PI: GALARZA, JOSE M Title: Broadly protective (universal) virus-like particle (VLP) based influenza vaccine

Received: 12/05/2012 FOA: PA12-088 Council: 05/2013

Competition ID: ADOBE-FORMS-B2 FOA Title: PHS 2012-02 OMNIBUS SOLICITATION OF THE NIH, CDC, FDA AND 

ACF FOR SMALL BUSINESS INNOVATION RESEARCH GRANT APPLICATIONS 

(PARENT SBIR [R43/R44])

1 R43 AI106145-01A1 Dual: HL,NR Accession Number:

IPF: 10004226 Organization: TECHNOVAX, INC.

Former Number: Department:

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

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Year 2:

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:

Jose Galarza Ph.D. TechnoVax Inc. PD/PI 

Diana Dalfo Ph.D Technovax Inc. Other (Specify)-Staff Scientist

Innocent Mbawuike Ph.D Baylor College of Medecine Co-Investigator

Ruben Donis Ph.D CDC Other (Specify)-Collaborator

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State Application Identifier

Applicant Identifier

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

5. APPLICANT INFORMATION * Organizational DUNS:

* Legal Name:

Department: Division:

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Person to be contacted on matters involving this application

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

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Small Business Organization Type Women Owned Socially and Economically Disadvantaged

If Revision, mark appropriate box(es).

9. * NAME OF FEDERAL AGENCY:

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11. * DESCRIPTIVE TITLE OF APPLICANT'S PROJECT:

2. DATE SUBMITTED

3. DATE RECEIVED BY STATE APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R) 

County / Parish:

Province:

Prefix:

What other Agencies?

Pre-application Application Changed/Corrected Application

12/05/2012

TechnoVax Inc.

765 Old Saw Mill River Rd.

Tarrytown Westchester

10591-6702

Mr. Hector

Munoz

Broadly protective (universal) virus-like particle (VLP) based influenza vaccine that can neutralize a broad 

spectrum of influenza A virus subtypes.

Yes No

USA: UNITED STATES

NY: New York

R: Small Business

National Institutes of Health

New Resubmission

Renewal Continuation Revision

8. * TYPE OF APPLICATION:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011 

b. Agency Routing Identifier

12. PROPOSED PROJECT:

* Start Date * Ending Date

07/01/2013 06/30/2015

* 13. CONGRESSIONAL DISTRICT OF APPLICANT

NY-018

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

* First Name: Middle Name:

* Last Name: Suffix:

Position/Title:

* Organization Name:

Department: Division:

* Street1:

Street2:

* City:

* Country: * ZIP / Postal Code:

* Phone Number: Fax Number:

* Email:

* State:

County / Parish:

Province:

Prefix:

Galarza Ph.D.

Dr. Jose M.

10591-6702

Tarrytown Westchester

765 Old Saw Mill River Rd.

CEO & Founder

TechnoVax Inc.

NY: New York

USA: UNITED STATES

	


 		

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

15. ESTIMATED PROJECT FUNDING 16. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE 

ORDER 12372 PROCESS?

a. YES a. Total Federal Funds Requested

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 penalities. (U.S. Code, Title 18, Section 1001)

19. Authorized Representative

* First Name: Middle Name:

* Last Name: Suffix:

* Position/Title:

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Department: Division:

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Street2:

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* Phone Number: Fax Number:

* Email:

* Signature of Authorized Representative * Date Signed

20. Pre-application

* 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.

County / Parish:

c. Total Federal & Non-Federal Funds

18. SFLLL or other Explanatory Documentation

Province:

b. Total Non-Federal Funds

Prefix:

0.00

0.00

* I agree

Mr. Hector

Munoz

Chief Financial & Corp. Development Officer

TechnoVax Inc.

765 Old Saw Mill River Rd.

Tarrytown Westchester

NY: New York

USA: UNITED STATES 10591-6702

Add Attachment Delete Attachment View Attachment

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DATE:

THIS PREAPPLICATION/APPLICATION WAS MADE 

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372 

PROCESS FOR REVIEW ON:

PROGRAM HAS NOT BEEN SELECTED BY STATE FOR 

REVIEW

PROGRAM IS NOT COVERED BY E.O. 12372; OR

Jose M. Galarza 12/05/2012

b. NO

d. Estimated Program Income

	


 		

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

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

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

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

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

 Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7

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

Research & Related Senior/Key Person-------------------------------------------------------------------------- 12

 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 15

Research & Related Budget - Year 1---------------------------------------------------------------------------- 29

Research & Related Budget - Year 2---------------------------------------------------------------------------- 32

 Budget Justification---------------------------------------- 35

Research & Related Budget - Cumulative Budget----------------------------------------------------------------- 37

Research & Related Budget - Consortium Budget (Subaward 1)---------------------------------------------------- 38

SBIR/STTR Information----------------------------------------------------------------------------------------- 46

 Prior SBIR Phase II Awards---------------------------------------- 48

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 49

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 51

 Introduction---------------------------------------- 52

 Specific Aims---------------------------------------- 53

 Research Strategy---------------------------------------- 54

 Vertebrate Animals---------------------------------------- 60

 Select Agent Research---------------------------------------- 62

 Bibliography & References Cited---------------------------------------- 63

 Consortium/Contractual---------------------------------------- 65

 Letters of Support---------------------------------------- 66

 Resource Sharing Plan---------------------------------------- 69

PHS 398 Checklist--------------------------------------------------------------------------------------------- 70

Table of Contents Page 3

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
County:

* ZIP / Postal Code:

* Country:

* State:

* City: 

Street2:

Organization Name:

Project/Performance Site Location(s)

Project/Performance Site Primary Location

* Street1:

Province:

TechnoVax Inc.

765 Old Saw Mill River Rd.

Tarrytown Westchester

NY: New York

USA: UNITED STATES

10591-6702

OMB Number: 4040-0010 

Expiration Date: 08/31/2011

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. 

DUNS Number:

* Project/ Performance Site Congressional District: NY-018

Project/Performance Site Location 1

Baylor College of Medecine

One Baylor Plaza

Houston

TX: Texas

USA: UNITED STATES

* ZIP / Postal Code: 77030-3411 TX-007

* Country:

Province:

* State:

* City: 

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District: 

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. 

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Performance Sites Page 4

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1. * Are Human Subjects Involved?

IRB Approval Date:

Human Subject Assurance Number:

2. * Are Vertebrate Animals Used?

IACUC Approval Date:

Animal Welfare Assurance Number

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?

4.d. If yes, please explain:

6. * Does this project involve activities outside of the United States or partnerships with international collaborators?

6.b. Optional Explanation:

7. * Project Summary/Abstract

11. Equipment

8. * Project Narrative

12. Other Attachments

RESEARCH & RELATED Other Project Information

Is the IACUC review Pending?

If no, is the IRB review Pending?

2.a. If YES to Vertebrate Animals

3. * Is proprietary/privileged information included in the application? 

4.a. * Does this project have an actual or potential impact on the environment? 

6.a. If yes, identify countries:

1 2 3 4 5 6

9. Bibliography & References Cited

10. Facilities & Other Resources

Yes No

1.a If YES to Human Subjects

Yes No

Yes No

If yes, check appropriate exemption number.

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

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1236-TVx_UFlu_Narrative_120512.pdf Add Attachment Delete Attachment View Attachment

1237-TVx_UFlu_Bibliography_120512.pdf Add Attachment Delete Attachment View Attachment

1238-TVx_Facilities and_Resources_120 Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

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Is the Project Exempt from Federal regulations?

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

5.a. If yes, please explain:

	


 		

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Other Information Page 5

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1. ABSTRACT / SUMMARY 

We propose to develop a broadly neutralizing, possibly universal influenza vaccine based on 

virus-like particles (VLPs) displaying remodeled HA molecules which present otherwise cryptic 

epitopes. These remodeled HAs will be expressed in forms lacking the dominant hypervariable 

epitopes and instead display distinctly conserved subdominant antigenic sites known to elicit an 

antibody response that will neutralize a broad spectrum of influenza viruses. The protective 

scope of current influenza vaccines is restricted to homologous viruses or closely related 

variants and vaccine efficacy wanes following the fast antigenic evolution of the influenza virus. 

Most protective antibodies target highly variable and dominant sites on the globular head of the 

HA molecule, although more conserved and less immune-recognized conformational antigenic 

sites are also present in the stem (HA2) and between the globular head (HA1) and stem 

portions of HA. Isolated human antibodies directed toward these sites have been found to 

neutralize a broad spectrum of influenza viruses. It seems reasonable therefore to prepare and 

test vaccines that display these highly conserved subdominant antigenic sites and determine if 

they stimulate a broad antibody response which is minimal in a natural influenza infection or 

following vaccination with formulations containing whole HA molecules. Incorporation of 

remodeled HA molecules into influenza virus-like particles (VLPs) should provide an excellent 

opportunity to develop a broadly neutralizing vaccine. VLPs are generated by the co-expression 

of four structural influenza proteins (M1, M2, HA and NA) and do not contain viral genetic 

material and are therefore unable to replicate or cause infection. VLPs displaying different 

remodeled HA molecules will be produced, characterized and tested for the presence of 

conserved epitopes by immunoprecipitation with specific antibodies recognizing these sites. 

The neutralizing activity of VLP immunized mice sera will be assessed by an in-vitro microneutralization assay using three antigenically distinct viruses. The protective efficacy and 

immunogenicity stimulated by a single or combined candidate vaccine will be further 

investigated in lethal challenge studies with three antigenically diverse viruses. Further 

development of the most promising VLP(s) will be pursued by a phase II SBIR proposal. 

Project Description Page 6

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Narrative 

Development of a broadly protective (universal) vaccine able to withstand antigenic 

variation and sustain efficacy for an extended time should have a major impact on 

influenza prevention. To achieve this goal, we propose to create virus-like particles 

(VLPs) displaying remodeled HA molecules revealing distinctly conserved subdominant 

antigenic sites known to elicit an antibody response that will neutralize a broad spectrum 

of influenza viruses. 

Public Health Relevance Statement Page 7

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Facilities and Resources at TechnoVac Inc. 

TechnoVax is located in Tarrytown, NY and occupies space in the BioMed Realty Trust campus, 

one of the largest biotech facilities in the state of NY. Most of the tenants are life science, 

biotech or pharmaceutical oriented businesses providing an excellent infrastructure, 

environment and synergy for the successful operation of companies. TechnoVax facilities 

comprise laboratory and offices space to accommodate our scientific team and management. 

Laboratories: 

Currently operating space for research and development comprises an area of approximately 

2000sq. ft. divided into two independent fully equipped laboratory facilities including a tissue 

culture room featuring negative pressure air circulation and HEPA filtration for handling BSL2+ 

agents (inspected and approved by the USDA). 

Equipment: 

TechnoVax laboratories are equipped with three biosafety cabinets, inverted and fluorescence 

microscopes, micro-centrifuges (standard and refrigerated), one Beckman ultracentrifuge, 

electrophoresis units (DNA/protein), plate washer, ELISA reader (colorimetric), ELISA reader 

(fluorescence and chemiluminescence), spectrophotometer, thermocycler machines, autoclave, 

refrigerators, -20°C and -80°C freezers, CO2 incubators, shakers, transilluminator, digital 

documentation system, protein purification system, five liter fermenter with control unit, pH 

meter, autoclave, developer, liquid N2 storage, etc. 

Two extension programs are currently planned for 1st quarter and 2nd half of 2013. 

Facilities Page 8

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Pase II Extension - cGMP Production: 

In order to produce cGMP vaccines for its own upcoming toxicology studies and clinical trials, 

TechnoVax is planning to lease additional space to set-up its own cGMP pilot plant. This 

production area will fully comply with FDA regulations (Guidance for the Industry CGMP for 

Phase 1 Investigational Drugs” under regulation 21 CRF 201.2(c) (referred to as phase 1 

investigational drugs); U.S. Department of Health and Human Services, Food and Drug 

Administration, July 2008) and will allow TechnoVax to produce, for itself and third parties, 

biological vaccines and materials for Phase I clinical testing. The pilot plant will be operational 

towards mid of 2013 and will occupy an area of ~500-1000 sq. ft. 

Phase III Extension: 

TechnoVax is also planning to expand its current laboratory, production and administrative 

space in end 2013 by leasing an adjacent area of ~1800sq. ft. 

Administration: 

Fully equipped office space with printers, fax and copier machines occupies another ~1,000 sq. 

ft. TechnoVax, Inc. personnel have access to the NYMC library and via internet to e-journals. In 

addition the BMRLandmark campus provides conference rooms and shared facilities. 

Qualifications: 

The core competencies of TechnoVax, in addition to vaccine research and development, 

include genetic engineering and recombinant protein production utilizing eukaryotic and 

prokaryotic expression systems. Expertise and capabilities on protein design and reengineering 

permits us to generate molecules with modified or altered properties such as incorporating two 

different antigens in the same VLP, expressing critical antigens with cryptic neutralizing epitopes 

and expressing otherwise dangerous pathogen antigens. Our scientific team combines 

knowledge from biology, structural biology, biochemistry, molecular biology, immunology and 

cell biology to investigate properties of new protein for vaccine development and other 

applications. Protein purification and characterization are routinely performed in our laboratory 

implementing efficient protein purification strategies and complementary 

analytical/characterization methodologies. Assay development is an important investigative toll 

that form part of our core capabilities. 

In addition, our company carries out animal studies to evaluate immunogenicity and efficacy of 

different vaccines and formulations. These preclinical evaluations require expertise on planning, 

study design, execution, data gathering and analysis all competencies that TechnoVax has 

been practicing since 2004. Furthermore, the combined experience of managers and scientists 

of TechnoVax covers a broad range of expertise including GLP and GMP manufacturing, 

preclinical and clinical pharmaceuticals development, formulations, regulatory issues, and 

project management. 

TechnoVax participates in collaborative research and development programs with academic 

institutions and companies as well as manages SBIR funded projects. Our combined expertise 

and experience prepare us well to lead a VLP vaccine development and manufacturing projects. 

Process Development: 

During the last eight years of operations conducting vaccine research and development, 

TechnoVax has gained significant experience in devising and implementing methods for making 

products from laboratory scale to 5L fermentation capacity. These undertakings involve 

developing strategies as well as processes to scale up production and evaluate effectiveness of 

methods following refinements of parameters, protocols and conditions in order to maximize 

Facilities Page 9

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
yield and quality of the manufacture product. As part of the company activities, our team 

performs molecular biology work on diverse cloning strategies, gene expression and 

optimization studies in prokaryotic and eukaryotic systems (bacteria, insect and mammalian 

cells.). Cell lines development for transient or stable production of vaccine products or 

recombinant proteins antigens essential for preclinical studies or assay development are 

routinely created, selected and evaluated in our laboratory applying specifically devised 

procedures. Multiple gene recombinant vaccines or single proteins are being produced utilizing 

vectored expression methodologies such as baculovirus recombinants/ insect cells expression 

system. Candidate products developed utilizing this system include influenza virus-like particle 

(VLP) vaccines for epidemic and pandemic strains as well as subunits proteins antigens (HA, 

NA, NP). Similarly, stable transfected CHO, Vero and MDCK cells have been developed to 

manufacture VLP vaccines in a continuous basis utilizing standard fermentation techniques. 

TechnoVax has established upstream and downstream processes for the production of VLP 

vaccines utilizing recombinant baculovirus/insect cell system or stable transfected mammalian 

cells. Process development from tissue culture flasks volumes to 5L fermentation scale up 

processes are currently performed in the laboratory. Tangential flow filtration (TFF) processing 

systems, chromatographic methods, and gradient centrifugation are part of our bio-separation, 

recovery and purification strategies implemented to obtain candidate VLP vaccine product. 

Quality Assurance: 

TechnoVax has a fully developed and comprehensive “Laboratory Quality Assurance Plan” 

used to participate in consortium managed biddings for government contracts. The overall 

objective of our quality assurance programs is to generate defensible data that will meet the 

quality objectives of the user. 

Other: 

Dr. Jose M. Galarza is an Adjunct Professor, lecturer and graduate advisor at the Department of 

Microbiology of New York Medical College. This Institution is a good resource to access new 

College graduate seeking positions. 

Facilities and Resources at Baylor College of Medicine 

Department of Molecular Virology and Microbiology 

Laboratory:

Sufficient laboratory space is available within the Department of Molecular Virology and 

Microbiology to carry out all proposed studies. Dr Mbawuike has his office and laboratory 

equipped for microbiological and immunological studies. Dedicated central tissue culture rooms, 

in situ technology and RT-PCR measurement rooms are part of each laboratory. He has free 

access to the facilities and equipment of the department of Molecular Virology and Microbiology. 

Dr. Mbawuike occupies rooms, 244E (600 sq. ft., main laboratory and office), 252E (146 sq. ft., 

gamma counter room) and 209C (200 sq. ft., postdoctoral fellows office and laboratory) and 

shares equipment storage space in rooms 264E (Cyrostat) and 212C (Flow Cytometer). The 

main laboratory has a dedicated tissue culture room with two laminar flow hoods and a two 

compartment CO2 incubator. Also, in situ technology and RT-PCR measurement rooms are part 

of each laboratory. 

Facilities Page 10

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Dr Mbawuike office is staffed with shared support staff: an administrator, two secretaries and a 

clerk. 

Animal Facilities: BCM BSL-3/3E Satellite Facility: 

This is a ~3700 square feet, 13 room facility (rooms K101 – K117). The facility was constructed 

for biosafety level-3 (BSL-3) work, using both traditional and enhanced level pathogens. Due to 

the increased level of awareness needed for work with BSL-3 agents, the facility operates as a 

satellite facility under the anagement of the Biological Safety Officer, who reports to the Office of 

Environmental Safety. Seven of the 13 rooms (rooms K104, K106, K107, K108, K116B, K116C, 

and K116D) are flexible lab suites designed to be used as either animal or tissue culture rooms 

depending on the needs of the investigators. The remaining 6 rooms are supportive in nature 

and include a dedicated equipment room (K114), 2 gown-in rooms (K101, K116), a gown out/ 

shower room (K116F), 2 decontamination/autoclave rooms (K103, K116E) and a clean exit 

room (K117). An internal portion of the facility (K116-K116F) comprised of 6 rooms (3 flexible 

lab suites [K116B, K116C, K116D], gown-in room [K116], the gown-out/shower room [K116F], 

and a decontamination/autoclave room [K16E]) constitutes ~1250 square feet and has the 

ability to operate as an “enhanced” BSL-3 facility, without affecting operations in the BSL-3 

suite. This enhanced section is referred to as the BSL-3E suite. The entire area including both 

the BSL-3 suite and BSL-3E suite is referred to as the BSL-3/3E Facility. The facility was 

designed to house small animal models, thus the design of the labs includes containment 

standards and requirements defined in the CDC/NIH publication “Biosafety in Microbiological 

and Biomedical Laboratories (BMBL)”, 5th Edition, 2007 for animal BSL-3 (ABSL-3) space plus 

enhancements (ABSL-3E) including. 

Data Analysis: 

Computers, laptops and servers fully equipped with various statistical (e.g. SAS, STATVIEW 

and SLIDEWRITE) and graphical and office processing software. 

Equipment: 

A new Beckman-Coulter MCL-XL Automated four color fluorescein activated cell sorting (FACS) 

instrument is located in Dr. Mbawuike’s laboratory for these studies. A multi-parameter research 

EPICS Model 753 Laser Instrument interfaced with a CICERO High Speed Cell Sorting System 

is available for our use. EXPO software for FACS data analysis is linked to the cytometers and 

to the ELISA plate reader. An ABI PRISM 7700Sequence Detection System (Applied 

Biosystems) for real time RT-PCR analysis is also available. Here is a list of other 

representative equipment available for the proposed research: Autoclaves, incubators, pipet 

plugged, centrifuges, spectrophotometers, automatic microtiter dilutor and dropper, phase 

contrast microscope, -70C freezers, Gamma counters with tape printout, vacuum pumps and 

gel destainer, micro-fractionaters, pH meters, sonifier, IBM compatible computers, manual 

multichannel pipettes, electrophoresis power supply, rotavaporator, portable compressors, -20C 

freezers, C02 freezers, electronic balances, liquid nitrogen tanks, ultracentrifuges, refrigerators. 

Vertis homogenizers, inverted microscopes, UV fluorescent microscope, light microscopes, 

scintillation counters, typewriters, Molecular Devices kinetic spectrophotometer and plate 

reader, MASH-11 cell harvester. Liquid nitrogen freezers and BioRad 600 confocal microscope. 

Facilities Page 11

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Attach Current & Pending Support

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Dr. Jose M.

Galarza Ph.D.

CEO & Founder

TechnoVax Inc.

765 Old Saw Mill River Rd.

Tarrytown Westchester

NY: New York

USA: UNITED STATES 10591-6702

PD/PI

PhD

1981

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OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Ph.D

Dr.

Research Scientist

Technovax Inc.

Westchester

USA: UNITED STATES

Diana

Dalfo

765 Old saw Mill River Rd.

Tarrytown

NY: New York

10591-6702

* Project Role: Other (Specify) Other Project Role Category: Staff Scientist

1

Degree Type: PhD

Degree Year: 2005

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Key Personnel Page 12

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED Senior/Key Person Profile (Expanded)

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Ph.D

Dr.

Associate Professor

Baylor College of Medecine

USA: UNITED STATES

Innocent

Mbawuike

One Baylor Plaza

Houston

TX: Texas

77030-3411

* Project Role: Co-Investigator Other Project Role Category:

2

Degree Type: PhD

Degree Year: 1984

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Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Ph.D

Dr. O.

Associate Director

CDC Influenza Division

Influenza Division, OID, NCIRD

USA: UNITED STATES

Ruben

Donis

1600 Clifton Rd.

Atlanta

GA: Georgia

30333-0000

* Project Role: Other (Specify) Other Project Role Category: Collaborator

3

Degree Type: PhD

Degree Year: 1986

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To ensure proper performance of this form; after adding 20 additional Senior/ Key Persons; please save your application, close the Adobe 

Reader, and reopen it.

	


 		

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Key Personnel Page 13

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
	


 		

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Key Personnel Page 14

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
BIOGRAPHICAL SKETCH 

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

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

NAME 

Galarza, Jose M. 

POSITION TITLE 

CEO, TechnoVax, Inc. 

Adjunct Associate Professor 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 

National College, Jose Hernandez, Buenos Aires BS 12/1971 Science 

University of La Plata, La Plata, Argentina DVM 09/1977 Doctor Veterinary Medic. 

University of La Plata, La Plata, Argentina Doctorate 11/1981 Microbiology 

Univ. of Utah Medical Center, Salt Lake City, UT Post-Doc 1986/1991 Virology/Mol. Biology 

Univ. of California Irvine, Irvine, CA Res. Scient 1991/1995 Microbi./Molec. Genetics 

A. Personal Statement 

I have broad scientific and leadership experience conducting research in the areas of virology, molecular 

biology, immunology, and vaccine development over the course of 30 years. As a post-doctoral researcher, 

I investigated the fundamental molecular mechanisms of negative-sense (flu, VSV) viruses and positivesense (polio) virus transcription/replication, its regulation and reconstitution of biochemical active 

complexes with recombinant proteins and synthetic templates. Furthermore, I studied the induction of 

cellular immune responses elicited by highly conserved recombinant viral proteins (CTL-vaccines) of 

rapidly evolving viruses. This academic scientific experience brought me to the pharma/biotech industry 

(Wyeth, currently Pfizer) as PI and leader where I pursue new lines of research and development in the 

field of vaccinology. During my tenure, I was involved in many multidisciplinary vaccine R&D projects, 

collaborations including influenza projects with external collaborators and a CRADA with the NIH to 

develop para-influenza and RSV vaccines, as well as regulatory and scientific advisory committee member 

but above all, in the management and leadership of a research team that ultimately discovered the 

formation of wild type and chimeric influenza virus-like particles (VLP), a platform technology for vaccine 

development. Furthermore, as member of the new technology for vaccine development advisory group 

participated in the evaluation of external and internal emerging technologies, HPV and Rotavirus VLPs, 

Alphavirus platform, Dengue vaccine development strategies, DNA based vaccines, conjugates, chimeric 

proteins as adjuvanted subunit vaccines, etc. 

In 2003, I co-founded TechnoVax, Inc. and became its CEO. The main focus of our company is the R&D 

of vaccines based on the virus-like particle (VLP) technology. My research accomplishments and 

experience in the field of vaccine R&D, particularly in the VLP area, together with my managerial and 

leadership skills equip me with the knowledge, technical expertise and managerial experience that are 

required to effectively execute and develop a VLP-based vaccine development project. Development of a 

successful broadly protective influenza vaccine able to sustain efficacy for an extended time will have a 

major impact in the prevention and control of influenza. This project, in collaboration with Dr. Mbawuike,

Baylor College of Medicine, generates extraordinary interest and enthusiasm. 

Positions and Honors

Positions and Employment 

 1982-1986 Research Assistant; Immunology and Virology Section of the Federal Research Council 

(CONICET), Argentina 

 1985-1986 Adjunct Associate Professor; Dept. of Microbiology, Northeast University, Formosa, 

Argentina 

 1986-1991 Postdoctoral Research Associate, Department of Cellular, Viral and Molecular Biology, 

University of Utah Medical Center, Salt Lake City, Utah 

Biosketches Page 15

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1991-1995 Research Scientist, Department of Microbiology and Molecular Genetics, College of 

Medicine, University of California, Irvine, CA 

1996-2002 Principal Scientist and Project Leader, Department of Vaccine Research and Development 

Wyeth, Pearl River, NY 

2003-Present President, CEO and Founder, TechnoVax, Inc. Tarrytown, NY 

Adjunct Associate Professor, Dept. of Microbiology and Immunology 

New York Medical College, Valhalla, NY 

Other Experience and Professional Memberships 

1982-1985 Instructor, Department of Agriculture and Natural Resources. Courses in Animal Virology; 

Formosa, Argentina. 

1985 Principal Organizer, Conference on Interferon; Northeast National University, Formosa, 

Argentina 

Memberships: American Association for the Advancement of Science (1989-present); American Society 

for Virology (1990-present); American Society for Microbiology; The New York Academy of 

Sciences (2003-present) 

2006-2011. NIH reviewer on study sections “Non HIV Microbial Vaccines”, “Bio-defense” and “Immunology 

and Virology Topics”. 

Honors

1982 Exchange Scholarship, University of Glasgow 

1986-1988 Organization of American States Fellowship 

Patents 

1. A Subunit Vaccine for Influenza Virus Containing NP Protein Produced in Sf9 Cells by Recombinant 

Baculovirus. Filing Date: July 1990; Inventors: Donald F. Summers, Jose M. Galarza, Innocent N. 

Mbawuike and Robert B. Couch 

2. Reconstituted Influenza A Transcriptase Reaction Using Overproduced Influenza Proteins. 

Filing Date: August 23, 1991 Serial Number: 07/748,999; Inventors: Donald F. Summers and Jose M. 

Galarza 

3. Nucleotide Sequence of Influenza A/Udorn/72 (H3N2) Genome. 

Filing Date: June 23, 2000; Inventors: Jose M. Galarza and Theresa Latham. 

4. Assembly of Wild Type and Chimeric Influenza Virus-Like Particles (VLPs). 

Filing Date: June 23, 2000; Inventors: Jose M. Galarza and Theresa Latham. 

 5. Influenza Virus-Like Particles (VLP) Compositions 

 Filing Date: April 30, 2007; Inventors: Jose M. Galarza and Demetrius Matassov. 

 6. Polyvalent Influenza Virus-Like Particles (VLP) Compositions 

 Filing Date: April 30, 2007; Inventors: Jose M. Galarza and Demetrius Matassov. 

7. Respiratory syncytial virus (RSV)-Like particles Vaccines Composed of Composed of chimeric 

RSV/influenza structural proteins. Inventors: J.M. Galarza and Demetrius Matassov, Filed June 2007; 

patent pending 

8. Monovalent or divalent wild type respiratory virus (RSV)-like particles vaccines bearing viral surface 

antigens that stimulate an immune response that protects humans against one or both of the RSV groups 

(RSV-A and RSV-B). Inventors: J. M. Galarza and Demetrius Matassov. Filed date: June 2007, patent 

pending. 

C. Selected Peer-reviewed publications

Most relevant to current application 

1. Latham, T., Galarza J.M., Formation of Wild-Type and Chimeric Influenza Virus-Like Particles (VLPs) 

Following Simultaneous Expression of Only Four Structural Proteins, 

Journal of Virology, 75: 6154-6165, 2001. 

Biosketches Page 16

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
2. Galarza, J.M., Latham, T., Cupo, A., Virus-Like Particle (VLP) Vaccine Conferred Complete Protection 

against a Lethal Influenza Virus Challenge, Viral Immunology, 18: 244-251, 2005 

3 Matassov, D., Cupo, A., Galarza J.M., A Novel Intranasal Virus-Like Particle (VLP) Vaccine Designed 

to Protect Against the Pandemic 1918 Influenza A Virus (H1N1), Viral Immunology, Vol. 20, 447-452, 

2007. 

Additional publications of importance to the field 

1. Galarza, J.M., Sowa, A., Hill, V.M., Skorko, R., Summers, D.F., Influenza A Virus NP Protein 

Expressed in Insect Cells by a Recombinant Baculovirus is Associated with a Protein Kinase Activity 

and Possesses Single Stranded RNA Binding Activity, Virus Research, 24: 91-106, 1992. 

2. Mbawuike, I.N., Galarza, J.M., Summers, D.F., Couch, R.B., Baculovirus-Expressed Influenza 

A/Udorn (H3N2) Nucleoprotein Induces Protective T-cell Immunity Against Influenza A/H3N2 and 

A/H1N1 in Mice, Vaccine Research, 3: 211-227, 1994. 

3. Neufelt, K., Galarza, J.M., Summers, D.F., Richards, O.C., Ehrenfeld, E., Identification of Terminal 

Adenylyl Transferase Activity of the Poliovirus Polymerase 3Dpol, Journal of Virology, 68: 5811-5818, 

1994. 

4. Shi, L., Peng, Q., Summers, D.F., Galarza, J.M., Influenza A Virus RNA Polymerase Subunit PB2 is 

the Endonuclease Which Cleaves Host Cell mRNA and Functions Only as Trimeric Enzyme, Virology, 

208: 38-47, 1995. 

5. Shi, L., Galarza, J.M., Summers, D.F., Recombinant-Baculovirus-Expressed PB2 Subunit of the 

Influenza A Virus RNA Polymerase Binds Cap Groups as an Isolated Subunit, Virus Research, 42:1-9, 

1996. 

6. Peng, Q., Galarza, J.M., Shi, L., Summers, D.F., Influenza A Virus RNA-Dependent RNA Polymerase 

Cleaves Influenza mRNA in vitro, Virus Research, 42: 149-158, 1996. 

7. Galarza, J.M., Peng, Q., Shi, L., Summers, D.F., Influenza A Virus RNA-Dependent RNA Polymerase: 

Analysis of RNA Synthesis in vitro, Journal of Virology, 70: 2360-2368, 1996. 

D. Research Support 

Ongoing Research Support 

2R44AI063830-03A1 (Galarza, PI) 05/01/2005 – 08/31/2012 

NIAID, NIH 

Title: Development of Influenza Virus-Like Particle (VLP) Vaccines 

The parent Phase I SBIR award created and tested in preclinical studies several VLP vaccine candidates 

designed to protect against highly pathogenic pandemic strains of the influenza virus such as H1N1-1918, 

H5N1 and H7N7 avian influenza viruses. The goal of this SBIR II is to construct and produce the VLP vaccines 

in Sf9 & mammalian cells. Protective efficacy against avian H5N1, H7N7 and human H1N1-1918 influenza 

viruses will be tested. 

Completed Research Support 

1 R43 AI063830-01 (Galarza, PI) 2005-2007 

NIH/NIAID, SBIR Grant to TechnoVax, Inc. 

Title: Influenza Virus-Like Particles as Vaccines 

Role: PI 

NIH Service Grant # 5R01A12316. (Summers, PI) 1991-1995 

Title: Influenza RNA-Dependent RNA Polymerase 

Role: Co-Investigator 

Biosketches Page 17

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1

BIOGRAPHICAL SKETCH 

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

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

NAME 

Dalfo, Diana 

POSITION TITLE 

Research Scientist 

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 

University of Barcelona (Spain) Bachelor 1999 Biology 

University of Barcelona (Spain) Master 2001 Genetics 

University of Barcelona (Spain) Ph. D. 2005 Genetics 

Columbia University (NY, USA) 

New York University (NY, USA) 

Postdoctoral 

Postdoctoral 

2005 

2007 

Medicine 

Developmental Genetics 

A. Personal Statement 

Our main goal is to produce highly immunogenic and non-infectious virus-like particle (VLP) vaccines 

using a cell-based manufacturing system. This technology allows the development of single or polyvalent 

vaccines that carry the antigen(s) to protect against influenza. These virus-like particles will contain a 

combination of different proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1) and M2 proteins. We 

aim to broad coverage against circulating viruses not included in the currently licensed vaccine and enhance 

vaccine potency for the elderly population. 

B. Positions Honors and Grants. 

Positions and Employment

1998 Undergraduate student, Department of Genetics, University of Barcelona 

1998-1999 Investigation contract to the European project, UE Biotechnology programme Bio4-97-2123, 

Department of Genetics, University of Barcelona 

1999-2000 Assistant Professor of Molecular Genetics, Department of Genetics, University of Barcelona 

2000-2003 Predoctoral fellowship from Ministerio de Educacion y Cultura of Spain 

2004 Collaboration fellowship with the Department of Genetics, University of Barcelona 

2005 Postdoctoral Research Scientist, Medicine Department, Columbia University, NY 

2007 Postdoctoral Fellow, Developmental Genetics Program, New York University, NY 

2011 Research Scientist, TechnoVax, Inc. 

Honors 

2003 University of Barcelona Travel Grant Award to participate at the 7th Evolutionary Biology meeting in 

 Marsella, France 

2010 Sackler Institute Travel Grant Award to participate at the C. elegans: Development and Gene 

 Expression Meeting, June 17-20 in Heidelberg, Germany 

2008 Reviewer for the journal Cell Research 

2008 Undergraduate Honors Thesis Committee member at Biology Department, NYU 

Grants 

2000 Spanish National Institute of Education and Culture grant for a stage of one month in the 

 international amphioxus laboratory, Observatoire Oceanologique du Banyuls, France 

2000-2003 Ph.D. fellowship supported by Spanish government (AP99) 

C. Publications 

Biographical Sketches for each listed Senior/Key Person 2 Page 18

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
2

1. Dalfo D., Cañestro C., Albalat R., Gonzàlez-Duarte R. (2001) Characterization of a microsomal retinol 

dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates. Enzymology and molecular 

Biology of Carbonil metabolism: 359-370. Edited by Elsevier Science (book chapter) 

2. Dalfo D., Cañestro C., Albalat R., Gonzàlez-Duarte R. (2001) Characterization of a microsomal retinol 

dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates. Chem-Biol Interact 130: 359-

370 

3. Dalfo D., Albalat R., Molotkov A., Duester G., Gonzàlez-Duarte R. (2002) Retinoic acid synthesis in the 

prevertebrates amphioxus involves retinol oxidation. Dev Genes Evol 212: 388-393 

4. Dalfo D., Permanyer J., Gonzàlez-Duarte R., Albalat R. (2003) SDR-RDH enzymes in lower chordates. An 

evolutionary approach into the retinoic acid metabolism. P. 12th Inter. Congress on Genes, gene families 

and isozymes 185-188 

5. Fuentes M., Schubert M., Dalfo D., et al. (2004) Preliminary observations on the spawning conditions of the 

European amphioxus (Branchiostoma lanceolatum) in captivity. J Exp Zoolog B Mol Dev Evol 302 (4): 384-

391 

6.

7. Dalfo D., Marques N., Albalat R. (2007) Analysis of the NADH-dependent retinaldehyde reductase activity 

of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol 

dehydrogenase family. FEBS J 274 (14): 3739-3752 

8. Setty Y., Dalfo D., Korta D., Hubbard EJ., Kugler H. (2011) A model of stem cell population dynamics: insilico analysis and in-vivo validation. Development (in press) 

9. D

10.

D. Research Support 

R01 DK068437 (NIH) PI: Dr. Blaner (Columbia University Medical School) 2003-2007 

TITLE: Characterization of β-carotene cleavage enzymes in the mouse. 

GOALS: Retinoic acid (RA) regulates relevant vertebrate physiological processes such as anterior-posterior 

pattern formation, cell proliferation, tissue differentiation, embryonic development and vision. The main source 

of RA derives from the enzymatic cleavage of dietary ß-carotenes. An excess of RA during early pregnancy 

has been associated with a significant increase in birth defects and in several kind of cancer. Understanding 

how RA is synthesized from the dietary ß-carotens would help to fight against cancer and thus, would have a 

significant effect in public health. 

ROLE: I was the primary member working on these studies. I conceived, designed, performed and analyzed all 

the experiments (including making ischemic/reperfusion in mouse model and all the biochemical studies). 

R01 GM061706 (NIH) PI: Dr. Hubbard (NYU, Medical School) 2005-2009 

TITLE: RNAi-based identification of genes involved in the germline proliferation in C. elegans 

GOALS: The goal of this project was to understand the development and molecular basis for soma-germline 

interactions that influence germline proliferation and differentiation. The germ cells of most animals, including 

mammals, proliferate extensively either before or during the initial phases of differentiation, building an 

adequate progenitor pool for adult gamete production. The conservation of disease pathways between C. 

elegans and higher organisms, together with its simplicity make of this organism an important model for human 

diseases. 

ROLE: I was the primary member working on these studies. I conceived, designed, performed and analyzed all 

the experiments. This study has led to 1 peer-reviewed research articles submitted, 1 book chapter, 2 public 

talks and 4 poster presentations in international conferences. 

R03 HD066005-01 (NIH) PI: Dr. Hubbard (NYU, Medical School) 2008-2010 

TITLE: TFGß signalling role in the control of germline proliferation in C. elegans

GOALS: Transforming growth factor ß (TGFß) signaling is a key player in development including axis 

formation, body patterning, tissue specification and morphogenesis. At the cellular level, TGFß exerts its 

effects through several different mechanisms such as inhibition of cell cycle progression, stimulation of 

Biographical Sketches for each listed Senior/Key Person 2 Page 19

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
3

differentiation and triggering of apoptosis. TGFß signaling has been best studied in C. elegans for its role in 

dauer formation (dauers are resistant to environmental conditions and do not age). However, a possible role 

for the TGFß pathway in cell proliferation in the C. elegans germ line has not been previously investigated 

ROLE: I was a key member of this project and I was responsible of the design, performance and analysis of all 

experiments. In addition, I was absolutely involved in the process of writing the application for the NIH R03 

grant. This study has led to 1 peer-reviewed research articles in preparation, 3 public talks and 4 poster 

presentations in international conferences. 

Biographical Sketches for each listed Senior/Key Person 2 Page 20

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
BIOGRAPHICAL SKETCH 

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

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

NAME 

Mbawuike, Innocent 

POSITION TITLE 

Associate Professor 

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 

Boston State College (Univ. Massachusetts) B.S. 1978 Biology 

Boston University Ph.D. 1984 Microbiology 

Georgetown University, Washington, D.C. Post-Doc 1984-1986 Immunology 

Baylor College of Medicine, Houston, Texas Post-Doc 1986-88 Viral Immunology 

 

A. Personal Statement 

The focus of my research in the past thirty years has been to define the role of cell mediated immune (CMI) 

responses in the control of influenza viruses in both human and animal models. We were among the first to 

characterize the human IFN-γ ELISPOT assay for influenza in humans and since extended that to RSV and 

smallpox. In recent collaborative work with Dr. Sette in La Jolla Institute for Allergy and Immunology, we 

identified repertoire of T-cell specificities for influenza A virus in humans using the IFN-γ ELISPOT assay. We 

have also developed influenza A/H5N1 and A/H7N7 VLP reagents, each expressing the M1, M2, HA and NA 

proteins of the respective viruses (collaboration with Dr. Galarza of Technovax) and demonstrated their ability 

to elicit responses IFN-γ and GrB ELISPOT assays in human PBMC. With our research groups’ participation in 

NIAID-sponsored clinical trials of influenza A/H5N1 and A/H7N7 vaccines and our expertise in influenza CMI, 

we are in a unique position to execute the proposed study successfully. In addition, an ongoing pandemic 2009 

H1N1 influenza study presents a unique opportunity to directly assess the role of CMI in protection against 

influenza disease. My laboratory remains the CMI site for evaluation of CTL responses to numerous 

biodefense agent vaccines (including smallpox and pandemic influenza viruses) being tested at the VTEU and 

VRPRU at BCM. With two long term collaborators (Galarza and Zheng) and clinical perspective from Dr. 

Couch (Consultant), our research team is well positioned to successfully execute this project and generate 

data to provide important insights into how CMI fights influenza in humans. 

B. Positions and Honors. 

Positions and Employment

1984-1986 Postdoctoral Fellow in Immunology, Department of Microbiology, Georgetown University School of 

Medicine and Dentistry, Washington, D.C. 

1986-1988 Research Associate, Influenza Research Center, Department of Microbiology & Immunology, 

Baylor College of Medicine 

1988-1991 Instructor (Viral Immunologist), Influenza Research Center, Department of Microbiology & 

Immunology, Baylor College of Medicine 

1991-1994 Research Assistant Professor, Department of Microbiology and Immunology, Baylor Coll. of Med 

1994-2004 Assistant Professor, Department of Molecular Virology and Microbiology, Baylor Coll. of Medicine

2004-Present Associate Professor, Department of Molecular Virology and Microbiology, Baylor Coll. of Med. 

Other Experience and Professional Memberships

Adhoc Reviewer, Immunity and Host-Defense (IHD) Study Section, NIAID, NIH, 2010

Member, Vaccines Against Microbial Diseases (VMD) Study Section, NIAID, NIH, 2005-2009. 

Member, Special Emphasis Panel for NIAID: Immune Defense Mechanisms at the Mucosa, 2009. 

Consultant, WHO Consultation on Immunological Assays To Evaluate Efficacy of Influenza Vaccines, 2005. 

Member, Special Review Committee for NIAID, "Multicomponent Vaccine Development, 1994. 

Biographical Sketches for each listed Senior/Key Person 3 Page 21

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Ad hoc Reviewer, Journal of Virology, 1997-present. 

Ad hoc Reviewer, Journal of Infectious Diseases, 1998-present. 

American Association of Immunologists 

American Association for the Advancement of Science 

American Society for Microbiology 

Clinical Immunology Society 

Honors

1992-1994 Shannon Fellow, National Institute on Aging, National Institutes of Health 

1994 National Science Foundation Award for the DNA LITERACY PROGRAM, 1994. 

1994 National Institute on Aging and the Gerontological Society of America Award to The Third 

 Annual Summer Training Course in Experimental Aging Research, San Francisco, CA 

1995 American Association of Immunologists Travel Award to the 9th International Congress of 

 Immunology 

2002 American Association of Immunologists Faculty Travel Award, Experimental Biology 

 

C. Selected Peer-reviewed publications 

Most relevant to current application

1. DiFabio S, Mbawuike IN, Fujihashi K, Couch RB, McGhee JR, Kiyono H. Quantitation of human influenzaspecific CTLs: Correlation of cytotoxicity and perforin synthesis with increased numbers of interferon gamma 

producing CD8+

 T cells. Int Immunol 6:11-19, 1994 (PMID: 8148319). 

2. Mbawuike, I.N., C. L. Acuna, K.C. Walz, R. L. Atmar, S.B. Greenberg and Robert B.Couch.1997. 

Cytokines and impaired CD8+ CTL activity among elderly persons and the enhancing effect of IL-12 . 

Mech. Age Dev. 94:25-39, 1997 (PMID: 9147358). 

3. Mbawuike, IN., K. Fujihashi, S. DiFabio, S. Kawabata, J. R. McGhee, R. B. Couch and H. Kiyono. 1999. 

Human IL-12 enhances interferon-γ- producing influenza memory CD8+ CTLs. J. Infect. Dis. 180:1477-

1486 (PMID: 10515806). 

4. Mbawuike, I., Zang, Y., and Couch, R.B., Humoral and Cell-Mediated Immune Responses of Humans to 

Inactivated Influenza Vaccine with or without QS21 Adjuvant Vaccine. Vaccine. 25(17):3263-9, 2007 

(PMID: 17280748). 

5. Bui, H-B, Peters, B., Assarsson, E., Mbawuike, I., and Sette, A. Ab and T cell epitopes of 

influenza A virus, knowledge and opportunities. PNAS 104: 246-251, 2006 (PMCID: PMC1765443).

6. Mbawuike IN, Zhang Y, Couch RB. Control of mucosal virus infection by influenza nucleoprotein-specific 

CD8+ cytotoxic T lymphocytes. Respir Res. 8:44-51, 2007 (PMCID: PMC1914056). 

7. Assarsson E, Bui HH, Sidney J, Zhang Q, Glenn J, Oseroff C, Mbawuike IN, Alexander J, Newman MJ, 

Grey H, Sette A. Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans. 

J Virol. 82(24):12241-51, 2008 (PMCID: PMC2593359). 

8. Zheng B, Zhang Y, He H, Marinova E, Switzer K, Wansley D, Mbawuike I, Han S. Rectification of ageassociated deficiency in cytotoxic T cell response to influenza A virus by immunization with immune 

complexes. J Immunol. 2007 Nov 1;179(9):6153-9. Free Article: 

http://www.jimmunol.org.ezproxyhost.library.tmc.edu/cgi/content/full/179/9/6153 

Additional recent publications of importance to the field

1. Mbawuike, I.N., Lange AR, Couch RB. Diminished influenza virus specific MHC class I-restricted cytotoxic 

T lymphocyte activity among elderly persons. Viral Immunol 6:55-64, 1992 (PMID: 8476508). 

2. Mbawuike, I.N., Piedra, P, Cate, T.R. and Couch, R.B. Cytotoxic T lymphocyte (CTL) response of 

unprimed infants to natural influenza A virus infection and live or inactivated vaccine, J. Med. Virol. 50:105-

111, 1996 (PMID: 8915874). 

3. Mbawuike, I.N. S. Pacheco, C. L. Acuna, K. C. Switzer, Y. Zhang and G. R. Harriman. Mucosal Immunity 

To Influenza Without IgA: An IgA Knockout Mouse Model.

. J. Immunol. 162: 2530-2537, 1999. Free Article: 

http://www.jimmunol.org.ezproxyhost.library.tmc.edu/cgi/content/full/162/5/2530 

Biographical Sketches for each listed Senior/Key Person 3 Page 22

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
4. Mbawuike, I.N. , J. Wells1

, R. Byrd, S. G. Cron, W. P. Glezen and P. A. Piedra. HLA-Restricted CD8+

CTL, IFN-γ and IL-4 Responses to Respiratory Syncytial Virus Infection in Infants And Children J. Infect. 

Dis. 183: 687-696, 2001 (PMID: 11181144). 

5. Mbawuike I.N., ZHANG, Y., WANG, Y and Song L. Cationic Liposome-Mediated Enhanced Generation Of 

Human HLA-Restricted RSV-Specific CD8+ CTL. J. Clin. Immunol. 22: 164-175, 2002 (PMID: 12078858). 

6. Zhang, Y, Y. Wang, X. Gilmore, K. Xu, M. Chen, P. Tebebi and I.N. Mbawuike. Apoptosis And Reduced 

Influenza A Virus Specific CD8+ T Cells in Aging Mice. Cell Death and Differentiation, 9: 651-660, 2002 

(PMID: 12032674). 

7. Orson, F., B. M. Kinsey, C. L. Densmore, T. Nguyen, Y. Wu, I. N. Mbawuike, P. R. Wyde. 

Protection against influenza infection by cytokine-enhanced aerosol genetic immunization. J. 

Gene Medi.: 8, 488-497, 2006 (PMID: 16389596). 

D. Research Support 

Ongoing Research Support

NO1-AI-030039 (Couch, PI) 08/01/2003-07/31/2010 

NIAID, NIH 

TITLE: Viral Respiratory Pathogens Research Unit 

GOALS: The major goal of this project is to examine the interaction of respiratory pathogens - viruses and 

bacteria. New methods to development protection will be explored. 

Role: Project PI and Director: “Epitope-Based Multi-Peptide Vaccines For Influenza” 

2R44AI063830-03A1 (Galarza, PI) 05/01/2005 – 08/31/2012 

NIAID, NIH 

Title: Development of Influenza Virus-Like Particle (VLP) Vaccines 

The parent Phase I SBIR award created and tested in preclinical studies several VLP vaccine candidates 

designed to protect against highly pathogenic pandemic strains of the influenza virus such as H1N1-1918, 

H5N1 and H7N7 avian influenza viruses. The goal of this SBIR II is to construct and produce the VLP vaccines 

in mammalian cells. Protective efficacy against avian H5N1, H7N7 and human H1N1-1918 influenza viruses 

will be tested in mice and ferrets. 

Role: Co-investigator 

 

1R01AI062917-01A1 (Zheng, Biao, Q, PI) 06/01/05-02/28/11 

NIAID, NIH 

TITLE: Fc Receptor Signaling in Vaccine Design for the Elderly 

The goal of this project is to study the mechanisms of immune modulation by manipulating Fc receptor 

signaling on antigen-presenting cells and lymphocytes. This proposal will try to identify novel immunization 

strategy to provide effective protection to immune compromised population against infectious diseases. 

Role: Co-investigator 

N01 AI25465 (Keitel, PI) 11/01/2007-10/31/2014 

NIAID, NIH 

Vaccine and Treatment Evaluation Units (VTEUs). The purpose of this contract is to strengthen and expand 

DMID’s capacity to conduct clinical trials of promising candidate vaccines and therapies for infectious diseases. 

The unit will also conduct clinical trials in larger populations and to safely test vaccines in specific vulnerable 

populations, such as infants and the elderly. 

Role: CMI Laboratory Director: To evaluate T cell immune responses to vaccines. 

Completed Research Support

1-U01-AI056447-01 (Subcontractor; Van Nest, PI; Dynavax) 08/01/2003-07/31/2007 

NIAID, NIH 

ISS-Linked NP Vaccine to Control Pandemic Flu Outbreak. The goal of this proposal is to develop a novel 

influenza vaccine which includes nucleoprotein (NP) linked to an immunostimulatory oligonucleotide (ISS) to 

induce protective immunity against divergent and potentially pandemic influenza strains. 

Role: Subcontractor to Dynavax

Biographical Sketches for each listed Senior/Key Person 3 Page 23

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1 R21 AI53454-01 (Mbawuike, PI) 09/01/2002-08/31/2005 

NIAID, NIH 

Vulnerability to Smallpox Due to Declining CTL Immunity. The major objectives of study are to compare the 

sero-prevalence and memory CD8+ CTL activity status against vaccinia in representative young adult and 

elderly populations, and to identify easily detectable and quantifiable surrogates of CTL competence 

against vaccinia that may be used as a surrogate of CTL immunity to smallpox infection. A secondary 

objective is to explore the immunological basis for complications from vaccinia vaccination. 

Role: PI 

R01 AG14351 (Mbawuike, PI) 04/01/1997-05/31/2003 

National Institute on Aging, NIH 

TH1 cytokines and impaired CD8+ CTL in elderly humans. The major goal of this project is to determine the 

mechanism of reduced CD8+ CTL responses in elderly humans and how to correct it. 

Role: PI 

RO1 AG10057 Mbawuike (PI) (Mbawuike, PI) 09/30/1996-08/31/2000

National Institute on Aging, NIH 

Influenza Nucleoprotein as a Probe for Low CTL in Aging. The major goals of this project are to test the 

hypothesis that the diminished ability of aged mice to clear influenza virus infection and recover from the 

disease is due to deficient CD8+

 cytotoxic T lymphocyte (CTL) activity and to identify the deficient mechanisms 

responsible for the deficient CTL activity. Novel influenza "T cell vaccines", namely, influenza plasmid NP DNA 

and recombinant NP baculovirus vaccines will be utilized as probes for CD8+

 CTL in aging. 

Role: PI 

NO1-A1-65316 (Glezen, PI) 09/30/96 - 12/31/03 

NIAID, NIH 

Maternal Immunization for the Prevention of Infectious Diseases in Neonates and Infants. The major goal of 

this project is to test vaccines in pregnant women to promote passive immunity in the neonate and young 

infant. The priority agents are RSV, group B streptococcus, pneumococci and Hemophilus influenzae type b. 

Role: Co-investigator 

Biographical Sketches for each listed Senior/Key Person 3 Page 24

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
 Principal Investigator/Program Director (Last, first, middle): Donis, Ruben O. 

PHS 398/2590 (Rev. 05/01) Page _______ 

BIOGRAPHICAL SKETCH

Provide the following information for the key personnel in the order listed for Form Page 2.

Follow the sample format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Ruben O. Donis, Ph.D. 

POSITION TITLE

eRA COMMONS USER NAME Associate Director, Influenza Division, CDC

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 Buenos Aires, Argentina M.V. 1972-1978 Veterinary Medicine

Cornell University, Ithaca, NY Ph.D. 1980-1986 Virology

St. Jude Children’s Research Hospital Post-Doc 1986-1989 Molecular Virology

A. Personal Statement 

The major emphasis of our research is to understand the virulence and evolution of influenza virus in avian 

and mammalian hosts to design public health intervention strategies. Reverse genetics and organismal 

approaches are being exploited to understand molecular determinants of genetic reassortment and its role 

on interspecies transmission and the emergence of pandemic influenza viruses. Studies on the virulence of 

influenza virus inform risk assessments and also prioritize development of vaccine candidates. An 

important focus of our work entails leveraging our expertise on antigenic drift of the hemagglutinin to 

develop structure-based approaches that expand the breath of immunity to seasonal and pandemic 

influenza vaccines. This information is being applied to development of broadly neutralizing therapeutic 

monoclonal antibodies and universal vaccines. 

B. Positions and Honors

Professional Positions 

1980-1981 Assistant Research Scientist, Division of Laboratories and Research, New York State Department of 

Health, Albany, New York 

1986-1989 Postdoctoral Fellow, St. Jude Children's Research Hospital, Memphis, Tennessee 

1989-2003 Assistant/Associate/Professor, Dept. of Veterinary and Biomedical Sciences, University of Nebraska 

2003-2006 Chief, Molecular Genetics Section, Influenza Branch, CDC 

2004-present Adjunct Professor, Dept. of Microbiology and Immunology, Emory University, Atlanta, Georgia 

2006-2012 Chief, Molecular Virology and Vaccines Branch, Influenza Division, CDC 

2012-present Associate Director, Influenza Division, CDC 

Honors and Awards 

1983-1986 Cornell University, NYS College of Veterinary Medicine Rotating Fellowship 

2007 Distinguished Service Award, United States Secretary of Health and Human Services 

2008 Leveraging Collaboration Award, Food and Drug Administration, US DHHS

2009 Charles Shepard Science Award, Centers for Disease Control and Prevention 

C. Representative Peer-Reviewed Publications (2009-2012) from a total of 153

1. Schat KA, Bingham J, Butler JM, Chen LM, Lowther S, Crowley TM, Moore RJ, Donis RO, Lowenthal JW (2012) 

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian 

influenza virus in chickens and ducks. PLOS One 7:e30960 

2. Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle 

AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox 

NJ, Anderson LJ, Rupprecht CE, Donis RO (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad 

Sci U S A 109:4269 

Biographical Sketches for each listed Senior/Key Person 4 Page 25

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
 Principal Investigator/Program Director (Last, first, middle): Donis, Ruben O. 

PHS 398/2590 (Rev. 05/01) Page _______ 

3. Yamada S, Shinya K, Takada A, Ito T, Suzuki T, Suzuki Y, Le QM, Ebina M, Kasai N, Kida H, Horimoto T, Rivailler 

P, Chen LM, Donis RO, Kawaoka Y (2012) Adaptation of a duck influenza A virus in quail. J Virol 86:1411 

4. Barlow PG, Svoboda P, Mackellar A, Nash AA, York IA, Pohl J, Davidson DJ, Donis RO (2011) Antiviral activity and 

increased host defense against influenza infection elicited by the human cathelicidin LL-37. PLOS One 6:e25333 

5. Chen LM, Rivailler P, Hossain J, Carney P, Balish A, Perry I, Davis CT, Garten R, Shu B, Xu X, Klimov A, Paulson 

JC, Cox NJ, Swenson S, Stevens J, Vincent A, Gramer M, Donis RO (2011) Receptor specificity of subtype H1 

influenza A viruses isolated from swine and humans in the United States. Virology 412:401 

6. Christman MC, Kedwaii A, Xu J, Donis RO, Lu G (2011) Pandemic (H1N1) 2009 virus revisited: an evolutionary 

retrospective. Infect Genet Evol 11:803 

7. Cox N, Donis R, Barr JR (2011) Exposure science for viral diseases: 2009 H1N1 pandemic influenza virus. J Expo 

Sci Environ Epidemiol 21:1 

8. Feeley EM, Sims JS, John SP, Chin CR, Pertel T, Chen LM, Gaiha GD, Ryan BJ, Donis RO, Elledge SJ, Brass AL 

(2011) IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog 7:e1002337 

9. Gustin KM, Maines TR, Belser JA, van Hoeven N, Lu X, Dong L, Isakova-Sivak I, Chen LM, Voeten JT, Heldens JG, 

van den Bosch H, Cox NJ, Tumpey TM, Klimov AI, Rudenko L, Donis RO, Katz JM (2011) Comparative 

immunogenicity and cross-clade protective efficacy of mammalian cell-grown inactivated and live attenuated H5N1 

reassortant vaccines in ferrets. J Infect Dis 204:1491 

10. Han T, Sui J, Bennett AS, Liddington RC, Donis RO, Zhu Q, Marasco WA (2011) Fine epitope mapping of 

monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface 

display. Biochem Biophys Res Commun 409:253 

11. Hossain MJ, Bourgeois M, Quan FS, Lipatov AS, Song JM, Chen LM, Compans RW, York I, Kang SM, Donis RO 

(2011) Virus-like particle vaccine containing hemagglutinin confers protection against 2009 H1N1 pandemic 

influenza. Clin Vaccine Immunol 18:2010 

12. Huang FF, Barnes PF, Feng Y, Donis R, Chroneos ZC, Idell S, Allen T, Perez DR, Whitsett JA, DunussiJoannopoulos K, Shams H (2011) GM-CSF in the lung protects against lethal influenza infection. Am J Respir Crit 

Care Med 184:259 

13. Isakova-Sivak I, Chen LM, Matsuoka Y, Voeten JT, Kiseleva I, Heldens JG, den Bosch H, Klimov A, Rudenko L, 

Cox NJ, Donis RO (2011) Genetic bases of the temperature-sensitive phenotype of a master donor virus used in 

live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2). Virology 412:297 

14. Karpala AJ, Bingham J, Schat KA, Chen LM, Donis RO, Lowenthal JW, Bean AG (2011) Highly pathogenic (H5N1) 

avian influenza induces an inflammatory T helper type 1 cytokine response in the chicken. J Interferon Cytokine 

Res 31:393 

15. Maines TR, Chen LM, Belser JA, Van Hoeven N, Smith E, Donis RO, Tumpey TM, Katz JM (2011a) Multiple genes 

contribute to the virulent phenotype observed in ferrets of an H5N1 influenza virus isolated from Thailand in 2004. 

Virology 413:226 

16. Maines TR, Chen LM, Van Hoeven N, Tumpey TM, Blixt O, Belser JA, Gustin KM, Pearce MB, Pappas C, Stevens 

J, Cox NJ, Paulson JC, Raman R, Sasisekharan R, Katz JM, Donis RO (2011b) Effect of receptor binding domain 

mutations on receptor binding and transmissibility of avian influenza H5N1 viruses. Virology 413:139 

17. Pierce CL, Williams TL, Moura H, Pirkle JL, Cox NJ, Stevens J, Donis RO, Barr JR (2011) Quantification of 

immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatographytandem mass spectrometry. Anal Chem 83:4729 

18. Robertson JS, Nicolson C, Harvey R, Johnson R, Major D, Guilfoyle K, Roseby S, Newman R, Collin R, Wallis C, 

Engelhardt OG, Wood JM, Le J, Manojkumar R, Pokorny BA, Silverman J, Devis R, Bucher D, Verity E, Agius C, 

Camuglia S, Ong C, Rockman S, Curtis A, Schoofs P, Zoueva O, Xie H, Li X, Lin Z, Ye Z, Chen LM, O'Neill E, 

Balish A, Lipatov AS, Guo Z, Isakova I, Davis CT, Rivailler P, Gustin KM, Belser JA, Maines TR, Tumpey TM, Xu X, 

Katz JM, Klimov A, Cox NJ, Donis RO (2011) The development of vaccine viruses against pandemic A(H1N1) 

influenza. Vaccine 29:1836 

19. Samson HC, Topliff CL, Donis RO, Kelling CL (2011) Comparison of viral replication and IFN response in alpaca 

and bovine cells following bovine viral diarrhea virus infection. Virology 413:111 

20. Spesock A, Malur M, Hossain MJ, Chen LM, Njaa BL, Davis CT, Lipatov AS, York IA, Krug RM, Donis RO (2011) 

The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 

proteins. J Virol 85:7048 

Biographical Sketches for each listed Senior/Key Person 4 Page 26

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
 Principal Investigator/Program Director (Last, first, middle): Donis, Ruben O. 

PHS 398/2590 (Rev. 05/01) Page _______ 

21. Xu J, Christman MC, Donis RO, Lu G (2011) Evolutionary dynamics of influenza A nucleoprotein (NP) lineages 

revealed by large-scale sequence analyses. Infect Genet Evol 11:2125 

22. Anderson T, Capua I, Dauphin G, Donis R, Fouchier R, Mumford E, Peiris M, Swayne D, Thiermann A (2010) FAOOIE-WHO Joint Technical Consultation on Avian Influenza at the Human-Animal Interface. Influenza Other Respi 

Viruses 4 Suppl 1:1 

23. Balish AL, Davis CT, Saad MD, El-Sayed N, Esmat H, Tjaden JA, Earhart KC, Ahmed LE, Abd El-Halem M, Ali AH, 

Nassif SA, El-Ebiary EA, Taha M, Aly MM, Arafa A, O'Neill E, Xiyan X, Cox NJ, Donis RO, Klimov AI (2010) 

Antigenic and genetic diversity of highly pathogenic avian influenza A (H5N1) viruses isolated in Egypt. Avian Dis 

54:329 

24. Carney PJ, Lipatov AS, Monto AS, Donis RO, Stevens J (2010) Flexible label-free quantitative assay for antibodies 

to influenza virus hemagglutinins. Clin Vaccine Immunol 17:1407 

25. Castleman WL, Powe JR, Crawford PC, Gibbs EP, Dubovi EJ, Donis RO, Hanshaw D (2010) Canine H3N8 

influenza virus infection in dogs and mice. Vet Pathol 47:507 

26. Davis CT, Balish AL, O'Neill E, Nguyen CV, Cox NJ, Xiyan X, Klimov A, Nguyen T, Donis RO (2010) Detection and 

characterization of clade 7 high pathogenicity avian influenza H5N1 viruses in chickens seized at ports of entry and 

live poultry markets in Vietnam. Avian Dis 54:307 

27. Floridia M, Tamburrini E, Anzidei G, Tibaldi C, Muggiasca ML, Guaraldi G, Fiscon M, Vimercati A, Martinelli P, 

Donisi A, Dalzero S, Ravizza M (2010) Declining HCV seroprevalence in pregnant women with HIV. Epidemiol 

Infect 138:1317 

28. Hossain MJ, Perez S, Guo Z, Chen LM, Donis RO (2010) Establishment and characterization of a Madin-Darby 

canine kidney reporter cell line for influenza A virus assays. J Clin Microbiol 

29. Lednicky JA, Villanueva JM, Burke SA, Shively R, Shaw MW, Daniels DE, Hamilton SB, Donis RO (2010) Validation 

of a method for preparing influenza H5N1 simulated samples. J Virol Methods 

30. Rivailler P, Perry IA, Jang Y, Davis CT, Chen LM, Dubovi EJ, Donis RO (2010) Evolution of canine and equine 

influenza (H3N8) viruses co-circulating between 2005 and 2008. Virology 408:71 

31. Song JM, Hossain J, Yoo DG, Lipatov AS, Davis CT, Quan FS, Chen LM, Hogan RJ, Donis RO, Compans RW, 

Kang SM (2010a) Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like 

particles. Virology 

32. Song JM, Kim YC, Barlow PG, Hossain MJ, Park KM, Donis RO, Prausnitz MR, Compans RW, Kang SM (2010b) 

Improved protection against avian influenza H5N1 virus by a single vaccination with virus-like particles in skin using 

microneedles. Antiviral Res 88:244 

33. Song JM, Kim YC, Lipatov AS, Pearton M, Davis CT, Yoo DG, Park KM, Chen LM, Quan FS, Birchall JC, Donis RO, 

Prausnitz MR, Compans RW, Kang SM (2010c) Microneedle delivery of H5N1 influenza virus-like particles to the 

skin induces long-lasting B- and T-cell responses in mice. Clin Vaccine Immunol 17:1381 

34. Steitz J, Barlow PG, Hossain J, Kim E, Okada K, Kenniston T, Rea S, Donis RO, Gambotto A (2010a) A candidate 

H1N1 pandemic influenza vaccine elicits protective immunity in mice. PLOS One 5:e10492 

35. Steitz J, Wagner RA, Bristol T, Gao W, Donis RO, Gambotto A (2010b) Assessment of route of administration and 

dose escalation for an adenovirus-based influenza A Virus (H5N1) vaccine in chickens. Clin Vaccine Immunol 

17:1467 

36. Stevens J, Chen LM, Carney PJ, Garten R, Foust A, Le J, Pokorny BA, Manojkumar R, Silverman J, Devis R, Rhea 

K, Xu X, Bucher DJ, Paulson J, Cox NJ, Klimov A, Donis RO (2010a) Receptor Specificity of Influenza A H3N2 

Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs. J Virol 

37. Stevens J, Chen LM, Carney PJ, Garten R, Foust A, Le J, Pokorny BA, Manojkumar R, Silverman J, Devis R, Rhea 

K, Xu X, Bucher DJ, Paulson JC, Cox NJ, Klimov A, Donis RO (2010b) Receptor specificity of influenza A H3N2 

viruses isolated in mammalian cells and embryonated chicken eggs. J Virol 84:8287 

38. Yang H, Chen LM, Carney PJ, Donis RO, Stevens J (2010) Structures of receptor complexes of a North American 

H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site. PLoS Pathog 6:e1001081 

39. Alvarez P, Mattiello R, Rivailler P, Pereda A, Davis CT, Boado L, D'Ambrosio E, Aguirre S, Espinosa C, La Torre J, 

Donis R, Mattion N (2009) First isolation of an H1N1 avian influenza virus from wild terrestrial non-migratory birds in 

Argentina. Virology 396:76 

Biographical Sketches for each listed Senior/Key Person 4 Page 27

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
 Principal Investigator/Program Director (Last, first, middle): Donis, Ruben O. 

PHS 398/2590 (Rev. 05/01) Page _______ 

40. Brooks WA, Alamgir AS, Sultana R, Islam MS, Rahman M, Fry AM, Shu B, Lindstrom S, Nahar K, Goswami D, 

Haider MS, Nahar S, Butler E, Hancock K, Donis RO, Davis CT, Zaman RU, Luby SP, Uyeki TM (2009a) Avian 

influenza virus A (H5N1), detected through routine surveillance, in child, Bangladesh. Emerg Infect Dis 15:1311 

41. Brooks WA, Alamgir AS, Sultana R, Islam MS, Rahman M, Fry AM, Shu B, Lindstrom S, Nahar K, Goswami D, 

Haider MS, Nahar S, Butler E, Hancock K, Donis RO, Davis CT, Zaman RU, Luby SP, Uyeki TM, Rahman M 

(2009b) Avian influenza virus A (H5N1), detected through routine surveillance, in child, Bangladesh. Emerg Infect 

Dis 15:1311 

42. Dong J, Matsuoka Y, Maines TR, Swayne DE, O'Neill E, Davis CT, Van-Hoven N, Balish A, Yu HJ, Katz JM, Klimov 

A, Cox N, Li DX, Wang Y, Guo YJ, Yang WZ, Donis RO, Shu YL (2009) Development of a new candidate H5N1 

avian influenza virus for pre-pandemic vaccine production. Influenza Other Respi Viruses 3:287 

43. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Sessions WM, Xu X, Skepner E, Deyde V, OkomoAdhiambo M, Gubareva L, Barnes J, Smith CB, Emery SL, Hillman MJ, Rivailler P, Smagala J, de Graaf M, Burke 

DF, Fouchier RA, Pappas C, Alpuche-Aranda CM, Lopez-Gatell H, Olivera H, Lopez I, Myers CA, Faix D, Blair PJ, 

Yu C, Keene KM, Dotson PD, Jr., Boxrud D, Sambol AR, Abid SH, St George K, Bannerman T, Moore AL, Stringer 

DJ, Blevins P, Demmler-Harrison GJ, Ginsberg M, Kriner P, Waterman S, Smole S, Guevara HF, Belongia EA, 

Clark PA, Beatrice ST, Donis R, Katz J, Finelli L, Bridges CB, Shaw M, Jernigan DB, Uyeki TM, Smith DJ, Klimov 

AI, Cox NJ (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating 

in humans. Science 325:197 

44. Jackson S, Van Hoeven N, Chen LM, Maines TR, Cox NJ, Katz JM, Donis RO (2009) Reassortment between avian 

H5N1 and human H3N2 influenza viruses in ferrets: a public health risk assessment. J Virol 83:8131 

45. Jadhao SJ, Nguyen DC, Uyeki TM, Shaw M, Maines T, Rowe T, Smith C, Huynh LP, Nghiem HK, Nguyen DH, 

Nguyen HK, Nguyen HH, Hoang LT, Nguyen T, Phuong LS, Klimov A, Tumpey TM, Cox NJ, Donis RO, Matsuoka 

Y, Katz JM (2009) Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird 

markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004. Arch Virol 154:1249 

46. Kang SM, Yoo DG, Lipatov AS, Song JM, Davis CT, Quan FS, Chen LM, Donis RO, Compans RW (2009) Induction 

of long-term protective immune responses by influenza H5N1 virus-like particles. PLoS One 4:e4667 

47. Lepus CM, Gibson TF, Gerber SA, Kawikova I, Szczepanik M, Hossain J, Ablamunits V, Kirkiles-Smith N, Herold 

KC, Donis RO, Bothwell AL, Pober JS, Harding MJ (2009) Comparison of human fetal liver, umbilical cord blood, 

and adult blood hematopoietic stem cell engraftment in NOD-scid/gammac-/-, Balb/c-Rag1-/-gammac-/-, and C.B17-scid/bg immunodeficient mice. Hum Immunol 70:790 

48. Matsuoka Y, Swayne DE, Thomas C, Rameix-Welti MA, Naffakh N, Warnes C, Altholtz M, Donis R, Subbarao K 

(2009) Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of 

influenza H5N1 viruses for mice. J Virol 83:4704 

49. Nguyen T, Davis CT, Stembridge W, Shu B, Balish A, Inui K, Do HT, Ngo HT, Wan XF, McCarron M, Lindstrom SE, 

Cox NJ, Nguyen CV, Klimov AI, Donis RO (2009) Characterization of a highly pathogenic avian influenza H5N1 

virus sublineage in poultry seized at ports of entry into Vietnam. Virology 387:250 

50. O'Neill E, Donis RO (2009a) Generation and characterization of candidate vaccine viruses for prepandemic 

influenza vaccines. Curr Top Microbiol Immunol 333:83 

51. O'Neill E, Donis RO (2009b) Generation and characterization of candidate vaccine viruses for pre-pandemic 

influenza vaccines. In: Compans R, Orenstein W (eds) Pandemic Influenza Vaccines. Springer-Verlag 

52. Prabakaran M, Ho HT, Prabhu N, Velumani S, Szyporta M, He F, Chan KP, Chen LM, Matsuoka Y, Donis RO, 

Kwang J (2009) Development of epitope-blocking ELISA for universal detection of antibodies to human H5N1 

influenza viruses. PLoS One 4:e4566 

53. Rodriguez A, Perez-Gonzalez A, Hossain MJ, Chen LM, Rolling T, Perez-Brena P, Donis R, Kochs G, Nieto A 

(2009) Attenuated strains of influenza A viruses do not induce degradation of RNA polymerase II. J Virol 83:11166 

54. Suguitan AL, Jr., Marino MP, Desai PD, Chen LM, Matsuoka Y, Donis RO, Jin H, Swayne DE, Kemble G, Subbarao 

K (2009) The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity 

and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus. Virology 395:280 

55. Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, 

Yammanuru A, Han T, Cox NJ, Bankston LA, Donis RO, Liddington RC, Marasco WA (2009) Structural and 

functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 

16:265 

Biographical Sketches for each listed Senior/Key Person 4 Page 28

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

2

Delete Entry

Dr. Jose M. Galarza Ph.D. PD/PI

Rev. Diana Dalfo PhD Staff Scientist

1 Research Associate (Master level) 12.00

1 Research Intern 12.00

TechnoVax Inc.

07/01/2013 06/30/2014 Budget Period 

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Detailed Budget - Year 1 Page 29

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2013 06/30/2014

TechnoVax Inc.

Misc. small equipment maintenance/replacement

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Detailed Budget - Year 1 Page 30

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
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.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1239-TVx_UFlu_Budget_Justification_120

Total Direct Costs

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2013 06/30/2014

TechnoVax Inc.

Budget Period 1

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Detailed Budget - Year 1 Page 31

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

2

Delete Entry

Dr. Jose M. Galarza Ph.D. PD/PI

Dr. Diana Dalfo PhD Staff Scientist

1 Research Associate 8.00

1 Research Intern 6.00

TechnoVax Inc.

07/01/2014 06/30/2015 Budget Period 

Add Attachment Delete Attachment View Attachment

Previous Period

2

	


 		

 !!""

Detailed Budget - Year 2 Page 32

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2014 06/30/2015

TechnoVax Inc.

Misc. small equipment maintenance/replacement

Add Attachment Delete Attachment View Attachment

Budget Period 2 Delete Entry

	


 		

 !!""

Detailed Budget - Year 2 Page 33

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
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.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1239-TVx_UFlu_Budget_Justification_120

Total Direct Costs

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2014 06/30/2015

TechnoVax Inc.

Budget Period 2

Add Attachment Delete Attachment View Attachment

Next Period

Delete Entry

	


 		

 !!""

Detailed Budget - Year 2 Page 34

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
BUDGET JUSTIFICATION 

GENERAL CONSIDERATIONS 

In order to conduct the proposed project consisting of producing VLP vaccines and 

testing these vaccines in animals we have planned to budget our efforts over a two 

years period. 

Scientific personnel salaries and related expenses represent 52% of TechnoVax 

expenses or 41% of Total Requested Funds. High salaries costs are driven by two main 

factors: 1) the project duration and 2) the higher cost of salaries in the New York metro 

area. To attract adequate talent TechnoVax needs to offer competitive salaries, however 

in order to maintain financial balance, our salaries are still about +30% lower than those 

offered at other larger biotech companies located within a 10 miles radius (e.g. 

Regeneron, Progenics, Acorda/Astellas, Pfizer, etc.). Therefore we believe that our 

budget includes adequate staffing level as well as reasonable other direct and indirect 

costs to support a successful outcome for this project. 

Our sub-awardee, Dr. Innocent Mbawuike at Baylor College of Medicine, will perform the 

animal studies and is budgeting $ or about 20% of total requested funds. This 

cost is justified by the fact that these studies will need to take place in BSL-2 and “BSL-3 

enhanced” level facilities.

TECHNOVAX PERSONNEL 

Dr. Jose M. Galarza, D.V.M., Ph.D., CEO and President of TechnoVax will serve as PI 

of the project. His primary role will be oversight of all aspect of the project and direct 

hands-on participation in the creation, production, characterization and testing of Flu 

VLP vaccines. The PI will maintain constant communication with the Program Director, 

collaborators and external advisors to address scientific issues, progress and evaluate 

results. PI will allocate 25% of his time to the execution of this project. Proportional 

annual salary requested is and for periods 1 and 2 respectively. 

Dr. Diana Dalfo, PH.D., is a TechnoVax Staff Scientist for VLP vaccines projects and 

will perform experiments and guide a Research Associate on the different aspects of the 

project. Dr. Dalfo will allocate 60% of her time to the execution of this project. 

Proportional annual salary requested is and for periods 1 and 2 

respectively. 

One Research Associate, M.S. (with 2-3 year experience), will be involved in cell 

culture maintenance, cloning, plasmid preparation and purification. Production and 

characterization of large scale DNA. Immunological assays for evaluating the immune 

response elicited by the Universal Flu VLP vaccine candidate (serum neutralization test). 

Research associate will allocate 100% of his/her time to the execution of this project. 

Annual salary requested is $ and $ for periods 1 and 2 respectively as a 

larger effort will occur during the first year. 

Research Intern (M.S. graduate), will provide technical support in the lab to the 

research team on a full time basis for 18 months. Annual salary requested is 

Budget Justification Page 35

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Fringe Benefits: Based on current payroll data, Fringe Benefits amount to 25% of Base 

Salary Costs. 

Salary and Costs increases: a 3% annual increase has been budgeted for salaries in 

period 2.

EQUIPMENT 

Miscellaneous Equipment: as current lab equipment is mostly fully utilized by current 

projects, various small equipment items will be needed for day to day operations (fridge, 

-20C freezer, etc.). has been budgeted annually to cover purchases and 

maintenance costs. 

TRAVEL 

Domestic Travel: $ in year 1 is budgeted for attending a major scientific conference 

and scientific meetings as follows: PI or Staff Scientist: ($ for registration 

and $ for travel) 

OTHER DIRECT COSTS 

Material and supplies: General laboratory supplies including plastic-ware, reagents, 

chemicals, tissue culture media and protein analysis and purification material, Western 

blot reagent, disposables are requested for vaccine production, characterization and 

testing. This total budget of $ over 2 years is based on a current comparable 

project for the production of an influenza VLP vaccine and can be broken down into two 

general categories comprising: 

 Year 1 Year 2 

Plasticware, glassware and misc.: 

Reagents, cell lines, Ab and chemicals: 

Total Material and supplies 

Equipment or Facility Rental/User Fees: Electron-Microscopy images will be needed 

after each production batch; we currently use City College of New York (CUNY) EM 

equipment for a cost of per session. 

INDIRECT COSTS 

F&A costs: as awarded in previous SBIR grants, we are requesting a 25% indirect cost 

rate applied to direct costs to cover F&A costs. 

FEE 

TechnoVax requests 6.99% fixed fee for re-investment into company infrastructure and 

Operating costs. 

Budget Justification Page 36

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Section A, Senior/Key Person

Section C, Equipment

RESEARCH & RELATED BUDGET - Cumulative Budget

Section D, Travel

Domestic

Section E, Participant/Trainee Support Costs

Foreign

Tuition/Fees/Health Insurance

Stipends

Travel

Subsistence

Other

Number of Participants/Trainees

1.

2.

3.

4.

5.

6.

1.

2.

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.

9.

10.

Totals ($)

Total Number Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Other 1

Other 2

Other 3

Section B, Other Personnel

Section J, Fee

Section I, Total Direct and Indirect Costs (G + H)

Section H, Indirect Costs

Section G, Direct Costs (A thru F)

4

	


 		

 !!""

Cumulative Budget Page 37

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 1

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2013 * End Date: 06-30-2014 Budget Period: 1

A. Senior/Key Person

Prefix * First Name Middle Name * Last Name Suffix * Project Role Base Salary

($)

Cal.

Months

Acad.

Months

Sum.

Months

* Requested

Salary ($)

* Fringe

Benefits ($)

* Funds Requested ($)

1. Dr. Innocent Mbawuike Co-PI 2.00

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Mime Type: Total Senior/Key Person

B. Other Personnel

* Number of

Personnel

* Project Role Cal.

Months

Acad.

Months

Sum.

Months

* Requested

Salary ($)

* Fringe

Benefits

* Funds Requested

($)

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Research Assistant 3.00

1 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 38

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 1

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2013 * End Date: 06-30-2014 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)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 39

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTIONS F-K, BUDGET PERIOD 1

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2013 * End Date: 06-30-2014 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

8. Other Supplies and Services

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. MTDC

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Arif Karim, 214-767-3261

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: 1243-BAYLOR_Budget

Justification_120512.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 40

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 2

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2014 * End Date: 06-30-2015 Budget Period: 2

A. Senior/Key Person

Prefix * First Name Middle Name * Last Name Suffix * Project Role Base Salary

($)

Cal.

Months

Acad.

Months

Sum.

Months

* Requested

Salary ($)

* Fringe

Benefits ($)

* Funds Requested ($)

1. Dr. Innocent Mbawuike Co-PI

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Mime Type: Total Senior/Key Person

B. Other Personnel

* Number of

Personnel

* Project Role Cal.

Months

Acad.

Months

Sum.

Months

* Requested

Salary ($)

* Fringe

Benefits

* Funds Requested

($)

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Research Assistant 3.00

1 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 41

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 2

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2014 * End Date: 06-30-2015 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)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 42

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - SECTIONS F-K, BUDGET PERIOD 2

* ORGANIZATIONAL DUNS:

* Budget Type: ❍ Project ● Subaward/Consortium

Enter name of Organization:

* Start Date: 07-01-2014 * End Date: 06-30-2015 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

8. Other Supplies and Services

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. MTDC

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Arif Karim, 214-767-3261

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: 1243-BAYLOR_Budget

Justification_120512.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 43

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 2

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

Tracking Number: GRANT11282977 OMB Number: 4040-0001

Expiration Date: 06/30/2011

Subaward 1 Page 44

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
BUDGET JUSTIFICATION – Baylor College of Medecine 

PERSONNEL 

Innocent N. Mbawuike, Ph.D. (BCM PI) (about 2 months/year FT effort) is Associate Professor of 

Molecular Virology and Microbiology. Dr. Mbawuike has extensive experience in the evaluation of 

immunological responses related to influenza in animal models and humans. As BCM co-investigator on 

this SBIR Phase I proposal, he will conduct immunogenicity studies with mice immunized with influenza 

VLP vaccines. He will supervise influenza challenge studies in mice to be conducted in the BSL-2 facility. 

Dr. Mbawuike will monitor the day to day experiments conducted at BCM and will be responsible for 

preparing reports and manuscripts for studies generated at this site. 

To be named. (Research Assistant I, about 3 months/year FT effort). An experienced senior research 

assistant will be hired or assigned to perform the animal immunization experiment and conduct virus 

and antibody assays under the supervision of Dr. Mbawuike. The research Assistant will participate and 

perform the influenza virus challenge studies in the BCM BSL-2 and BSL-3 facilities. 

OTHER DIRECT COSTS 

Animal purchase and housing: Approximately 250 young adult mice will be purchased for 

($ each). The cost for housing animals in the biohazard suite of BCM vivarium is based on $ 

for mice (4 mice per cage) per diem respectively. It is estimated that the animals will be housed for 

approximately 60 days during each experiment for a cost of 

Supplies: General Laboratory supplies including cell culture reagents and plastic ware were requested 

for laboratory tests. Laboratory test include virus titration, virus neutralization and hemagglutination 

inhibition (HI) for specimens obtained from mice and ELISA assays for antigen-specific IgG, IgA and IgM 

antibodies. A budget of $ is allocated over the two year period. 

INDIRECT COSTS 

As per negotiated rate of 56.50% with DHHS. 

Subaward 1 Budget Justification Attachment Page 45

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
SBIR/STTR Information OMB Number: 4040-0001 

Expiration date: 06/30/2011 

* Program Type (select only one)

* SBIR/STTR Type (select only one)

* 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?

* 2. Does this application include subcontracts with Federal laboratories or any other Federal Government agencies?

* 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 

* 4. Will all research and development on the project be performed in its entirety in the United States?

* 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? 

* 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)?

* 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:

Questions 1-7 must be completed by all SBIR and STTR Applicants:

If no, provide an explanation in an attached file.

Administration at its web site: http://www.sba.gov 

SBIR STTR

Both (See agency-specific instructions to determine whether a particular agency allows a single submission for both SBIR and STTR)

Phase I Phase II

Fast-Track (See agency-specific instructions to determine whether a particular agency participates in Fast-Track)

Yes

No

10

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

* 1b. Anticipated Number of personnel to be employed at your organization at the time of award.

	


 		

 !!""

SBIR/STTR Information Page 46

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
* 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. 

* Attach File:

* 9. Will the Project Director/Principal Investigator have his/her primary employment with the small business at the time of award?

* 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?

* 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?

SBIR-Specific Questions:

STTR-Specific Questions:

SBIR/STTR Information

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.

Questions 10 and 11 apply only to STTR applications. If you are submitting ONLY an SBIR application, leave questions 10 and 11 blank.

1234-TVx_Commercialization_Hist Add Attachment Delete Attachment View Attachment

Yes

No

Yes

No

Yes

No

Yes

No

	


 		

 !!""

SBIR/STTR Information Page 47

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
COMMERCIALIZATION HISTORY 

TechnoVax has received one SBIR Phase II award in 2009: 

Grant Number: 

Project Title: Development of Influenza Virus-Like Particle (VLP) Vaccines 

As requested, a commercialization plan was provided at the time of the grant application. 

Currently this project is on-going and is in pre-clinical phase. 

TechnoVax has not received any other SBIR Phase II award since 2009. 

Prior SBIR Phase II Awards Page 48

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application

* Phone Number: Fax Number:

Email:

OMB Number: 0925-0001 

Province:

Prefix:

Dr. Jose

M.

Galarza

Ph.D.

No Yes

No Yes

Mr. Hector

Munoz

Chief Financial & Corp. Development Officer

765 Old Saw Mill River Rd.

Tarrytown

Westchester

USA: UNITED STATES

NY: New York

10591-6702

	


 		

 !!""

Clinical Trial & HESC Page 49

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

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://stemcells.nih.gov/research/registry/. 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.

PHS 398 Cover Page Supplement

No Yes

Cell Line(s):

	


 		

 !!""

Clinical Trial & HESC Page 50

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your 

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

OMB Number: 0925-0001 

2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application 

(for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments: 

 Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

1240-TVx_UFlu_INTRODUCTION_ Add Attachment Delete Attachment View Attachment

1241-TVx_UFlu_SPECIFIC_AIMS_ Add Attachment Delete Attachment View Attachment

1242-TVx_UFlu_Research_Plan_ Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

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List of Research Plan Attachments Page 51

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
1. INTRODUCTION 

This is a resubmission of a proposal to develop a broadly protective (universal) influenza VLP vaccine. 

We thank the reviewers for their insightful comments. We utilize VLP technology, since it permits 

modifications that would prevent virus replication for a viral vaccine. Broadly neutralizing antibodies 

have been isolated from humans and are directed against domains in the stem and other less 

immunogenic regions of HA. The isolation of such broadly neutralizing antibodies indicates that they 

are immunogenic and our goal is to determine if neutralizing antibody can be generated by VLPs 

modified to eliminate the immunodominant, hypervariable epitopes comprising the head region of HA. If 

so, they could be used in the case of an influenza pandemic, as a supplement to current vaccines to 

broaden protection as well as elicit longer duration of immunity. Previous reviewers found our proposal- 

“innovative”, “highly significant”, “a rational approach”, “outstanding expertise in influenza vaccinology”, 

“outstanding environment”. However, weaknesses identified by the prior reviewers are discussed here 

and changes were made in the research plan. “Proposal is over ambitious for the two year project 

period”: We have experience producing and characterizing VLP vaccines. We utilize CHO cells, which 

express the M1 and M2 structural proteins of the virus to which HA and NA are transfected to simplify 

and shorten VLP production time. We express the VLPs in CHO cells, which allows for adequate yield 

for characterization, immunological analysis with the broadly neutralizing human antibodies that we 

have assessed, as well as for immunogenicity and efficacy studies in animals. “If the remodeled HA 

do not perform as expected the rest of the experiment cannot be completed”: We show as 

preliminary data that at least some of our remodeled HAs can be produced and have elicited 

neutralizing antibodies. However, to mitigate risk and investigate structural factors enhancing 

immunogenicity, we have designed several different constructs including 5TA which models a wild type 

HA, able to fold properly and form trimers but contains specific protease cleavage sites which allow for 

removal of the globular domain by protease treatment after the VLP is produced and purified exposing 

conserved subdominant epitopes in the HA2 (stem) and the remainder of HA1. Thus we expect to be 

able to produce, characterize and test this VLP as well as others. “Acknowledge but no alternate 

plans are presented”: VLPs are composed of a scaffold of the M1 and M2 proteins to which various 

remodeled HAs and NAs can be incorporated. To strengthen the design process, we have enlisted Dr. 

Zhongtoa Zhang a structural biologist/protein chemist in the Department of Biochemistry, New York 

Medical College (see letter). “The preliminary results are not described clearly, and as presented, 

do not provide convincing evidence of feasibility for this project”: We have revised the 

preliminary data section and provide a more detail explanation of what is presented in the sera 

neutralization assay shown in figure 4. “The VLP vaccine may be expensive and laborious to 

produce” Initially, until the candidates that afford broad protection are identified, we will transiently 

transfect remodeled HA plasmids into a cell line that constitutively expresses the M1/M2 scaffold, 

allowing VLP production. Once candidates are identified, a constitutively producing vaccine cell line can 

be created. In fact, this strategy should be faster, less laborious, and more cost-effective than current 

methods of manufacturing seasonal flu vaccines. Rationale for using ELISA to quantify antibodies 

is not clear” “ELISA primarily measures binding Ab, which presumably these vaccines will not 

induce”: Our first method to evaluate antibody response is virus neutralization, ELISA is proposed as a 

secondary method to determine whether other antibodies are being elicited and can be measured by 

ELISA, particularly using virus as target antigen. The revised Research Plan addresses other 

reviewers’ comments. 

Introduction Page 52

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
2. SPECIFIC AIMS 

We propose to develop a broadly neutralizing influenza vaccine utilizing virus-like particles that 

display on their surfaces remodeled HA molecules. Most antibodies against HA target 

predominantly immunodominant but highly variable antigenic sites on the globular portion of the 

molecule, whereas the conserved subdominant antigenic sites, particularly those in the stem 

region are poorly recognized by the immune system. Recently identified human antibodies 

directed against these conserved antigenic sites have been isolated, and have demonstrated 

the ability to neutralize a broad range of influenza virus strains. It is reasonable, therefore to 

envision developing a vaccine that primarily displays highly conserved conformational antigenic 

sites able to elicit an antibody response recognizing these sites and obtain protection against a 

broad spectrum of antigenically distinct influenza viruses. To attain this goal and test this 

hypothesis, we propose to re-engineer influenza HA molecules and display these structures on 

the surface of influenza virus-like particles (VLPs). We will create remodeled HA by removing 

regions of the molecule that form the immune-dominant and highly variable antigenic sites and 

repositioning subdominant conserved and broadly protective antigenic regions. Broadly 

neutralizing influenza VLP vaccines displaying on their surfaces such remodeled HA molecules 

that reveal the subdominant conserved antigenic sites will be produced in mammalian cells. 

The immunogenicity and efficacy elicited by candidate VLP vaccines revealing remodeled HA 

molecules will be assessed in an influenza mouse model. To achieve these goals, we propose 

the following specific aims: 

Specific aim #1: Construct several remodeled HA expression constructs devoid of highly 

variable immunodominant antigens sites but preserving conserved subdominant antigenic 

regions and sub-clone them into an appropriate plasmid for VLP creation. Produce and 

characterize virus-like particles (VLPs) displaying remodeled HA molecules on their surfaces. 

Evaluate the presence of conserved subdominant antigenic sites in HA molecules displayed on 

purified VLPs by immuno-precipitation using antibodies that exclusively recognized these sites. 

Specific aim #2: Assess the immune response elicited in mice following immunization with 

candidate VLPs displaying alternative remodeled HA protein on their surfaces. The capacity of 

immunized mouse sera to neutralize different influenza A virus strains will be measured by an 

in-vitro micro-neutralization assay. Those VLP vaccines capable of inducing broadly 

neutralizing antibodies, as demonstrated by partially or completely neutralizing a panel of 

antigenically distinct influenza viruses, will be further tested in in-vivo efficacy and 

immunogenicity studies in mice alone and in combination with VLPs containing unaltered HA to 

detect synergy or competition. 

Specific aim #3: Conduct efficacy and immunogenicity studies of single or combination HA 

remodeled VLP vaccines in a lethal influenza mouse model. Groups of immunized mice will be 

challenged with lethal doses of at least three antigenically distinct influenza viruses (HA5, HA1 

and HA3). Further testing of the most promising candidate vaccines in ferrets will be pursued in 

a Phase II SBIR proposal, which will also seek enabling data for an IND application with the 

FDA. 

Specific Aims Page 53

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
3. RESEARCH PLAN 

3.1. Significance and Impact 

We propose to develop a broadly protective virus-like particle (VLP) based influenza vaccine that can 

neutralize a spectrum of influenza A virus subtypes by eliminating the major epitopes on HA to expose the 

more highly conserved universal and less available epitopes in the molecule. 

Seasonal influenza infections continue to pose a significant burden in the US and around the world. Influenza 

A viruses cause respiratory infections that afflict more than 60 million Americans of all ages every year causing 

some 200,000 hospitalizations and about 36,000 deaths per year [1,2]. In addition to seasonal circulating 

viruses, pandemic strains periodically emerge with dire consequences [3,4]. For example, the H5N1 avian 

virus, continues to circulate in birds and causes occasional human infection with high mortality [5,6]. If this 

virus acquires the capacity to effectively transmit amongst humans, the consequences are likely to be severe 

[7,8]. In recent studies, airborne transmission of the H5N1 occurred in ferrets after four mutations in the HA 

and one mutation of the PB2 protein[9,10]. In addition, outbreak of flu in chickens, as happened recently in 

Mexico, could disrupt conventional production of vaccines. 

The rapid antigenic evolution of influenza virus driven by the continuous accumulation of mutations (antigenic 

drift) or by gene swapping through reassortment (antigenic shift) overcomes the immune responses elicited by 

natural infection or vaccination. This necessitates periodic reformulation of vaccines with the predominant 

circulating strains and annual re-immunization to upgrade vaccine composition and to improve efficacy. 

Furthermore, the large number of avian and mammalian species susceptible to influenza virus provides 

additional sources of emerging virus that have the potential to give rise to a pandemic. Creation of a vaccine 

able to protect against both drifting viruses and those emerging from gene-shifting has been a major objective 

in the influenza vaccine field. Significant efforts have been applied to develop a universal vaccine, but 

achieving it has been elusive. However, the recent identification of broadly neutralizing antibodies from infected 

individuals [11,12] provides new possibilities for the design of vaccines able to elicit broad neutralizing 

protection. These broadly neutralizing antibodies predominantly bind to highly conserved sites on the stem 

regions of the hemagglutinin (HA), blocking virus infection by the steric inhibition of membrane fusion rather 

than receptor binding. Proper presentation of these highly conserved but subdominant (cryptic) antigenic sites 

in a vaccine composition could elicit broadly neutralizing antibodies. To achieve this goal, we propose to utilize 

VLPs displaying remodeled HAs that mainly exhibit these conserved epitopes. Development of a broadly 

protective (universal) vaccine able to withstand antigenic variation and sustain efficacy for an extended time 

should have a major impact on influenza prevention. Used not only as a stand-alone vaccine, but also as a 

component of seasonal vaccines, it could reduce both the incidence and severity of infection. 

3.2. Innovation 

We have shown that we can produce Virus-Like Particles (VLPs) with high immunogenicity and efficacy by 

expressing 4 flu proteins M1, M2, HA and NA simultaneously [31, 20, 21]. Also, we have produced VLPs with 2 

different HAs in the same particle as well as chimeric molecules indicating the versatility of the system. The 

dominant immunogenic epitopes in HA are located in the globular domain necessary for binding to cellular 

receptors. If these are removed the virus would no longer be infectious and able to multiply. Current concepts 

suggest that these domains block immunogenic recognition of other regions of the HA molecule by steric 

hindrance and by immunodominance. The discovery of broadly neutralizing antibodies in formerly infected 

individuals indicates that these other epitopes are immunogenic and elicit a broad and effective immune 

response [11, 13]. We have designed a series of modified HA molecules to express in our VLP expression 

system. Preliminary studies with two of these reengineered HAs indicate that they are incorporated into VLPs 

produced in CHO cells, and elicit a immune response after immunization as tested in micro-neutralization 

assays of flu virus. 

While these VLPs are produced by transient transfection in CHO cells expressing the M1 and M2 proteins, we 

have also created another VLP production system using continuous cell lines suitable for laboratory 5 liters 

scale up. Here we describe our preliminary findings and our proposal to produce several differently modified 

HAs to assess assembly, immunogenicity and efficacy that they would elicit against diverse flu strains. This 

cannot be done with egg based or cell based virus production methods. 

Research Strategy Page 54

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Our rationale for creating a broadly protective vaccine is based on 

developing VLPs displaying remodeled HA molecules (VLP-HA-Rem) that 

expose widely conserved subdominant antigenic sites while devoid of 

dominant and hypervariable epitopes. Conserved antigenic sites in the HA 

molecule may comprise linear or conformational determinants (epitopes); 

and both configurations would be suitable targets for specific antibody 

elicitation. Most of the HA conserved epitopes map to the HA2 fragment or 

the HA1 and HA2 interface, distant from the receptor binding site. The 

stem region of the trimeric HA is formed by the HA2 fragments of each 

monomer and contains the fusion peptide. Antibodies directed to this 

region should neutralize virus entry by blocking membrane fusion 

sterically, as shown with the recently identified broadly neutralizing human 

antibodies [11, 12]. To better display these sites, we remodeled the HA 

molecules by deleting regions of the HA1 fragments containing dominant 

variable sites, rearranging disulfide bridges and/or introducing hinge 

linkers to reposition the highly conserved epitopes and making them 

predominant to elicit production of broadly protective neutralizing antibodies. These remodeled HAs are 

incorporated and displayed on the surface of native influenza virus-like particles [31] 

VLP vaccines are highly immunogenic because of their particulate nature and display of a repetitive array of 

antigens (polyvalency). Structured macromolecular antigens such as VLPs facilitate cross-linking of multiple 

antigen receptors enhancing activation of B lymphocyte and priming T helper cells. It is anticipated that a VLPHA-Rem vaccine will stimulate a robust and long lasting immune response and hopefully broad protection. To 

our knowledge, although attempts to develop a universal vaccine are being pursed[14-16], including the most 

recent work that grafted A-helix peptide on a icosahedral particle [30], the strategy proposed here for creating 

this type of vaccine is novel and if successful, could have a significant impact on the prevention and control of 

influenza. 

3.3. Approach 

Progressive accumulation of mutations, due to the high error 

frequency of the influenza polymerase, drives the rapid 

antigenic evolution of the HA molecules of these viruses. 

Furthermore, gene swapping between influenza A strains by 

reassortment brings about sudden and more pronounced 

antigenic changes (antigenic shift). These mechanisms allow 

influenza viruses to evade antibody responses elicited by 

natural infection or vaccination. Therefore, development of an 

influenza vaccine able to overcome antigenic evolution and 

afford broad and sustained protection is one of the most 

desirable goals in influenza vaccine development. Here, we 

propose a strategy to develop a broadly protective vaccines 

based on VLPs displaying on their surface remodeled HA 

molecules that primarily present highly conserved subdominant 

antigenic sites. Human antibodies directed at these sites have demonstrated the ability to neutralize a broad 

spectrum of antigenically distinct influenza viruses [11]. 

To generate remodeled HA, we have re-designed five distinct HA genes, which represent alternative structural 

conformations of the HA molecule. Computer and structural analysis was used to guide remodeling, molecular 

design and sequence selection [11,17-19]. Four of these constructs contain alternative truncations of the HA1 

fragment, preserving or repositioning disulfide bridges and signal domains (Fig.1 & 5). The 3TA construct also 

contains a 12aa peptide linker that bridges non-deleted portions of the HA1, which connects to the HA2 

fragment allowing for conformational epitopes to form (Fig. 1). A construct, 5TA comprises a full length HA 

molecule containing two unique protease cleavage sites that, following protein folding and display on the VLP 

vaccine, will allow for the enzymatic cleavage of the top portions of the molecule to expose the conserved 

determinants in the HA2-stem and stem/HA1 interphase (Fig. 2). Evaluation of their ability to be incorporated 

into VLPs and the potency and breadth of the immune response they generate should further enable universal 

vaccine design. 

Research Strategy Page 55

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
TechnoVax has pioneered research in 

influenza virus-like particles (VLPs) 

(Latham T. and J. M. Galarza, 2001, J. 

Virol. 75:6154-6165) and the development 

of vaccines based on this technology. VLP 

vaccines have demonstrated 

immunogenicity and have been shown to 

provide protection against an otherwise 

lethal influenza virus challenge when 

administered via either intranasal or 

intramuscular routes [20,21] including 

100% protection against a lethal challenge 

by influenza A/Vietnam/1203/2004 (H5N1) 

virus. VLP vaccines are produced in cell-based recombinant systems as structural and biochemical mimics of 

the wild type virus. These VLPs lack viral genetic material and are unable to replicate or cause infection. 

Vaccine inactivation is not required, better maintaining antigenic epitopes and immunogenicity. Because of the 

flexibility, speed and safety of the technology, vaccines can be generated rapidly and without risk of 

disseminating infectious material. Other VLP vaccines have proven successful in preventing HPV caused 

cervical cancers (Guardasil, Cervarix) and hepatitis B induced liver disease [22,23]. Our flu VLP manufacturing 

system is based on generating stably engineered mammalian cell lines. Candidate VLP vaccines are produced 

utilizing engineered cell lines that express the polypeptides required for the self-assembly and release of viruslike particles from the cell surface. Our plan is to produce the VLP vaccines in stably transfected cells (CHO) 

following the expression of M1-M2-NA and remodeled HAs. This approach eliminates the need for vectors or 

viruses to drive gene expression, minimizing downstream purification and safety concerns. We have produced 

H5N1 VLP using this strategy (figure 3). Additionally, we have created CHO and Vero lines that constitutively 

express the M1 and M2 proteins, which form a scaffold for VLP assembly. Addition of remodeled HA and NA 

genes would allow for the generation of VLPs displaying remodeled HA revealing conserved epitopes common 

to multiple subtypes. Furthermore, utilization of these cell lines for vaccine development will facilitate the 

regulatory process, as these cells are being extensively used in the biopharmaceutical industry for the 

manufacturing of many FDA approved products. 

3.3.1 Preliminary Studies 

Our initial studies were performed 

with two of the synthesized 

remodeled HAs to determine 

whether immunizing mice with 

VLPs displaying these molecules 

elicited antibodies capable of 

neutralizing a homologous 

influenza virus strain. Two 

remodeled HAs (3TA and 5TA-see 

fig.1 and 2) derived from the 

influenza A/Vietnam/1023/2004 

(H5N1) were subcloned into our 

expression vector that also 

contains the M1, M2 and the NA 

construct. Design of this HA was 

based on the results reported with 

an identified human broadly 

neutralizing antibody [11] Our 

construct was transiently 

transfected into CHO cells together 

with a plasmid expressing the M1 and M2 proteins. Five days post-transfection, VLP material was collected 

from the culture medium, purified by ultracentrifugation and analyzed by Western blot utilizing an HA2 fragment 

(stem region) specific antibody. We detected these proteins in the purified material (Fig. 1 and 2), and then 

immunized four mice with each VLP preparation (3TA-VLP and 5TA VLP-protease treated) twice, two weeks 

Fig. 3

Research Strategy Page 56

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
apart via intramuscular route, without adjuvant. Three weeks after the booster immunization, serum samples 

from each VLP immunized group were pooled and tested in an in-vitro micro-neutralizing assay [21] for their 

ability to inhibit the replication of a homologous influenza virus strain (Flu A-reassortant VN-H5N1-PR8/CDCRG reference strain) in MDCK cells. Pre-immunization serum samples and inactivated-VN-H5N1-PR/CDC-RG 

reference strain immunized mice were used as negative and positive controls. Both VLP constructs, (3TA and 

5TA VLPs) induced antibodies able to neutralize the homologous virus strains (Fig. 4 panels 5TA and 3TA) 

suggesting that these structures displayed the proper conformation of protective antigenic sites necessary for 

inducing an antibody neutralizing response. These preliminary studies indicates that it is feasible to remodel/reengineer HA molecules by deleting the dominant antigenic regions and after immunizing mice elicit a 

neutralizing response which reduced flu virus infection of MDCK cells. While these results do not allow a 

candidate to be selected, they do provide proof of concept of the approach. In pursue of this objective, we 

propose three specific aims that are outlined below. 

3.3.2 Experimental Design and Methods 

Aim 1: Generate alternative constructs of remodeled HA 

and sub-clone them into appropriate construct for VLP 

creation. Produce and characterize virus-like particles 

(VLPs) displaying remodeled HA molecules on their 

surfaces. Evaluate the presence of conserved 

subdominant antigenic sites in HA molecules displayed 

on purified VLP by immune-precipitation using antibodies 

that exclusively recognized these sites.

In addition to the constructs described in the preliminary 

data, we plan to subclone into our expression vector 

three additional remodeled HA molecules (1TA, 2TA and 

4TA) (Fig. 5). These constructs represent alternatively 

remodeled HAs, which differ in the size of HA1 deletion, retained sequences and cysteine residues involved in 

disulfide bridges. 1TAs N-terminal sequence contains D17 to Y23 to promote the formation of an inter-chain 

disulfide bond between C20-C483 and an HA1 deletion 24-240 while retaining residues 241 to 346 linked to 

the complete sequence of the HA2 fragment, which includes the trans-membrane and cytoplasmic domains. 

The 1TA construct also contains a mutation of C290 to G to prevent potential interference with C294-C318 

disulfide bond formation, (aa numbering based on Uniprot KB database reference Q6DQ33). The 2TA 

construct encompasses a similar structure with a smaller deletion within the HA1 fragment. The 4TA construct 

encompasses the entire HA2 fragment compressing the extracellular (347-520), transmembrane, and 

cytoplasmic (521-568) domains (Fig. 5). 

Virus-Like Particle (VLP) Production 

Cell lines (CHO and Vero) that constitutively express the M1 and M2 proteins of the influenza virus have been 

constructed and will be used here. These M1 and M2 proteins are the scaffold elements for the assembly and 

release of VLP vaccines. To produce VLPs displaying remodeled HA molecules on their surfaces, these cells 

will be initially transiently transfected with plasmid vectors expressing one or another remodeled HA and NA 

genes. Those VLP constructs that demonstrate broadly protective effectiveness in the animals studies 

proposed below will be used to generate stably transfected cell lines for the continuous production of these 

type of vaccines 

VLP Vaccine Purification and Characterization 

Experimental VLPs will be purified from the culture medium five days post-transfection. We will first separate 

cells from the VLP bulk material and then concentrate the vaccine material to 1/20 of its original volume by 

tangential filtration. Concentrated vaccine material will be further purified by gradient ultracentrifugation. 

Alternative chromatographic methods including affinity and size exclusion processes are currently under 

development. Vaccine characterization and assessment of purity will be analyzed by Western blot, Coomassie 

blue and silver staining. The total protein content in the VLP vaccine will be determined using the Bradford 

assay and the HA content quantified by ELISA using a purified rHA5 as standard references and an anti-HA2 

specific antibody. The presence of a specific conserved and broadly neutralizing epitope in the purified VLPremodeled HA vaccines will be further assessed using immune-precipitation (IP) assays with two alternative 

Research Strategy Page 57

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
antibodies, one that recognizes a unique conserved site on the HA stalk- nAbs to the fusion peptide (kindly 

provided by our collaborator Dr. Ruben Donis, CDC-[11]-see letter) and a second polyclonal that exclusively 

reacts with the stem portion of the HA2 fragment. This analysis will confirm not only the presence of the 

expected remodeled HA on the VLP structure but also the presence of active antigenic sites important in 

eliciting a broadly protective response. Laboratory expression of other VLPs with non-optimized production 

systems has shown yields in the range of 50-100 mg/liter. 

Aim 2: Assess the immune response elicited in mice following immunization with candidate VLPs displaying 

alternative remodeled HA proteins on their surfaces. The capacity of immunized mouse sera to neutralize 

different influenza A virus strains will be measured by an in-vitro micro-neutralization assay. Those VLP 

vaccines capable of inducing broadly neutralizing antibodies, as demonstrated by partially or completely 

neutralizing a panel of antigenically distinct influenza viruses will be further tested by in-vivo efficacy and 

immunogenicity studies in mice. 

Those VLP constructs that show a positive Western blot for remodeled HA and reactivity in IP with at least one 

of the two testing antibodies will be further evaluated for the induction of neutralizing antibodies in mice. 

Groups of five BALB/C mice, eight weeks old, will be immunized twice, two weeks apart, via the intramuscular 

route with single VLP-remodeled HA or combination of VLP-HA-Rem vaccines. Three weeks after the booster, 

serum samples will be collected, pooled and tested in a micro-neutralization assay [21]. Serum samples from 

mice immunized with inactivated virus (see preliminary data) and pre-VLP immunization will serve as positive 

and background controls, respectively. To assess elicitation of neutralizing antibodies (nAbs), serum samples 

from immunized mice and controls will be incubated with homologous [influenza A/Vietnam/1203/2004 (H5N1) 

and Indonesia/05/2005 (H5N1), both PR8 reassortants and BSL2 agents] and heterologous viruses [influenza 

A/PR/8/34 (H1N1), A/swine/Iowa/30 (H1N1) A/Udorn/72 (H3N1), A/HK/68 (H3N2) and 

A/Mallard/Netherland/12/2000 (H7N7). Contemporaneous circulating influenza viruses [A/Perth/16/2009 

(H3N2) and A/California/07/2009 (H1N1), also available in our laboratory, could be used to further establish the 

neutralizing ability of the most promising vaccines. Selected viruses include representatives of the two major 

phylogentic groups, within the 16 HA subtypes, and correlates with the basic structure of the stalk of HA [13-

15]. Those VLP-Rem-HA vaccines that neutralize homologous and heterologous virus (cross-subtype 

neutralization) will be further tested for protective efficacy and immunogenicity in lethal challenge mouse model 

experiments. We expect that anti-HA nAbs will block infection by disrupting membrane fusion rather than 

preventing receptor binding, which is the prevailing mechanism and is readily overcome by antigenic variation. 

Aim 3: Conduct efficacy and immunogenicity studies 

of single or combination of VLP-HA Rem vaccines in a 

lethal influenza mouse model. Groups of immunized 

mice will be challenged with lethal doses of at least 

three influenza viruses subtypes (HA5, HA1 and 

HA3). Further testing of the most promising vaccine 

candidates in ferrets will be pursued in a Phase II 

SBIR proposal, which will also seek enabling data for 

an IND application with the FDA. 

Those VLP-Rem-HA vaccines that demonstrate 

elicitation of broadly nAbs in aim 2 will be tested for 

protective efficacy. These studies are designed to measure the ability of the vaccine to afford protection 

against a lethal challenge with at least three different influenza A virus subtypes. One example of the tree 

experiments follows: Forty-five female BALB/c mice (6-8 weeks old) will be randomized into 3 experimental 

treatment groups of 15 mice each; 1) VLP-HA Rem experimental vaccine, 2) Inactivated virus immunized 

control, and 3) placebo. Mice in all groups will receive two immunizations two weeks apart. The dose of the 

VLP-Rem-HA vaccine will be determined based on the results from aim 2. Blood samples for immunogenicity 

analysis will be collected before vaccination and three weeks after the booster shot. Subsequently, mice in all 

groups will be challenged with a lethal dose (10LD50) of test virus; one for each of the tree experiemtns: 

[A/Vietnam/1203/04 (H5N1), A/swine/Iowa/30 (H1N1), A/Udorn/72 (H3N2)]. 

Efficacy: The protective efficacy afforded by the VLP-Rem-HA or control vaccines will be evaluated by 

Research Strategy Page 58

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
monitoring clinical symptoms of influenza disease and protection from death. Body weight measurements and 

clinical signs of disease will be monitored daily for 18 days post-challenge at which time percent survival within 

each group will be recorded. Animals that experience greater than 20% body weight decrease or show severe 

symptoms of influenza such as labored breathing or paralysis will be euthanized. All challenge experiments will 

be performed in the animal facility of Baylor College of Medicine. This Institution has a CDC certified BSL3 

containment facility, which is required to conduct experiments with influenza A/Vietnam/1203/04 (H5N1). 

Challenge experiments with A/swine/Iowa/30 (H1N1), A/Udorn/72 (H3N2) or related strains will require BSL2 

containment, also available at Baylor. We will comply with all institutional, local, state and federal regulations 

for the execution of this work. 

Immunogenicity: The level of serum antibody elicited by the VLP-HA Rem and control immunizations will be 

evaluated by micro-neutralization [25] and ELISA assays [21, 24]. A hemagglutination inhibition (HAI) test will 

not be performed because VLP-HA Rem vaccines were designed to block membrane fusion rather than 

receptor binding, which is measured in HAI. ELISA will measure antibody titers elicited by the vaccine that 

should recognize subdominant conserved sites present in the wild type virus. Furthermore, the antineuraminidase response elicited by VLP vaccination will be evaluated by measuring the neuraminidase 

inhibition activity of serum samples [26]. Evaluation of the humoral immune response is very important 

because virus-neutralizing antibodies are the correlate of protection from influenza. Recombinant H5 protein or 

influenza A reassortant VN-H5N1-PR8/CDC-RG reference virus will be used for assessing the humoral 

response. Serum samples from two mice of each group will be pooled to assure sufficient material for the 

serological determinations which will be performed prior to immunization (pre-immunization sample) and before 

challenge (post-immunization sample.) The IgG subtypes will be assessed by ELISA and characterized as to 

the type of immune response (Th1/Th2) elicited by VLP- HA Rem vaccine and virus control. This study will 

provide information on the magnitude of the systemic response elicited by the VLP-HA Rem vaccine, in 

comparison to inactivated virus vaccines. 

Cell-mediated immunity assays will further evaluate the magnitude of the effector T-cell population stimulated 

by the different VLP-Rem-HA vaccines and controls. Interferon γ (IFN-γ) production in splenocytes of five mice 

per group will be measured by ELISPOT assay [27]. Reassortant virus or purified antigen will be used to restimulate splenocytes during the performance of the ELISPOT assay. There is considerable precedence for the 

significance of this measurement, which not only provides further information about the type of immune 

stimulation elicited, but also reflects on the induction of effector T-cell function [28,29]. Also, nasal and 

bronchial lavage will be collected to assess the presence of antibody (methods above) in the mucosal surface. 

3.4. Potential Problems and Alternative Solutions 

While our constructs elicit nAbs, they may not be sufficiently potent to be clinically useful as standalone 

vaccines. If necessary we will test whether VLPs enhance protection elicited by a standard flu vaccine. Testing 

this vaccine together with an adjuvant also will be considered. Proper folding and expression of all remodeled 

HAs is a concern; however, WB and IP assays aim to prove that VLPs display remodeled HA revealing 

conserved antigenic sites. Also, Dr. Zhang, a structural biologist and protein chemist in the Department of 

Biochemistry, New York Medical College, will use a structural and computational modeling approach to create 

new remodeled HA or improve the properties of the one being study. Selection of the HA5 molecule for 

remodeling is based on the data obtained with the isolated human broadly nAbs [11]. This work shows that the 

most broadly neutralizing Ab does not block infection with all 16HAs; therefore we expect to recreate several 

conserved epitopes in one or more of the VLP constructs in order to cover more serotypes. Furthermore, other 

HAs (e.g. H3, H7), which were no neutralized by the human antibody could be remodeled to broaden the 

neutralizing power of a VLP vaccine. Success with this strategy will provide the foundation to implement a 

similar approach to develop a broadly 

protective flu B vaccine. Completion of these 

studies and identification of candidate 

vaccines will provide the data to support a 

Phase II SBIR application. 

3.5. Timelines 

As per the Activities & Milestones chart. 

Research Strategy Page 59

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Vertebrate Animals 

Immunogenicity and efficacy studies proposed in this application will be carried out in the animal facility 

of Baylor College of Medicine, which includes a BSL-3E Enhanced Satellite Facility. This enhanced 

BSL-3 facility is designed to house small animal models with containment standards and requirements 

defined in the CDC/NIH publication “Biosafety in Microbiological and Biomedical Laboratories (BMBL)”, 

5th Edition, 2007 for animal BSL-3 (ABSL-3) space plus recommended enhancements (ABSL-3E). The 

facility is certified by the CDC and APHIS Agricultural Select Agents Programs and the BCM Select 

Agent Registration on file with the CDC. Dr. Mbawuike, Co-Investigator in this project and his staff 

have access to the BCM BSL-3/3E Select Agent facility and have completed the required BSL-3 

training program offered by UTMB Environmental Health Safety Office. Dr. Mbawuike has completed 

federally mandated security risk assessment, and has been added to the Select Agent Registration on 

file with the CDC. Work with the influenza A/Vietnam/1203/04 (H5N1) will be carried out in this facility 

by Dr. Mbawuike and his team. 

Other influenza viruses plan to be used in these studies [influenza A/Swine/Iowa/30 (H1N1) virus, 

A/Udorn/72 (H3N2), or possibly A/HK/68 (H3N2), A/Mallard/Netherland/12/2000 (H7N7),

A/Perth/16/2009 (H3N2) and A/California/07/2009 (H1N1)], are BSL-2 agent and the BCM Facility also 

meet requirement for handling this agent. 

The facility is fully accredited by the American Association for Laboratory Animal Science (AALAS). 

Veterinarians and staff provide oversight of animal health and well being guidance and assistance with 

veterinary medical and surgical techniques, and the services of disease surveillance, diagnosis and 

treatment, animal husbandry and nutrition, zoonosis control, hazard containment, and equipment and 

room sanitation. Additional staff assistance includes consultation with researchers on handling, 

restraint, anesthesia, analgesia and euthanasia. 

Animals will be euthanized in accordance to the IACUC approved guidelines established by the 

American Veterinary Medical Association, so as to minimize pain or discomfort in the animal, which 

results in the rapid unconsciousness followed by cardiac or respiratory arrest and ultimate loss of brain 

function. Euthanasia of animals is expected if animals demonstrate these clinical conditions (not 

inclusive): rapid, shallow, and labored respiration, prolonged inappetence, pyrexia, loss of 20-25% body 

weight, and central nervous system disturbances. 

Mouse model 

BALB/c mice (6-8 weeks old, female) will be used to evaluate immunogenicity and efficacy of VLP-HA 

Rem vaccines. Groups of mice (as described in the research plan) will receive two doses of VLP-HA 

Rem or controls via IM route. Prior to any procedures, animals will be anesthetized with a combination 

of ketamine (70mg/kg) and xylazine (6mg/kg) (~volume 100μl) administered by intramuscular injection. 

Blood samples will be collected from the submandibular vein (as suggested by a prior reviewer). Mice 

will be euthanized by inter-peritoneal (IP) administration of sodium pentobarbital (50-90mg/kg) and 

cervical dislocation as a secondary procedure to ascertain death. 

Prior to the efficacy studies (Aim: 3), we will assess the immune response elicited in mice following 

immunization with experimental VLP–HA Rem vaccines (Aim: 2). Immunization and serum samples 

collection will be performed at Baylor and micro-neutralization test at TechnoVax, Inc. 

The total number of mice proposed in these studies is: ~250 (25 mice for aim 2 and 225 for aim 3) 

Vertebrate Animals Page 60

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Statistical Analysis 

In the design of this study, considerations for determining animal sample size were based on several 

factors to minimize the number of animals required to achieve the objectives of the study while 

maintaining the statistical significance of the data. The appropriate number of animals for aim 3 was 

based on the following factors: the difference in means between two groups (effect size), standard 

deviation (variability of data), significance level (α) and power (1- β). The significance level was set at 

alpha = 0.05 and the desired power was set at 80% (β = 0.2). The other factors (effect size and 

deviations) were based on our previous studies. Student t test (two tailed, unpaired) in the Excel 

program will be used to compare antibody levels between two groups. In addition, ANOVA test will 

used to perform simple analysis of variance to test the means of antibody titers among several sample 

groups. For all analyses, a P value of less than 0.05 derived from a two-tailed test will be considered 

significant. 

Vertebrate Animals Page 61

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Select Agents Research 

Avian influenza H5N1 challenge studies will be conducted BCM BSL-3E Enhance Satellite 

Facility located in the basement of Garage 6. This enhanced BSL-3 facility is designed to house 

small animal models with containment standards and requirements defined in the CDC/NIH 

publication “Biosafety in Microbiological and Biomedical Laboratories (BMBL)”, 5th Edition, 2007 

for animal BSL-3 (ABSL-3) space plus recommended enhancements (ABSL-3E). The facility is 

certified by the CDC and APHIS Agricultural Select Agents Programs and the BCM Select 

Agent Registration on file with the CDC. Dr. Mbawuike and his staff have access to the BCM 

BSL-3/3E Select agent facility and have completed the required BSL-3 training program offered 

by UTMB Environmental Health Safety Office. Dr. Mbawuike has an approved rDNA and/or 

HA/HC protocol, have completed federally mandated security risk assessment, and have been 

added to our Select Agent Registration on file with the CDC 

Experiments with the Influenza A/Swine/Iowa/30 (H1N1), and A/Udorn/72 (H3N2), will be 

performed at the Baylor College of Medicine Animal Facility under BSL-2 bio-containment. 

Select Agent Research Page 62

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
Bibliography 

1. NIFID, http://www.nfid.org/influenza/index.html. 2008. 

2. CDC, http://www.cdc.gov/flu/weekly/. 2011. 

3. Dawood, F.S., et al., Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N 

Engl J Med, 2009. 360(25): p. 2605-15. 

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Infect Dis, 2007. 195(7): p. 1018-28.

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6. Kandun, I.N., et al., Three Indonesian clusters of H5N1 virus infection in 2005. N Engl J Med, 

2006. 355(21): p. 2186-94. 

7. Sorrell, E.M., et al., Predicting 'airborne' influenza viruses: (trans-) mission impossible? Curr 

Opin Virol, 2011. 1(6): p. 635-42. 

8. Fouchier, R.A., A. Garcia-Sastre, and Y. Kawaoka, Pause on avian flu transmission studies. 

Nature, 2012. 481(7382): p. 443. 

9. Imai, M., et al., Experimental adaptation of an influenza H5 HA confers respiratory droplet 

transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature, 2012. 486(7403): p. 420-8.

10. Herfst, S., et al., Airborne transmission of influenza A/H5N1 virus between ferrets. Science, 

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11. Sui, J., et al., Structural and functional bases for broad-spectrum neutralization of avian and 

human influenza A viruses. Nat Struct Mol Biol, 2009. 16(3): p. 265-73.

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MBio, 2010. 1(1). 

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Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature, 1981. 

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18. Wilson, I.A., J.J. Skehel, and D.C. Wiley, Structure of the haemagglutinin membrane 

glycoprotein of influenza virus at 3 A resolution. Nature, 1981. 289(5796): p. 366-73.

19. Kwong, P.D. and I.A. Wilson, HIV-1 and influenza antibodies: seeing antigens in new ways. Nat 

Immunol, 2009. 10(6): p. 573-8.

20. Galarza, J.M., T. Latham, and A. Cupo, Virus-like particle (VLP) vaccine conferred complete 

protection against a lethal influenza virus challenge. Viral Immunol, 2005. 18(1): p. 244-51.

21. Matassov, D., A. Cupo, and J.M. Galarza, A novel intranasal virus-like particle (VLP) vaccine 

designed to protect against the pandemic 1918 influenza A virus (H1N1). Viral Immunol, 2007. 20(3): p. 

441-52.

22. Schiller, J.T., Frazer, I. H., and D.R. Lowy, Human papillomavirus vaccines. In: Plotkin SA, 

Orenstein WA, Offit PA. eds Vaccines (Saunders Elsevier) 5th Ed., 2008: p. 243-257.

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27. Anthony, D.D. and P.V. Lehmann, T-cell epitope mapping using the ELISPOT approach. 

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28. Barouch, D.H. and N.L. Letvin, CD8+ cytotoxic T lymphocyte responses to lentiviruses and 

herpesviruses. Curr Opin Immunol, 2001. 13(4): p. 479-82.

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stem of influenza virus hemagglutinin. Jour. of Virol, 2012, Vol. 86 (21): p. 11686-11697.

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References Cited Page 64

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
RESOURCES SHARING PLAN 

It is the intent of TechnoVax to share the data generated during this research with NIH 

and by publishing in peer reviewed journals. Prior to disclosure, protection of patentable 

information will be established.

Resource Sharing Plan Page 69

Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.
PHS 398 Checklist

OMB Number: 0925-0001 

1. Application Type:

From SF 424 (R&R) Cover Page. The responses provided on the R&R cover page are repeated here for your reference, as you answer 

the questions that are specific to the PHS398.

* Type of Application:

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2. Change of Investigator / Change of Institution Questions

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* Inventions and Patents:

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4. * Program Income

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and 

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If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name, 

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Principal Investigator/Program Director (Last, first, middle): Galarza, Jose, M.