice, a positive result would broaden 

the potential applicability of GvaX12®to both sexes. 

Contact PD/PI: Liu, Yingru

Research Strategy Page 84
Given that vaginal IgG Abs are likely derived from the circulation, we will also compare the response to s.c. 

immunization with GvaX12® (with appropriate controls) because would expect this route to induce 

predominantly serum IgG Ab, as well as vaginal IgG Ab as reported previously (22). If this should be sufficient 

to generate protection against gonococcal challenge, it would be important to determine whether a Th1/IFN

response were also induced, because we previously demonstrated this effect to be essential for protective 

immunity (see Phase I data above). However, GvaX12®is intended for local mucosal, not systemic application, 

because systemic release of IL-12 can be toxic. Therefore, an alternative Th1-driving adjuvant for systemic 

immunization with OMV may be necessary; however, that possibility is beyond the scope of this application. 

We realize that the mouse model cannot fully predict the efficacy of the proposed vaccine in humans, which 

can only be determined by means of clinical trials. N. gonorrhoeae is an exclusively human pathogen and nonhuman primates are not susceptible to infection, with the exception of chimpanzees (33). However, despite its 

limitations the mouse model is the only available animal model for studying the response of an intact 

mammalian immune system to N. gonorrhoeae, and we have used it to good effect for this purpose for several 

years. Importantly it provides a means to elucidate the type(s) of immune response necessary for protective 

immunity against gonococcal infection, and may also identify potential surrogate markers of protection. Thus it 

can test hypotheses that cannot be tested in humans and help to frame the questions that will need to be 

addressed in humans. 

Task 1B: Determine the basis of cross-protective immunity against antigenically diverse clinical 

isolates of N. gonorrhoeae 

We have found that i.vag. immunization with GvaX12® containing OMV derived from any of three widely used 

“laboratory strains” (FA1090, MS11, and FA19) induces protection against challenge, not only against the 

homologous strain, but also against each other, despite their known antigenic differences (see above). In 

addition, serum and vaginal Ab showed strong cross-reactivity in ELISA against heterologous gonococcal 

strains (see above). Although these findings were wholly unexpected, they have been repeatedly reproduced. 

However, the extent to which these extensively subcultured strains are representative of naturally circulating N. 

gonorrhoeae strains is uncertain. Therefore, to further investigate these findings we will determine whether Ab 

cross-reactivity and associated cross-protection against challenge extends to antigenically diverse, minimally 

passaged clinical isolates of N. gonorrhoeae (in collaboration with Dr. Marcia Hobbs, University of North 

Carolina, Chapel Hill; letter attached). 

Our preliminary findings showed: 1) that immunization of mice i.vag. with GvaX12® containing OMV prepared 

from strain FA1090 also protects against subsequent challenge (with two minimally passaged clinical isolates 

provided by Dr. Hobbs): and 2) that serum and vaginal Ab react in ELISA with these isolates, similar to what 

we observed with heterologous lab strains (data not shown). Although we can extend these experiments to 

include additional clinical isolates, the time and effort involved in such an extension would severely limit the 

number of strains that could be tested. Therefore we propose the following process: 

1. First, we will perform screening assays by ELISA on a larger number (at least 30) of clinical isolates 

from diverse geographic locations in order to determine whether serum and vaginal Abs induced by 

immunization of mice with OMV from the laboratory strains cross-react with these clinical isolates in 

comparison to the homologous strains. For this purpose, we will immunize mice i.vag. with GvaX12® 

containing OMV prepared from gonococcal strains FA1090, MS11, and FA19 (at least 20 mice each), 

as described in Aim 1A above, and collect serum and vaginal wash 2 weeks after the last dose. 

Terminal bleeding (from the inferior vena cava or subclavian vein) of the mice will be performed under 

anesthesia in order to obtain as much serum as possible. Samples will be pooled to yield 5-10ml of 

pooled serum and 20ml or more of vaginal wash. Because this is a screening procedure and we 

already know that control immunizations are not protective and do not induce Ab, control serum and 

vaginal wash will be obtained from unimmunized mice. ELISA plates will then be coated with whole 

cells of N. gonorrhoeae, including strains FA1090, MS11, and FA19 as well as the clinical isolates, 

freshly grown overnight on “chocolate agar” plates incubated at 37°C in 5% CO2 as described 

previously. Concentrations of gonococci in each harvested culture will be adjusted to the same level by 

OD at 600nm. The pooled serum and vaginal wash from immunized and control mice will be serially 

diluted and incubated on the plates in quadruplicate for 4-6 hours. Bound antibodies will be detected by 

Contact PD/PI: Liu, Yingru

Research Strategy Page 85
incubation with alkaline phosphatase-conjugated anti-IgG or anti-IgA reagents (Southern 

Biotechnology), followed by appropriate substrate development. 

2. We will then perform western blots to identify gonococcal Ag bands detected by the Abs. For this 

purpose small-scale OMV preparations will be made from each isolate (by shearing cell suspensions in 

lithium acetate buffer and applying differential centrifugation as we have done previously (13), their 

protein concentrations will be determined by the Pierce bicinchoninic acid method (34). OMV (10-50μg 

protein) will be boiled in SDS sample buffer (with 2-mercaptoethanol), separated on SDSpolyacrylamide (10%) gels, blotted to nitrocellulose membranes and probed with suitably diluted serum 

or vaginal wash pools from immunized or control mice. Bound Ab will be detected using peroxidaseconjugated anti-IgG or anti-IgA reagents and chemiluminescence. A replicate gel will be stained with 

Coomassie blue to reveal the protein bands. Pre-stained MW markers will be included on each gel. 

3. We will then test selected clinical isolates (up to 6, chosen at random, to include strains that do not

react with the Ab in immunized mouse serum or vaginal wash, if any) in challenge-protection 

experiments. For this purpose, 2 groups (A and B) of 12 mice will be immunized i.vag. with GvaX12® 

containing OMV from strains FA1090, MS11 or FA19, as described above (Aim 1A). Two control 

groups (C and D) of 12 mice will be unimmunized. One month after immunization, one immunized and 

one control group (A and C) will be challenged i.vag. with the selected clinical isolate (transformed to be 

streptomycin-resistant by Dr. Hobbs), and the other two groups (B and D) will be challenged with the 

immunizing strain (FA1090/MS11/FA19). The course of infection will be monitored by vaginal swabbing 

and plating as described above. The recovery of gonococcal CFU and the numbers of mice remaining 

infected will be analyzed statistically by 2-way ANOVA and Kaplan-Meier methods, respectively, as 

described above. Each experiment will be repeated to verify the result. 

Outcomes, potential difficulties, further experiments/alternatives, and significance/interpretation for 

Task 1B 

We will first relate the ability of clinical isolates to react in ELISA with Ab in serum and vaginal washes of i.vag.-

immunized mice to their porin and Opa types as determined by Dr. Hobbs. On the basis of limited information 

pertaining to the laboratory strains (FA1090, MS11, and FA19 are known to express different porB and opa

genes), we do not expect to find any close concordance, but given that porin and Opa are major surface 

proteins, this will be examined. Examination of the sera and vaginal wash by western blots against OMV 

proteins should confirm lack of reactivity against bands corresponding to porin and Opa proteins, as noted 

already for FA1090, MS11, and FA19. However, caution is warranted because protein Ags are denatured in 

SDS-PAGE and may not be fully restored on blotting to nitrocellulose membranes, therefore any Ab reactive 

with them could be missed. An alternative approach would be to separate OMV proteins in non-denaturing 

gels for blotting; however the MW of the protein bands cannot be readily assigned and identification of the 

porin and Opa bands would be uncertain. It is also possible that the culture conditions used to prepare OMV, 

both for immunization and for Ab analysis, will affect the expression of various Ags, e.g., those regulated by 

iron or anaerobiosis. Thus it is important to grow N. gonorrhoeae under standard conditions; however, these 

conditions can be altered in light of findings to determine if, for instance, Ags selectively expressed under ironlimited or anaerobic conditions are relevant. 

It is possible that Abs against lipooligosaccharide (LOS) contribute to protective immunity. Interest currently 

focuses on the 2C7 epitope which is expressed by >95% of clinical isolates under phase-variable control (35, 

36). We will check the expression of 2C7 in the various strains of N. gonorrhoeae, using 2C7 Ab obtained from 

Dr. Peter Rice (University of Massachusetts; letter attached), to determine whether protective immunity and Ab 

reactivity accord with its expression. Otherwise, determining LOS type and structure is beyond the scope of 

the current proposal, and would require further collaborations with those who have the necessary expertise. 

The more important question is whether i.vag. immunization with OMV from the laboratory strains induces 

protection against naturally occurring strains of N. gonorrhoeae. Although this question cannot be 

comprehensively assessed within this proposal, we will determine whether protection is shown against at least 

6 randomly chosen clinical isolates, including any that do not recognize Ab in ELISA and/or western blot 

assays. If protection is dependent on vaccine-induced Ab that recognize the challenge strain, then any clinical 

isolates that fail to react with Ab should not be protected against, and a finding to the contrary would challenge 

the hypothesis. Conversely, if strains are found that are not protected against despite recognizing vaccineinduced Ab, this would negate the hypothesis that these Ab determine protective immunity. If we are to go 

Contact PD/PI: Liu, Yingru

Research Strategy Page 86
forward with a vaccine based on gonococcal OMV it will be important to know if there is a frequent occurrence 

of strains that are not protected against. If so, then a broader-based vaccine, perhaps incorporating OMV from 

multiple strains, or including selected additional Ags (as in the recently introduced 4CMenB vaccine against N. 

meningitidis) would become necessary. 

If in Aim 1A it is found that i.n. immunization with GvaX12® results in protective immunity against homologous 

challenge (similar to that found with i.vag. immunization) and generates Ab (as detected by ELISA and western 

blotting against the homologous strain) it will be important to determine: 1) whether these Ab also recognize Ag 

in the clinical isolates; and 2) whether protective immunity against them is induced by this route of 

immunization. It is possible that different patterns of Ab reactivity may be induced by the two routes (because 

the nasal passages contain organized lymphoid tissue where mucosal immune responses are initiated, 

whereas such tissues are absent from the genital tract (30, 37)). If so, these differences could help elucidate 

the contribution of different Ab specificities to gonococcal immunity. 

Task 1C: To determine whether immunization with GvaX12® containing detergent-extracted OMV 

induces protective immune responses against N. gonorrhoeae 

A potential disadvantage of native OMV as a vaccine immunogen arises from reactogenicity caused by the 

presence of LOS. This effect has been observed with meningococcal OMV vaccines that are administered 

parenterally, although the mucosal routes of administration that we propose for our gonococcal OMV vaccine 

might prove less reactogenic. However, to forestall this potential problem, we will evaluate the ability of 

detergent-extracted gonococcal OMV (deOMV) to induce protection against N. gonorrhoeae, and to induce 

responses similar to those observed with native OMV. 

For this purpose, we will modify our standard OMV (FA1090) preparation by extraction with 1% sodium 

deoxycholate in 1mM EDTA, 0.1M Tris buffer pH 8.5, followed by chromatography on a size-exclusion column 

(38). Mice will be immunized i.vag. with 2 doses (2-week interval) of GvaX12® containing deOMV (50μg 

protein) as described above. Control mice will be immunized with deOMV plus blank particles, and a 

comparison group of mice will be immunized with GvaX12® containing native OMV. Samples of serum and 

vaginal wash will be collected at 2 weeks after immunization for Ab assay. One month later mice will be 

challenged with N. gonorrhoeae FA1090, and the course of infection will be monitored by vaginal swabbing 

and plating, as described above. Further samples of serum, vaginal wash, and ILN will be collected at 

termination, for evaluation of Ab and cytokine responses, as described above. 

We will compare protection against challenge, as well as Ab and cytokine responses induced by immunization 

with deOMV plus IL-12 particles with those induced by native OMV plus IL-12 particles, and with those induced 

by deOMV administered with blank particles. If the response to deOMV plus IL-12 particles is significantly less 

than that induced by native OMV plus IL-12 particles, we will repeat the experiment using increased doses of 

deOMV (e.g., 100-200μg protein), or by administering 3 or more immunizations. If i.n. immunization is 

successful with native OMV (Aim 1A above), we will further determine whether it works with deOMV. 

Outcomes and significance for Task 1C 

No difficulty is expected in performing experiments to evaluate the efficacy of deOMV as a vaccine immunogen 

in comparison with native OMV. On the basis of reports that meningococcal deOMV are less immunogenic 

than native OMV (attributed to the reduced LOS content), we may find that gonococcal deOMV are also less 

immunogenic; if so, we will determine whether increased dose or frequency of immunization can compensate 

adequately. The significance of successful results with deOMV may lie with diminished toxicity as revealed by 

the studies proposed in Aim 2 below 

3.2.2 Specific Aim 2: Conduct studies of Rodent pharmacokinetics and reproductive toxicology

A. Task 2A: determine the bioavailability and pharmacokinetics of IL-12 following intravaginal and 

intranasal administration of GvaX12® in mice. 

The objective of this study is to gain insight into the bioavailability, pharmacokinetics and potential acute 

toxicity of IL-12 following a single i.vag. or intranasal (i.n.) administration of GvaX12® or as a bolus intravenous 

(i.v.) dose of IL-12 to female mice. The study design is as follows: 

Contact PD/PI: Liu, Yingru

Research Strategy Page 87
Table 1 study design 

Group Test Item

Dose 

Route Dose Level (mg/kg)

Dose Rate

(mL/kg)

Dose 

Concentration

(mg/mL)

No. of Animals

Females

1 GvaX12®

[1μg IL-12

+ OMV (40μg protein)]

i.vag. IL-12: 0.04

OMV protein: 1.6 1.6 IL-12: 25OMV protein: 1.0 12

2 GvaX12®

[1μg IL-12

+ OMV (40μg protein)] i.n. IL-12: 0.04 *OMV protein: 1.6 1.6 IL-12: 25OMV protein: 1.0 12

3 IL-12 [1μg]

soluble i.v. 0.04 4.0 0.01 12

* Dose of IL-12 given i.n. is based on i.vag. dose. If higher levels are found to be needed by this route (Specific Aim 1A), 

the dose used for these studies will be modified accordingly. 

The following parameters and end-points will be evaluated in this study: clinical signs (i.e., hunched posture, 

ruffled fur, lethargy, loss of appetite, cool to touch), body weights, and toxicokinetic parameters (see below). 

Samples will be collected according to the following table: 

 Table 2 Schedule for Sample Collection 

Group Subgroup No. of 

animals

Sample Collection Time Points

(post start of dose)

5 

min

15 

min

30 

min 1h 2h 4h 6h 12h 16h 24h

1

A 3 - - X - - - - X

T

- - -

B 3 - - - X - - - X

T

- -

C 3 - - - - X - - - X

T

-

D 3 - - - - - X - - - X

T

2

A 3 X - - - - X

T

- - - -

B 3 - X - - - - X

T

- - -

C 3 - - X - - - - X

T

- -

D 3 - - - X - - - - - X

T

3

A 3 X - - - - X

T

- - - -

B 3 - X - - - - X

T

- - -

C 3 - - X - - - - X

T

- -

D 3 - - - X - - - - - X

T

X = Sample collected via saphenous vein puncture ; XT= Sample collected via terminal bleed on 

anesthetized mice from the inferior vena cava or subclavian vein; 

- = Not applicable.

Blood will be collected into serum separation tubes (SST) tubes by saphenous vein puncture (target of 0.1 mL) 

or via the abdominal aorta (maximum possible volume) while under isoflurane anesthesia (terminal samples 

only). An additional terminal blood sample (target volume up to 1 mL) will be collected of 1 spare animal and 

used as a pre-dose sample. Samples will be frozen immediately over dry ice and transferred to a freezer set to 

maintain -20C until use. Levels of IL-12 will be measured by ELISA. Tmax, (time of maximum serum 

concentration), T1⁄2 (half-life), Cmax (maximum serum concentration), Cmax/D (maximum serum concentration 

divided by dose) and AUC (area under the curve) will be calculated. Levels of other pro and anti-inflammatory 

cytokines (e.g., IFNTNF, IL-10 and TGF) will also be measured. 

Outcomes, potential difficulties, alternatives and significance For Task 2A 

The primary outcome measure for the pharmacokinetic study will be the circulating level of IL-12 over time, 

according to the schedule in Table 2. Secondary measures include circulating levels of IFN. The absolute 

bioavailability will be calculated using the dose-corrected area under curve (AUC) for non-intravenous 

administration divided by the AUC for intravenous administration, according to classical pharmacology 

modeling. However, based on our previous experience we do not expect significant systemic exposure of IL-12 

by either the i.n. or i.vag. route. Because the generation of IFN is a well-known effect of IL-12, we expect 

systemic IL-12 exposure also will result in increased IFN. The results will inform starting doses for 

subsequent IND enabling non-clinical studies in rodents. 

Contact PD/PI: Liu, Yingru

Research Strategy Page 88
No technical difficulties are anticipated in the performance of these studies. All the methods are in place and 

have been used previously. Extensive experience with GvaX12® administered i.vag. in doses up to 2μg of 

cytokine per mouse given at 2-day intervals for up to 5 doses, revealed no adverse effects. Whether similar 

doses given intranasally will result in adverse or toxic effects is unknown. GvaX12®is not designed for 

intravenous administration; however, standard pharmacokinetic and toxicological evaluations require 

comparisons with this route. Mice will be inspected at least twice daily for the following signs: hunched posture, 

ruffled fur, lethargy, loss of appetite, coolness to touch, and body weight. Animals displaying signs of distress 

after GvaX12® administration will be reported and monitored closely. A combination of 2 or more signs without 

evidence of recovery within 12h, or >10% loss of body weight, will be recorded as adverse reaction and the 

affected animals will be euthanized. 

B. Reproductive toxicology studies in rats. 

IL-12 is a pro-inflammatory cytokine. Because the proposed vaccine may be used in women of child-bearing 

age, reproductive toxicity is a concern. Therefore, early reproductive toxicology testing is proposed. Studies, 

including a GLP embryo-fetal development study and a study for effects on pre- and post-natal development in 

rats will be conducted at Comparative Biosciences. Complete study designs can be found in Appendix 1. If the 

i.n. route is found to be effective, reproductive toxicology will be delayed until our type C meeting (see below). 

Outcomes, potential difficulties, and alternatives for Task 2B

GvaX12® might induce untoward effects at the proposed dose levels. However, a major goal of encapsulation 

is to limit systemic exposure and side effects. Therefore, we believe such a finding is unlikely. However, 

significant reproductive toxicity would trigger a no-go decision for the continued development of this product. 

We have identified several back-up drug substances as substitute for IL-12 (e.g., anti-IL-10 antibody), which 

are under development separately. 

3.2.3 Specific Aim 3: To prepare and submit a briefing package, written questions and formal request for a 

pre-pre-IND (Type C) meeting with the FDA

The successful accomplishment of Aims 1 and 2 will incentivize and prioritize the development of our 

prophylactic gonorrhea vaccine. The early input of the FDA will be invaluable in guiding our IND enabling 

scale-up and toxicology study designs. Regulatory Professionals is already assisting TherapyX

inc in the 

preparation and submission of GvaX12® IND-related materials. The master services agreement and SOW 

related to the proposed studies is included in Appendix 2. The consultants at Regulatory Professionals have 

extensive expertise in the preparation of briefing packages, meeting requests, clinical (IND) and marketing 

applications for various drug formulations, including biologics and polymeric drug delivery systems. A briefing 

package and meeting request will be prepared and submitted along with specific questions in the areas of 

clinical, nonclinical, and Chemistry, Manufacturing and Control (CMC). The aim is to confirm FDA requirements 

for data to support Phase I/II clinical studies, primarily with respect to safety. Ultimately, the questions will be 

determined by the data collected from the studies conducted in Aims 1 and 2 and by our discussion with 

toxicology consultants at Regulatory Professionals. 

Outcomes, potential difficulties, and significance 

The FDA encourages investigators to initiate contact with the agency as early in the drug development process 

as possible. The stated goal is to allow investigators the opportunity to consider FDA recommendations in 

planning preclinical and clinical development programs. TherapyX

inc has recently completed a pre-pre-IND 

(Type C) meeting with the FDA for a separate project involving oral administration of TGF-loaded particles 

(TGFNanoCap®). The preparation for this meeting included the creation of a briefing package which included 

specific questions requesting the FDA’s opinion of the company’s proposed Non-Clinical (toxicology), Quality 

(CMC), and Clinical (early clinical trial design) study plans. We expect that the preparation of the briefing 

package for this program will follow the same developmental path. If circumstances dictate, additional 

consulting expertise can be obtained. We do not expect there to be any difficulties that cannot be overcome 

with the assembled team of advisors. 

Contact PD/PI: Liu, Yingru

Research Strategy Page 89
TIMELINE

Contact PD/PI: Liu, Yingru

Research Strategy Page 90
Vertebrate Animals \

VERTEBRATE ANIMALS

TherapyX

Inc. has an inter-institutional agreement in place with the University at Buffalo (UB) 

regarding these and other animal studies. All in vivo murine studies will be performed in the 

Laboratory Animal Facilities of the Jacobs School of Medicine and Biomedical Sciences at 

UB (Animal Welfare Assurance Number A3354-01). The care and use of laboratory animals 

at UB is in accordance with the principles and standards set forth in the Principles for Use 

of Animals (NIH Guide for Grants and Contracts), the Guide for the Care and Use of 

Laboratory Animals (DHEW, PHS, NIH Publ. No. 80-23, Rev 1978), the provision of the 

Animal Welfare Acts (P.L. 89-544 and its amendments), NYS laws for animal welfare and 

other applicable laws and regulations. Compliance is validated by the UB IACUC and 

regular inspections by USDA and NYS inspecting veterinarians. Assurances and the 

composition of the Institute Animal Care and Use Committee are on file with the Office of 

Protection from Research Risks, Office of the Director, NIH. 

1. Proposed use of animals

Inbred wild-type mice (BALB/c) of 2-6 months age at the start and of female sex will be 

used. Small volumes of blood (50-100μl) will be obtained from the saphenous vein, or 

from the retro-orbital sinus under anesthesia. Larger volumes of blood will be collected 

as a terminal procedure under anesthesia. Spleens, genital tracts, lymph nodes, and 

other organs will be obtained from mice killed by cervical dislocation under anesthesia. 

IACUC approval for these studies is in place (protocol # MIC19074Y). For the 

gonococcal vaginal challenge model, 2-month old female mice will be injected 

subcutaneously with 0.5mg of 17- estradiol (Premarin) on days –2, 0, and +2 relative 

to gonococcal challenge, then injected once daily i.p. with streptomycin sulfate (1.2mg) 

and vancomycin HCl (0.6mg), and provided drinking water containing trimethoprim 

sulfate (0.04g/100ml). On day 0, the mice are inoculated vaginally with N. gonorrhoeae 

(approx. 5x106 CFU) suspended in PBS containing 0.5mM CaCl2, 1mM MgCl2, and 1% 

gelatin. Colonization is monitored daily for up to 14 days (or until the infection is 

cleared) by vaginal swabbing and plating. Animals are then euthanized and terminal 

blood samples and tissues harvested for laboratory investigation. These experiments 

will be performed in a separate BSL2 suite, and used bedding, cages, and wastes will 

be autoclaved or disinfected prior to disposal or re-use. Carcasses will be treated as 

infectious waste. 

2. Justification

Mice are chosen for in vivo investigation of immune responses to N. gonorrhoeae 

because they constitute a well-established model for immunological studies, there is 

considerable background information on their immune systems, and a wide range of 

high-quality immunochemical and cellular reagents is available. It is not possible to 

investigate immune responses to infection other than by means of a live animal 

infection model, and the mouse vaginal gonococcal challenge model is the only 

available model of genital gonococcal infection. Human infection studies of this nature 

would be unethical. In-bred animals are required for in vitro cell culture experiments. 

Groups of animals used for gonococcal challenge (12) are as small as possible 

consistent with yielding statistically valid data. 

Contact PD/PI: Liu, Yingru

Vertebrate Animals Page 91
Vertebrate Animals \

Anticipated mouse numbers:

Year 1 

Aim 1A. To determine an optimal route of immunization: Intravaginal vs 

Intranasal.

We will compare the efficacy of optimized intravaginal dose of GvaX12®+ OMVs with the 

intranasal route. Groups will consist of 12 mice. 

1. Intra-vaginal route: GvaX12® + OMVs (12 BALB/c) 

2. Intra-nasal route: GvaX12® + OMVs (12 BALB/c) 

3. Sham intra-vaginal (12 BALB/c) 

4. Sham intra nasal (12 BALB/c) 

5. Blank (control) microparticles + OMVs intranasal (12 BALB/c) 

6. GvaX12® alone intranasal (12 BALB/c) 

 Six groups X 12 per group = 72 X up to 3 repeats = 216 BALB/c mice 

Aim 1B. To determine the basis of cross-protective immunity against 

antigenically diverse clinical isolates of N. gonorrhoeae. 

We will: (I) determine whether cross-reactivity of Ab and associated cross-protection 

against challenge extends to antigenically diverse, minimally passaged clinical isolates 

of N. gonorrhoeae; and (II) identify by means of proteomics technologies the 

gonococcal Ags detected by Abs induced in mice immunized with gonococcal OMV 

plus GvaX12®

For each clinical isolate examined: 

1. Groups A and B immunized with FA1090/MS11/FA1 (24 BALB/c mice) 

2. Groups C and D Unimmunized mice (24 BALB/c mice) 

 Four groups x 12 mice per group = 48 mice x up to 2 repeats = 96 BALB/c mice 

 X 6 clinical isolates to be examined = 576 mice 

Total Mice in Year 1.... 216 + 576 = up to 792 mice

Year 2 

Aim 1C. To determine whether immunization with detergent-extracted OMV (plus 

GvaX12®) induces protective immune responses against N. gonorrhoeae 

Mice will be immunized by the intravaginal route with detergent-extracted OMV + 

GvaX12® and challenged with the homologous gonococcal strain. Groups will consist of 

12 mice 

1. GvaX12®+ deOMV (12 BALB/c) 

2. Blank ms + de OMV (12 BALB/c) 

3. GvaX12® + native OMV (12 BALB/c) 

Contact PD/PI: Liu, Yingru

Vertebrate Animals Page 92
Vertebrate Animals \

Three groups x 12 mice per group = 36 mice, x up to 6 repeats with doses adjusted as 

necessary = 216 mice

Repeat experiments using intranasal immunization (if successful) = 216 mice 

Aim 2A. Determine the bioavailability and pharmacokinetics of IL-12 following 

intravaginal/intranasal administration of IL12/ms vaccine in mice. 

To goal of this aim is to obtain preliminary data on bioavailability and pharmacokinetics 

of IL-12 following a single intravaginal/intranasal or bolus intravenous dose of IL-12 to 

BALB/c mice 

1. Group 1 intra-vaginal (12 BALB/c) 

2. Group 2 intra-nasal (12 BALB/c) 

3. Group 3 soluble IL-12 given intravenously (12 BALB/c) 

Three groups x 12 mice per group = 36 mice x up to 2 repeats = 72 BALB/c mice 

Total in Year : 216 + 216 + 72 = 604 mice 

Aims 2B and 2C. Reproductive Toxicity in rats. 

If necessary (i.e., if we need to use intra-vaginal administration), we will conduct routine 

reproductive toxicology, arriving at a potentially no-go decision node. This work will be 

subcontracted to Comparative Biosciences and completed under their IACUC (see Appendix 3 

for details). 

Aim 2B: Embryonic-Fetal Development Study 

109 Sprague Dawley rats will be used at Comparative Biosciences, Sunnyvale, CA, to 

investigate potential effects on embryonic-fetal development. These are standard animals 

and standard numbers required for these routine reproductive toxicity assessments (See 

Appendix 3).

Aim 2C Effects on pre and post-natal development 

260 Sprague Dawley rats will be used at Comparative Biosciences, Sunnyvale, CA, to 

investigate potential effects on pre- and post-natal development. These are standard animals 

and standard numbers required for these routine reproductive toxicity assessments (See 

Appendix 3).

Total: 109 + 260 = 369 animals 

3. Veterinary care and facilities

The UB Laboratory Animal Facility is directed by full-time professional veterinarians and 

staffed by trained, qualified personnel, all of whom may be called upon for advice and 

assistance at any time. The facility is fully accredited by the American Association for 

the Accreditation of Laboratory Animal Care, and complies with NIH policy, the Animal 

Contact PD/PI: Liu, Yingru

Vertebrate Animals Page 93
Vertebrate Animals \

Welfare Act, and all applicable federal, state, and local laws. Mandatory training is 

provided for all laboratory and scientific personnel who are involved in procedures. 

Veterinary staff at UB include Dr. Lisa Martin, Director, Dr. Sasha Black, Clinical 

Veterianrian, and Dr. Jennifer Peirick, Clinical Veterinarian. 

4. Provisions for animal welfare 

Mice will be housed and maintained according to the prescribed standards for this 

species, under the supervision of the UB Laboratory Animal Facility. For blood 

collection from the saphenous vein or retro- orbital sinus, mice will be anesthetized by 

inhalation of isofluorane for 1 minute. For terminal bleeding from the subclavian vein, 

mice will be anesthetized with xylazine plus ketamine (1.5mg/100g and 10mg/100g, 

respectively, i.p.) and the animals will be killed (by cervical dislocation) while under 

anesthesia. 

5. Euthanasia

Mice will be killed at the termination of experiments by cervical dislocation under 

anesthesia, or by CO2 asphyxiation, or by overdose of sodium pentobarbital injected i.p. 

All of these are consistent with the recommendations of the Panel on Euthanasia of the 

American Veterinary Medical Association. 

Contact PD/PI: Liu, Yingru

Vertebrate Animals Page 94
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Services; 2015. 

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breaking the chain of transmission. Geneva, Switzerland: WHO; 2007. 

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Infections. 2013;89(Suppl 4):iv63-iv8. doi: 10.1136/sextrans-2013-051225.

6. Bolan GA, Sparling PF, Wasserheit JN. The emerging threat of untreatable gonococcal infection. New 

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8. Broutet N, Fruth U, Deal C, Gottlieb SL, Rees H. Vaccines against sexually transmitted infections: The 

way forward. Vaccine. 2014;32:1630-7. doi: 10.1016/j.vaccine.2014.01.053. 

9. Gottlieb SL, Deal CD, Giersing B, Rees H, Bolan G, Johnston C, Timms P, Gray-Owen SD, Jerse AE, 

Cameron CE, Moorthy VS, Kiarie J, Broutet N. The global roadmap for advancing development of vaccines 

against sexually transmitted infections: Update and next steps. Vaccine. 2016;34:2939-47. doi: 

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10. Wetzler LM, Feavers IM, Gray-Owen SD, Jerse AE, Rice PA, Deal CD. Summary and 

recommendations from a National Institute of Allergy and Infectious Diseases (NIAID) workshop on “Gonorrhea 

Vaccines: the Way Forward”. Clinical and Vaccine Immunology. 2016;23:656-63. doi: 10.1128/cvi.00230-16.

11. Jerse AE, Bash MC, Russell MW. Vaccines against gonorrhea: current status and future challenges. 

Vaccine. 2014;32:1579-87. doi: 10.1016/j.vaccine.2013.08.067. PubMed PMID: 24016806; PMCID: 4682887. 

12. Zhu W, Chen CJ, Thomas CE, Anderson JE, Jerse AE, Sparling PF. Vaccines for gonorrhea: can we 

rise to the challenge? Frontiers in Microbiology. 2011;2:124. Epub 2011/06/21. doi: 10.3389/fmicb.2011.00124. 

13. Feinen B, Jerse AE, Gaffen SL, Russell MW. Critical role of Th17 responses in a murine model of 

Neisseria gonorrhoeae genital infection. Mucosal Immunology. 2010;3:312-21. doi: 10.1038/mi.2009.139. 

PubMed PMID: 20107432; PMCID: 2857675. 

14. Liu Y, Russell MW. Diversion of the immune response to Neisseria gonorrhoeae from Th17 to Th1/Th2 

by treatment with anti-TGF-β antibody generates immunological memory and protective immunity. mBio. 

2011;2: (00095-11). doi: 10.1128/mBio.00095-11; PMCID: 3101786. 

15. Liu Y, Feinen B, Russell MW. New concepts in immunity to Neisseria gonorrhoeae: innate responses 

and suppression of adaptive immunity favor the pathogen, not the host. Frontiers in Microbiology. 2011;2:52. 

doi: 10.3389/fmicb.2011.00052; PMCID: 3153028. 

16. Liu Y, Islam E, Jarvis GA, Gray-Owen S, Russell MW. Neisseria gonorrhoeae selectively suppresses 

the development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-β-dependent 

mechanisms. Mucosal Immunology. 2012;5:320-31. doi: 10.1038/mi.2012.12; PMCID: 3328619. 

17. Liu Y, Liu W, Russell MW. Suppression of host adaptive immune responses by Neisseria gonorrhoeae: 

role of interleukin 10 and type 1 regulatory T cells. Mucosal Immunology. 2014;7:165-76. Epub 6/12/13. doi: 

10.1038/mi.2013.36. PubMed PMID: PMID: 23757303; PMCID: 3812424. 

18. Liu Y, Egilmez NK, Russell MW. Enhancement of adaptive immunity to Neisseria gonorrhoeae by local 

intravaginal administration of microencapsulated IL-12. Journal of Infectious Diseases. 2013;208:1821-9 doi: 

10.1093/infdis/jit354. PubMed PMID: 24048962; PMCID: 3814831. 

19. Liu Y, Hammer L, Liu W, Hobbs MM, Zielke RA, Sikora AE, Jerse AE, Egilmez NK, Russell MW. 

Experimental vaccine induces Th1-driven immune responses and protection against Neisseria gonorrhoeae in 

a murine model. Mucosal Immunology. 2016;(MS submitted). 

20. Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, Santos CA, 

Vijayaraghavan K, Montgomery S, Bassett M, Morrell C. Biologically erodable microspheres as potential oral 

drug delivery systems. Nature. 1997;386:410-4. doi: 10.1038/386410a0. 

Contact PD/PI: Liu, Yingru

References Cited Page 95
21. Conway TF, Hammer L, Furtado S, Mathiowitz E, Nicoletti F, Mangano K, Egilmez NK, Auci DL. Oral 

Delivery of Particulate Transforming Growth Factor Beta 1 and All-Trans Retinoic Acid Reduces Gut 

Inflammation in Murine Models of Inflammatory Bowel Disease. J Crohns Colitis. 2015;9(8):647-58. doi: 

10.1093/ecco-jcc/jjv089. PubMed PMID: 25987350; PMCID: PMC4817304. 

22. Plante M, Jerse A, Hamel J, Couture F, Rioux CR, Brodeur BR, Martin D. Intranasal immunization with 

gonococcal outer membrane preparations reduces the duration of vaginal colonization of mice by Neisseria 

gonorrhoeae. Journal of Infectious Diseases. 2000;182:848-55.

23. Holst J, Oster P, Arnold R, Tatley MV, Naess LM, Aaberge IS, Galloway Y, McNicholas A, O'Hallahan J, 

Rosenqvist E, Black S. Vaccines against meningococcal serogroup B disease containing outer membrane 

vesicles (OMV): lessons from past programs and implications for the future. Human Vaccines and 

Immunotherapeutics. 2013;9:1241-53. doi: 10.4161/hv.24129. 

24. Jerse AE. Experimental gonococcal genital tract infection and opacity protein expression in estradioltreated mice. Infection and Immunity. 1999;67:5699-708.

25. Russell MW, Moldoveanu Z, White PL, Sibert GJ, Mestecky J, Michalek SM. Salivary, nasal, genital, 

and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the 

cholera toxin B subunit. Infection and Immunity. 1996;64:1272-83.

26. Bergquist C, Johansson EL, Lagergård T, Holmgren J, Rudin A. Intranasal vaccination of humans with 

recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory 

tract and the vagina. Infection and Immunity. 1997;65:2676-84.

27. Rudin A, Riise GC, Holmgren J. Antibody responses in the lower respiratory tract and male urogenital 

tract in humans after nasal and oral vaccination with cholera toxin B subunit. Infection and Immunity. 

1999;67:2884-90.

28. Wu H-Y, Abdu S, Stinson D, Russell MW. Generation of female genital tract antibody responses by 

local or central (common) mucosal immunization. Infection and Immunity. 2000;68:5539-45.

29. Zielke RA, Sikora AE. Isolation of cell envelopes and naturally released membrane vesicles of 

Neisseria gonorrhoeae. Current Protocols in Microbiology. 2014;34:4A3.1-4A3 .17. doi: 

10.1002/9780471729259.mc04a03s34. 

30. Wu H-Y, Nguyen H, Russell MW. Nasal lymphoid tissue (NALT) as a mucosal immune inductive site. 

Scandinavian Journal of Immunology. 1997;46:506-13. 

31. Wu H-Y, Nikolova EB, Beagley KW, Eldridge JH, Russell MW. Development of antibody-secreting cells 

and antigen-specific T cells in cervical lymph nodes after intranasal immunization. Infection and Immunity. 

1997;65:227-35.

32. Wu H-Y, Russell MW. Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the 

common mucosal immune system. Immunol Res. 1997;16:187-201. doi: 10.1007/BF02786362. 

33. Arko RJ. Animal models for pathogenic Neisseria species. Clinical Microbiology Reviews. 1989;2, 

Suppl.:S56-S9. 

34. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, 

Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85.

35. Gulati S, McQuillen DP, Mandrell RE, Jani DB, Rice PA. Immunogenicity of Neisseria gonorrhoeae

lipooligosaccharide epitope 2C7, widely expressed in vivo with no immunochemical similarity to human 

glycosphingolipids. Journal of Infectious Diseases. 1996;174:1223-37.

36. Chakraborti S, Lewis LA, Cox AD, St. Michael F, Li J, Rice PA, Ram S. Phase-variable heptose I glycan 

extensions modulate efficacy of 2C7 vaccine antibody directed against Neisseria gonorrhoeae

lipooligosaccharide. Journal of Immunology. 2016;196:4576-86. doi: 10.4049/jimmunol.1600374. 

37. Russell MW, Mestecky J. Humoral immune responses to microbial infections in the genital tract. 

Microbes and Infection. 2002;4:667-77.

38. Frasch CE, van Alphen L, Holst J, Poolman JT, Rosenqvist E. Outer membrane protein vesicle 

vaccines for meningococcal disease. In: Pollard AJ, Maiden MCJ, editors. Methods in Molecular Medicine, vol 

66: Meningococcal Vaccines: Methods and Protocols. Totowa, NJ: Humana Press Inc.; 2001. p. 81-107.

Contact PD/PI: Liu, Yingru

References Cited Page 96
Contact PD/PI: Liu, Yingru

School of Medicine Division of Infectious Diseases ResearchCampus Box 7031 8309 Medical Biomolecular Research Bldg. 

The University of North Carolina at Chapel Hill  Chapel Hill, NC  27599-7031 

Administrative Office: (919) 843-0693  FAX: (919) 843-1015 

Department of Medicine 

Division of Infectious Diseases 

August 22, 2016 

Dr. Yingru Liu 

Senior Research Scientist 

TherapyX Inc. 

Farber 138 

3435 Main St.

Buffalo, NY 14214 

Dear Yingru, 

I am pleased to continue my enthusiastic support of your work with Dr. Michael Russell at the 

University of Buffalo in preparation for the new SBIR Phase II grant application entitled 

“Experimental Gonococcal Vaccine.” As you know, the development of new treatment and 

immunoprophylaxis strategies for genital tract infection with Neisseria gonorrhoeae is urgently 

needed, as increasing gonococcal antimicrobial resistance threatens the continued utility of 

current antibiotic treatments. Your preliminary success with administration of IL-12 

microspheres during primary infection with N. gonorrhoeae strain FA1090 and protection 

against re-infection with the homologous strain, well-characterized lab strains and more recent 

clinical isolates from the archives of our UNC strain collection is very encouraging. 

For a subcontract in the new grant, I agree to provide up to 30 clinical isolates of N. gonorrhoeae

from a variety of geographic locations to screen for reactivity with serum and vaginal antibodies 

from vaccine-protected mice. In addition, we will transform up to 6 strains in my laboratory to 

streptomycin resistance for use in the murine model of female genital tract infection. Should 

your studies lead to the development of a product for Phase I clinical trial studies in humans, you 

know that we will be anxious to collaborate using the experimental human infection model of 

gonococcal urethritis, which is up and running here at the University of North Carolina at Chapel 

Hill. 

Here’s wishing you and Mike and your colleagues good luck with the grant. I look forward to 

tracking your continued success demonstrating the potential for a gonococcal vaccine, at long 

last! 

Sincerely, 

Marcia M. Hobbs, Ph.D. 

Professor of Medicine, Microbiology & 

Immunology and Allied Health Sciences 

Letters of Support Page 97
Contact PD/PI: Liu, Yingru

August 8th, 2015

Dominick L. Auci, Ph.D. 

Vice President Research and Development 

TherapyX

Inc

Dear Dr. Auci, 

I am happy to provide advice and technical support with regard to the preparation 

and characterization of PIN microspheres for the studies outlined in your NIAID SBIR 

phase II application entitled “Experimental Gonococcal Vaccine.”

I have been working in the area of drug delivery, particularly with bioerodable 

oral polymeric particles for the delivery of biological macromolecules and small 

molecule drugs, for the past 3 decades. The Phase Inversion Nanoencapsulation 

technology was invented in my laboratory and has been tested for efficacy in numerous 

mammalian models over the past decade. 

I believe the proposed studies represent a very exciting opportunity to bring the 

PIN particle technology closer to the clinic. I am looking forward to continuing our 

productive collaboration with TherapyX

Inc. and Dr. Egilmez. 

Sincerely yours, 

Edith Mathiowitz, Ph.D. 

Professor of Medical Science and Engineering 

Director of Biotechnology Graduate Program, 

Member, Center for Biomedical Engineering, 

Department of Molecular Pharmacology, Physiology and Biotechnology 

Box G-B393 

Brown University, Providence, RI 02912 

Tel: (401) 863-1358 

Fax: (401) 863-1753 

edith_mathiowitz@brown.edu

Brown University 171 Meeting Street Providence, RI 02912 

DEPARTMENT OF MOLECULAR PHARMACOLOGY, BROWN PHYSIOLOGY, AND BIOTECHNOLOGY

Letters of Support Page 98
Page 1 of 2

August 21st, 2016 

Dr. Dominick Auci, MBA, Ph.D. 

Vice President, Research and Development 

TherapyX

Inc

138 Farber Hall 

3435 Main Street 

Buffalo, NY 14214 

Dear Dr. Auci, 

Re: "Experimental Gonococcal Vaccine." 

Dear Dr. Auci, 

Please accept this letter as indication that Comparative Biosciences, Inc. is 

committed to work on a fee for service basis with TherapyX

Inc. as indicated in the 

grant application to support the development of nanoencapsulated IL-12, 

Comparative Biosciences, Inc, Sunnyvale, CA is pleased to offer you assistance with 

your rodent toxicology and safety studies as you develop your therapeutic drug 

delivery system. Work on the referenced application will be valid for the duration of 

the grant award provided that the actual work is in compliance with the approved 

research proposal 

CBI is a preclinical contract research organization with nonclinical safety assessment 

location in Sunnyvale, CA that delivers comprehensive drug development and 

toxicology services. We will support this grant application by providing preclinical 

safety and toxicology testing. Comparative Biosciences, Inc. has a nearly twentyyear history of providing assistance to companies performing preclinical studies. 

Included among the members of our scientific staff are eight individuals with doctoral 

degrees, two board-certified pathologists, a laboratory animal veterinarian and two 

ophthalmologists also; and we have a full time, independent quality assurance unit. 

We provide state of the art toxicology, pharmacokinetics, pharmacology, and efficacy 

studies, particularly in the area of wound healing, repair and dermal burns. Our 

histopathology laboratory provides GLP and research histology and pathology, 

including special stains, immunohistochemistry, in situ hybridization, and antibody 

cross reactivity studies. We specialize in working with small innovative companies 

but also work with larger drug companies and academic organizations. 

We have a fully equipped rodent and large animal facility that will be an excellent 

asset as you pursue your studies. (OLAW Assurance No. A4269-01). As you know, 

Contact PD/PI: Liu, Yingru

Letters of Support Page 99
Contact PD/PI: Liu, Yingru

Page 2 of 2

your individual protocols will be reviewed by our duly constituted IACUC committee 

and receive its approval before your actual study can commence. Our IACUC has in 

place a duly approved IACUC proposal your studies. There are separate rodent and 

large animal holding rooms and a fully equipped surgery room and general 

procedure rooms, separate necropsy, and clean/dirty corridors with a pass through 

cage washer and autoclave as well as toxicology-related specialty equipment. We 

are AAALAC accredited, registered with the USDA, FDA, OLAW, have a duly 

constituted IACUC, and comply with the NIH Guide. Procedure rooms are 

completely equipped with gas anesthesia, surgical microscope and oxygen chamber. 

Comparative Biosciences, Inc. occupies a 16,000 sq ft facility in Sunnyvale, 

California. The facility includes an 8100 sq ft vivarium and a 1500 sq ft histology 

laboratory, with the balance dedicated to support functions (offices, conference 

rooms, archive room, etc). The vivarium includes a surgery, procedure room, 

gowning room, cage washing and service area, and 10 animal rooms. All 

histopathology is performed on-site, including a variety of special stains, 

immunohistochemistry, cryosectioning, plastic and paraffin embedding and 

sectioning, morphometry. 

CBI is very experienced in the preparation of animals for toxicology studies. Our 

histology laboratory has considerable expertise in the preparation of burn tissue 

specimens, immunohistochemistry, histomorphometry, histopathologic assessments, 

photomicroscopy and report preparation. 

We thank you for your interest in our facility and look forward to working with you on 

this project. 

With kindest regards, 

Carol Meschter, DVM, PhD, DACVP 

Institutional Official 

CEO

Comparative Biosciences, Inc. 

Letters of Support Page 100
I Regulatory Professionals, Inc.

Dr. Dominick Auci, MBA, Ph.D. 

Vice President, Research and Development 

TherapyX

inc

 

138 Farber Hall 

3435 Main Street 

Buffalo, NY 14214 

Dear Dr. Auci, 

August 8, 2016 

Regulatory Professionals, Inc. (RPI) is happy to express its commitment to TherapyX

inc as 

indicated in their phase II SBIR application entitled " Experimental Gonococcal Vaccine."

RPI is happy to continue its long-standing relationship with TherapyX

inc that has included 

support for its other programs, as well as on-going assistance on this product aimed at genital 

tract infection. We will continue to provide consultation services and strategic assistance with 

associated toxicology program design, CMC (chemistry, manufacturing and controls) strategies, 

as well as preparation of type C meeting briefing packages and related questions in preparation 

for IND submission. 

RPI is well qualified to provide the services as indicated in this phase II application. RPI is an 

experienced team of regulatory professionals dedicated to helping our clients advance the 

development and registration of their products with worldwide health authorities (US, Canada, EU 

and Japan). Our focus is to provide strategic and tactical support in all aspects of regulatory affairs, 

including nonclinical, CMC and clinical development for drugs, biologics, devices and combination 

products. Additionally, RPI has extensive knowledge base in the areas of regulatory affairs, 

electronic submission preparation, GXP compliance, technical and medical writing, 

advertising/promotional labeling.

We here at RPI are very excited about the potential of your vaccine and look forward to continuing 

working with TherapyX

inc

.

President and CEO 

Regulatory Professionals, Inc. 

8000 Jarvis Avenue, Suite 100 

Newark, CA 94560 

Tel: (408) 263-6861 xl04 

Fax: (408) 263-1231 

8000 Jarvis Avenue, Suite 100, Newark, CA 94560 I 408.263.6861 I Page 1 of 1 

Letters of Support Page 101

Contact PD/PI: Liu, Yingru