PI: Brooks, Benjamin Delbert Title: High-throughput, multiplexed characterization and modeling of 

antibody:antigen binding, with application to HSV

Received: 09/06/2016 FOA: PA16-302 Council: 01/2017

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

ACF FOR SMALL BUSINESS INNOVATION RESEARCH GRANT APPLICATIONS 

(PARENT SBIR [R43/R44])

1 R43 AI132075-01 Dual: GM Accession Number: 3968518

IPF: 10005562 Organization: WASATCH MICROFLUIDICS

Former Number: Department:

IRG/SRG: ZRG1 IMST-K (14)B AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Animals: N 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Benjamin Brooks Ph.D WASATCH MICROFLUIDICS PD/PI 

Chris Bailey-Kellog Dartmouth College MPI

GARY COHEN University of Pennsylvania MPI

Roselyn Eisenberg University of Pennsylvania MPI

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

demonstrates good grantsmanship, time has passed since the grantee applied. The samples may not 

reflect the latest format or rules. NIAID posts new samples periodically:

https://www.niaid.nih.gov/grants-contracts/sample-applications

The text of the application is copyrighted. The awardee provided express permission for NIAID to post

this grant application and summary statement for educational purposes. The awardee allows you to use

the material (e.g., data, writing, graphics) they shared in the applications for nonprofit educational 

purposes only, provided the material remains unchanged and the principal investigators, awardee 

organizations, and NIH NIAID are credited.

Freedom of Information Act (FOIA). NIAID is strongly committed to protecting the integrity and

confidentiality of the peer review process. When NIH responds to FOIA requests for grant applications and

summary statements, the material will be subject to FOIA exemptions and include substantial redactions. 

NIH must protect all confidential commercial or financial information, reviewer comments and

deliberations, and personal privacy information.

Note on Section 508 Conformance and Accessibility. We have reformatted this sample to improve

accessibility for people with disabilities and users of assistive technology. If you have trouble accessing the 

content, contact the NIAID Office of Knowledge and Educational Resources at deaweb@niaid.nih.gov.
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

3. DATE RECEIVED BY STATE State Application Identifier

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

❍ Pre-application ● Application ❍ Changed/Corrected

Application

b. Agency Routing Number

2. DATE SUBMITTED

2016-09-01

Application Identifier c. Previous Grants.gov Tracking Number

5. APPLICANT INFORMATION Organizational DUNS*: 

Legal Name*: WASATCH MICROFLUIDICS

Department:

Division: Wasatch Microfluidics

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Person to be contacted on matters involving this application

Prefix: Prof. First Name*: Benjamin Middle Name: Last Name*: Brooks Suffix: 

Position/Title:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email: 

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

7. TYPE OF APPLICANT* R: Small Business

Other (Specify):

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

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

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

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

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

9. NAME OF FEDERAL AGENCY*

National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

High-throughput, multiplexed characterization and modeling of antibody:antigen binding, with application to HSV

12. PROPOSED PROJECT

Start Date* Ending Date*

07/01/2017 06/30/2018

13. CONGRESSIONAL DISTRICTS OF APPLICANT

UT-002
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

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

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: Benjamin Middle Name: Delbert Last Name*: Brooks Suffix: Ph.D

Position/Title: Vice President of Research

Organization Name*: WASATCH MICROFLUIDICS

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested* $

b. Total Non-Federal Funds* $0.00

c. Total Federal & Non-Federal Funds* $

d. Estimated Program Income* $0.00

16. IS APPLICATION SUBJECT TO REVIEW BY STATE

EXECUTIVE ORDER 12372 PROCESS?*

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

DATE:

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

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

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

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

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

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

● I agree*

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

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE

Prefix: First Name*: Josh Middle Name: Last Name*: Eckman Suffix: 

Position/Title*: President

Organization Name*: Wasatch Microfluidics

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

Signature of Authorized Representative*

Josh Eckman

Date Signed*

09/06/2016

20. PRE-APPLICATION File Name: Mime Type:

21. COVER LETTER ATTACHMENT File Name:Cover_Letter_PA-16-302.pdf Mime Type: application/pdf
424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

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

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

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

Research & Related Other Project Information------------------------------------------------------------------ 6

 Project Summary/Abstract(Description)----------------------------------------------------- 7

 Project Narrative------------------------------------------------------------------------- 8

 Facilities & Other Resources-------------------------------------------------------------- 9

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

 Other Attachments------------------------------------------------------------------------- 14

 CBK_-_Personnel_Justification_PA-16-302------------------------------- 14

 SBC_000348985--------------------------------------------------------- 15

Research & Related Senior/Key Person-------------------------------------------------------------------------- 16

Research & Related Budget Year - 1---------------------------------------------------------------------------- 39

Budget Justification------------------------------------------------------------------------------------------ 42

Research & Related Cumulative Budget-------------------------------------------------------------------------- 45

Research & Related Budget - Consortium Budget (Subaward 1)---------------------------------------------------- 46

Research & Related Budget - Consortium Budget (Subaward 2)---------------------------------------------------- 51

Total Direct Costs Less Consortium F&A------------------------------------------------------------------------ 56

SBIR STTR Information----------------------------------------------------------------------------------------- 57

 Research & Development Outside the U.S.--------------------------------------------------- 59

 Prior SBIR Phase II Awards---------------------------------------------------------------- 61

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 62

PHS 398 Research Plan----------------------------------------------------------------------------------------- 64

 Specific Aims----------------------------------------------------------------------------- 65

 Research Strategy------------------------------------------------------------------------- 66

 Multiple PD/PI Leadership Plan------------------------------------------------------------ 72

 Bibliography & References Cited----------------------------------------------------------- 73

 Letters of Support------------------------------------------------------------------------ 75

Contact PD/PI: Brooks, Benjamin Delbert

Table of Contents Page 3
Contact PD/PI: Brooks, Benjamin Delbert

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

Project/Performance Site Location(s)

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

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

organization.

Organization Name: Wasatch Microfluidics

Duns Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*: UT-002

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

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

organization.

Organization Name: Dartmouth College

DUNS Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*:

Project/Performance Site Location 2 ❍ I am submitting an application as an individual, and not on behalf of

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

organization.

Organization Name: Trustees of the University of Pennsylvania

DUNS Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*: PA-002

 Page 4
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

File Name Mime Type

Additional Location(s)

Contact PD/PI: Brooks, Benjamin Delbert

 Page 5
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

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

1.a. If YES to Human Subjects

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

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

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

IRB Approval Date:

Human Subject Assurance Number

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

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ❍ Yes ❍ No

IACUC Approval Date:

Animal Welfare Assurance Number

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

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

4.b. If yes, please explain:

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

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

❍ Yes ❍ No

4.d. If yes, please explain:

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

5.a. If yes, please explain:

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

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* Abstract_PA-16-302.pdf

8. Project Narrative* Narrative_PA-16-302.pdf

9. Bibliography & References Cited References_v2_PA-16-302.pdf

10.Facilities & Other Resources Facilities_PA-16-302.pdf

11.Equipment Equipment_PA-16-302.pdf

12. Other Attachments CBK_-_Personnel_Justification_PA-16-302.pdf

SBC_000348985.pdf

Contact PD/PI: Brooks, Benjamin Delbert

 Page 6
Abstract. All therapeutic antibodies and most vaccines critically depend on the ability of 

antibodies to specifically recognize particular antigens; consequently, detailed characterization of 

antibody:antigen binding can provide invaluable information to understand and guide 

development. Unfortunately, due to the time and expense required, atomic resolution structure 

determination is typically used sparingly, late in a development process or for a small number of 

different antibodies or antigen variants. We seek to enable earlier and larger-scale, but still 

detailed, characterization and modeling of antibody:antigen binding, applicable to panels of 

antibodies that could result from screening polyclonal samples or engineered libraries, along with 

panels of antigens that could result from attempts to understand and account for diversity across 

populations. While not at atomic resolution, our approach will still allow residue-level localization 

of specific epitopes for specific antibodies, as well as group-level identification of functionally 

similar antibodies and their associated binding regions on the antigen. The approach will be 

enabled by a unique integration of a powerful experimental platform, the high-throughput 

multiplexed Wasatch Surface Plasmon Resonance (SPR), with powerful computational methods 

to design and analyze binding experiments. Studies of glycoprotein D (gD) of herpes simplex virus 

(HSV) will provide a solid foundation for developing, testing, and applying the technology to better 

understand critical differences across antibodies and antigenic variation. Ultimately, the 

approaches developed here will allow researchers to leverage extensive epitope characterization 

data generated with Wasatch’s SPR instrument in order to broadly and deeply characterize the 

basis for antibody:antigen recognition in wide-ranging vaccine and therapeutic antibody discovery 

and development programs. 

Contact PD/PI: Brooks, Benjamin Delbert

Project Summary/Abstract Page 7
Project Narrative

Detailed characterization of antibody:antigen binding is fundamental to understanding and 

potentially improving mechanisms of action of biotherapeutics and vaccines. Here, in order to 

support such characterization for large panels of related antibodies and antigen variants, 

computational design and analysis methods will be integrated with a high-throughput multiplexed 

experimental platform, enabling the overall grouping of antibodies by binding preferences as well 

as the detailed localization of particular antibody epitopes. By enabling a rich analysis at much 

higher throughput than traditional structural studies, this approach promises to better drive 

discovery and development of vaccines and therapeutic antibodies.

Contact PD/PI: Brooks, Benjamin Delbert

Project Narrative Page 8
Facilities, 

Wasatch Microfluidics, University of Pennsylvania, and Dartmouth College have the necessary facilities to 

complete this research and development project. A brief summary of their equipment and facilities follows: 

Wasatch Microfluidics Facilities 

General: The Wasatch facility has 5,000 sq.ft. of modern laboratory, manufacturing and office space in Salt 

Lake City, UT with room to grow up 2,000 sq. ft. if necessary.

Laboratory: Wasatch Microfluidics has 2,000 sq.ft. of research and development labs with lab benches, fume

hoods, purified water, desk space and network connections. Our manufacturing and engineering labs are 

equipped with electro-optical test, signal processing tools, and ample power and workspace for the proposed 

work. 

Computer: Wasatch has personal computers for all of the personnel. Lab computers are also used to run the 

detection systems and for data analysis. Wasatch also has Visual Studio and a cloud-based, Team Foundation 

Server (TFS) for all software projects to handle versioning, feature, and bug tracking. 

Office: Our new facility has 3,000 sq.ft. of office space including a reception area, break room and conference 

room. Individual offices are equipped with standard office furniture as well as phone and internet jacks. 

Machine Shop: Wasatch has access an agreement to a small machine shop for rapid prototyping.

Libraries: The Library at the University of Utah is 15 minutes from the Company’s facility. 

Wasatch also has an embedded employee at the University of Pennsylvania Dental school in the labs 

of Gary Cohen and Roselyn Eisenberg.

Dartmouth College, Bailey-Kellogg Facilities

Laboratory:

N/A

Computer:

The Bailey-Kellogg group has an extensive array of individual and shared computing resources. Each group 

member has a laptop and a workstation. For computationally demanding applications, the group uses the 

Computer Science department’s GridIron compute cluster, which thanks to a recent NSF infrastructure grant 

currently includes over 100 high-performance nodes totaling over 1200 cores, with over 4000GB of distributed 

RAM and a high-throughput filesystem with over 200TB of storage. We also have shared access to the 

Discovery cluster maintained by the university; Discovery is a world-class Linux supercomputer with over 2400 

cores (constantly expanding), supported by staff dedicated to research computing. Dartmouth provides a 

campus-wide wired network with a gigabit backbone and minimum 100 megabit service to researchers. 

Dartmouth also covers the entire campus with wireless access points.

Office:

The new wing of the Computer Science building houses an office for the PI, shared (two-person) offices for his 

postdocs and graduate students, and a 900 ft2 computer lab / meeting space for his group.

Clinical:

N/A

Contact PD/PI: Brooks, Benjamin Delbert

Facilities & Other Resources Page 9
University of Pennsylvania, Cohen/Eisenberg Facilities 

Laboratory: The labs are in the Levy Building, rooms 212, 215, 218, 225 & 233.The labs are 400 to 600 sq.ft 

each, close to each other and adequate to house the investigators and the support staff. The Department of 

Microbiology provides secretarial services, microbiology kitchen and dishwashing facilities as well as 

incubation and cold rooms, common equipment rooms and a darkroom. The library is located in the adjacent 

Evans building. 

Clinical: N/A 

Office 

There are four offices available. Rooms 213 and 216 are for Drs. Cohen and Eisenberg respectively. Rooms 

217 and 233 have desks available for students, postdocs and Research Specialists. Room 214 is equipped

Contact PD/PI: Brooks, Benjamin Delbert

Facilities & Other Resources Page 10
with a PC and Macintosh computer for common use. It also has a color printer and scanner for common use. 

Multiple Macintosh and PC computers are available in each of our labs and offices. All are equipped with 

programs for routine and scientific applications. We have black and white and color printers, scanners and 

LCD projectors. 

Scientific Environment 

Our laboratory space is in adjoining rooms near the offices within approximately 2500 square feet of space. 

Our confocal microscope is housed in a specially equipped darkroom. Room 233 contains clean air hoods and 

C02 incubators and microscopes, etc., for cell and virus culture. We maintain a baculovirus facility within the 

Microbiology department of the Dental School in Room 225. Equipment includes 2 New Brunswick Celligen 

Bioreactors for insect culture under precise conditions of pH, gases and temperature. These are equipped with 

5L vessels and are used routinely for preparing larger amounts of protein. We also have multiple specially 

designed spinners for preparing smaller batches of insect cells; There are 2 low temperature incubators 

dedicated to insect cell-baculovirus incubation, a cell culture hood, tangential flow equipment, centrifuge, 

microscope and FPLC for protein purification. There are multiple CORE facilities in the Levy Building including 

FACS, EM, Confocal microscope etc. The University of Pennsylvania and Wistar provide access to multiple 

CORE facilities. We have all of the equipment and facilities needed to carry out the research in this grant. The 

Wasatch Continuous Flow Microspotter (CFM) coupled to the Ibis MX96 imager is now in our laboratory on 

long term loan.

Contact PD/PI: Brooks, Benjamin Delbert

Facilities & Other Resources Page 11
Contact PD/PI: Brooks, Benjamin Delbert

Facilities & Other Resources Page 12
Equipment 

Wasatch Microfluidics, Dartmouth College, and University of Pennsylvania have the necessary equipment to 

complete this research and development project. 

Wasatch Microfluidics Equipment. 

Lab equipment: Wasatch is equipped with centrifuges, biosafety cabinet, CO2 incubators, vacuum pumps, 

and an Leica, inverted microscope with phase contrast, brightfield, and fluorescent capabilities (expandable if 

necessary). For storage, Wasatch has access to a cryogenic dewar, refrigerators, and a –80 oC freezer. 

Wasatch has access to three CFMs for cell or biomolecule printing. Wasatch owns two IBIS MX96 SPR 

imagers (one collocated at UPenn in Cohen/Eisenberg lab) and prototypes of our current SPR sensors. 

Chemistry equipment: Chemical/fume hoods are available. A water bath, digital thermometers, pH meters, 

centrifuges, water purification, and other basic lab equipment are also located in the facility. 

Microfabrication equipment: Two microscopes with attached cameras/video are available in the lab. The 

lab also has a small manufacturing area for producing the PDMS printheads which includes: injection molding 

setup, ovens, vacuum chambers, vacuum pumps, and mold making tools. Specialized chemistry facilities 

required to test the MFCA / SPR platform application testing is available. Wasatch has an established 

cleanroom manufacturing facility for the CFM printheads, which can also be used for some R&D efforts when a 

clean area is necessary. Wasatch also has a small milling machine. 

Office, computer, and general business space are also available to all participants in this project. 

University of Pennsylvania, Cohen and Eisenberg Equipment. 

Within the Dental School Microbiology department (housed in the Leon Levy Building) we have: two 

ultracentrifuges: a Beckman L8M70 and an Optima L90K ultracentrifuge, a Sorvall RC26+ high speed 

centrifuge, two Beckman GPR centrifuges, several microcentrifuges, ultra-low temperature freezers, liguid 

nitrogen apparatus, FPLC equipment, including a variety of columns, monitors and fraction collectors, 

Pharmacia spectrophotometer, SDSPAGE gel equipment, Biotek Synergy Bioassay plate reader, for ELISA, 

Fluorescence& Luminescence and a new Li-Cor Odyssey Infrared imaging system. We have a full tissue 

culture laboratory, including three biological hoods, and 4 incubators. We have developed a full state-of-the-art 

Baculovirus laboratory to grow insect cells for recombinant baculovirus infection that enables us to prepare and 

purify large amounts proteins. The equipment includes two Celligen bioreactors, 2 low temperature incubators, 

tangential flow apparatus, etc. We have a full SPR laboratory that includes a BiaCore T100 Biosensor & 

BIACore 3000, and a MicroCal ITC200. In our laboratory, we have the use of the WM Continuous Flow 

Microspotter (CFM) coupled to the Ibis MX96 imager for doing biosensor studies of larger numbers of samples 

to augment the BIAcore 3000.The Levy building has a state of the art FACS facility, a JOEL EM and a Leica 

histochemistry facility. All are available to UPENN members. We have access, within the Levy Bldg, and on 

campus to most other equipment needed to perform the proposed studies. 

Contact PD/PI: Brooks, Benjamin Delbert

Equipment Page 13
Personnel Justification 

Prof. Chris Bailey-Kellogg (.25 Summer Months) will oversee all computational components of 

the project, advising the Dartmouth PhD student and working together with the Wasatch 

programmer. We will research, design, develop, and implement methods to analyze Wasatch 

SPR data in order to define antibody communities and localize antibody epitopes. We will work 

with the other investigators to apply the methods to the study of antibody:antigen binding in 

HSV. 

Dartmouth PhD student (TBD, 9 Calendar Months) will lead the research effort into new 

methods for analyzing Wasatch SPR data for antibody:antigen binding analysis. 

Contact PD/PI: Brooks, Benjamin Delbert

CBK_-_Personnel_Justification_PA-16-302 Page 14
Contact PD/PI: Brooks, Benjamin Delbert

SBIR.gov SBC Registration Control ID Form

 SBC CONTROL ID SBC_000348985

 FIRM INFORMATION

 Company Wasatch Microfluidics, LLC

 Address 4909 BROWN VILLA COVE

 City SALT LAKE

CITY

 State UT Zip 84123-

 TIN/EIN DUNS 

 Company URL 

 Number of Employees: 5

 Is this SBC majority-owned by multiple venture capital operating companies,

 hedge funds, or private equity firms?

 No

 What percentage (%) of the SBC is majority-owned by multiple venture capital

 operating companies, hedge funds, or private equity firms?

 0%

 SBC CONTROL ID SBC_000348985

Powered by TCPDF (www.tcpdf.org)

Generated June 17, 2013 | U.S. Small Business Association | SBIR.gov

SBC_000348985 Page 15
Contact PD/PI: Brooks, Benjamin Delbert

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Benjamin Middle Name Delbert Last Name*: Brooks Suffix: Ph.D

Position/Title*: Vice President of Research

Organization Name*: WASATCH MICROFLUIDICS

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

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

Degree Type:  Degree Year: 

Attach Biographical Sketch*:             File Name: B_BrooksBioSketch_v2.pdf

Attach Current & Pending Support:   File Name: Pending_SUpport_BB.pdf

 Page 16
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

PROFILE - Senior/Key Person

Prefix:  First Name*: Chris Middle Name  Last Name*: Bailey-Kellog Suffix: 

Position/Title*: Professor

Organization Name*: Dartmouth College

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

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

Degree Type:  Degree Year: 

Attach Biographical Sketch*:             File Name: cbk-biosketch.pdf

Attach Current & Pending Support:   File Name: cbk-support.pdf

PROFILE - Senior/Key Person

Prefix:  First Name*: GARY Middle Name H Last Name*: COHEN Suffix: 

Position/Title*: Professor

Organization Name*: University of Pennsylvania

Department:

Division:

Street1*: UNIVERSITY OF PENNSYLVANIA

Street2: DEPT OF MICROBIOLOGY

City*: PHILADELPHIA

County:

State*: PA: Pennsylvania

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 191046030

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

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

Degree Type:  PHD Degree Year: 

Attach Biographical Sketch*:             File Name: GHC_Biosketch_April_SBIR.pdf

Attach Current & Pending Support:   File Name: GC_Support_PA-16-302.pdf

 Page 17
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

PROFILE - Senior/Key Person

Prefix:  First Name*: Roselyn Middle Name J Last Name*: Eisenberg Suffix: 

Position/Title*: PROFESSOR OF MICROBIOLOGY

Organization Name*: University of Pennsylvania

Department:

Division:

Street1*: Department of Pathobiology

Street2: School of Veterinary Medicine

City*: Philadelphia

County:

State*: PA: Pennsylvania

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 191046002

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

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

Degree Type:  PHD,BA Degree Year: 

Attach Biographical Sketch*:             File Name: RJE_HSV_Biosketch_SBIR.pdf

Attach Current & Pending Support:   File Name:

 Page 18
OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

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

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

NAME: Benjamin D. Brooks

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

POSITION TITLE: Vice President of Research & Development

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

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

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

University of Wyoming, Laramie, WY B.S. 08/1999 Mathematics

University of Wyoming, Laramie, WY MBA 08/2002 Business Administration

University of Wyoming, Laramie, WY PhD 12/2008 Molecular Biology

Scripps Research Institute, San Diego, CA Visit. Grad 

Student

12/2007 Immunology

University of Utah, Salt Lake City, UT Postdoc 10/2009 Pharmaceutics

A. Personal Statement

Wasatch Microfluidics’ is in the product development stage for engineering a label-free, Surface Plasmon 

Resonance based biosensor with integrated, dual-switching, next generation, flow cells for bioanalytical 

characterization of drug candidates. In 2011, while working on a microarray project at Grainger/Brooks lab at 

the University of Utah in Salt Lake City, Utah, using traditional pin printing, I began to work on Wasatch’s 

Continuous Flow Microspotter (CFM) for printing biomolecules. After the completion of my postdoctoral 

fellowship, I joined Wasatch to head their R&D efforts to facilitate both the label-free and the cell printing 

product development. My scientific expertise in developing immunoassays, surface functionalization, and 

surface property measurement will be invaluable for these products. In addition to my scientific skills and 

knowledge, this project will also draw on my MBA and practical business experience in driving technology 

towards solving real world clinical problems, a challenge I welcome. Lastly, I have extensive experience

managing software project both academically (see reference #5 in the SPR, Protein Microarrays, and 

Immunoassays section) and commercially.The team driving this project forward involves world class 

academicians, clinicians, and business professionals within Wasatch Microfluidics as well as in Gary Cohen’s 

group at UPenn and Biosensor Tools, all well prepared to create the foundation for a quantum leap in 

biosensor design and cell printing for biochemical screening applications through the development of a highthroughput, 3D printer with integrated imaging systems. 

B. Positions and Honors

Positions

2012 – Present VP, Research and Development, Wasatch Microfluidics, Inc., Salt Lake City, UT

2009 – 2012 Postdoctoral Fellow, Dept. of Pharmaceutical Chemistry, University of Utah, Salt Lake City, 

UT

2006 – 2008 Visiting Graduate Student, The Scripps Research Institute, Sand Diego, CA

2000 – 2003 Instructor, Dept. of Computer Science and Business Technology, Casper College, Casper, 

WY

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 19
Honors

2003 Gamma Sigma Delta Honor Society of Agriculture

1995 Mortar Board and Golden Key Honor Societies

C. Contribution to Science

My contributions to science fall into four categories; immunoassays, nanoparticles, cell printing, and antibiotic 

release. During my graduate work, I had the privilege of working on multiple immunoassays formats including, 

ELISAs, both traditional and nanoparticle based, immunoblotting, and protein microarrays, in an attempt to 

develop an immunoassay to prion protein. During my postdoc, I had the privilege to work for Dr. David 

Grainger, an expert in surface chemistry and Dr. Amanda Brooks, an expert in drug delivery. In the 

Grainger/Brooks lab, I managed multiple graduate student projects relating to nanoparticles and 

immunoassays. As a new investigator at Wasatch, I work currently on SPR microarrays and cell printing.

SPR, Protein Microarrays, and Immunoassays

The development of a high-throughput, SPR sensor drives a majority of my current research and will represent 

a major advancement in label-free characterization of protein binding. At Wasatch we are combining our 

advanced microfluidics with industry standard SPR technologies to improve throughput over current 

technologies. Prototypes of this technology predict a major advancement in the drug discovery and 

development field. 

1. “Surface Plasmon Resonance Arrays for Antibody Characterization.” Label-free biosensor methods in 

drug discovery. Label-Free Biosensor Methods in Drug Discovery (Book Chapter in Springer Protocols 

Book Series.) [2015: 35-76]. SN Davidoff, NT Ditto, AE Brooks, JW Eckman, BD Brooks.

2. 

3. “The Importance of Epitope Binning for Biological Drug Discovery.” Current Drug Discovery Technologies.

[2014/06, 11(2):109]. BD Brooks.

4. “High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge!” 

Drug Discovery Today. [2014/08, 19(8): 1040–1044] BD Brooks, AR Miles, YN Abdiche.

5. “Identification of problems developing an ultrasensitive immunoassay for the ante mortem detection of the 

infectious isoform of the CWD-associated prion protein.” Journal of Immunoassay and Immunochemistry.

30 (2), 135-149. BD Brooks, A Brooks, SS Wulff, RV Lewis

Cell Printing

Cell printing represents an emerging field in drug discovery and development. Controlled delivery of cells 

would represent a major advancement for the screening and toxicity characterization of drug development. 

Wasatch’s 3D flow cells allows scientist the ability to print cells in a submerged environment and in a 

microarray format. Numerous advances in this area are forthcoming.

1. “Maximizing fibroblast adhesion on protein-coated surfaces using microfluidic cell printing.” RSC Advances 

5 (126), 104101-104109. SN Davidoff, D Au, BK Gale, AE Brooks, BD Brooks.

2. “The Submerged Printing of Cells onto a Functionalized Surface Using a Continuous Flow Microspotter.” 

Journal of Visualized Experiments. [2014, 86: e51273-e51273]. SN Davidoff, BD Brooks.

3. “Comparison of Submerged and Unsubmerged Printing of Ovarian Cancer Cells.” Biomedical sciences 

instrumentation. [2015, 51: 24-30]. SN Davidoff, D Au, S Smith, AE Brooks, BD Brooks.

Antimicrobial Controlled Release

Antibiotics remain one of the greatest advances in modern medicine. With the emergent threats of antibiotic 

resistance, the field has been reinvigorated. Controlled and local release will become an important tenant of 

future delivery of the drug. I have been on the forefront of pushing for localized, surface delivery of drugs with 

an emphasis on bone applications.

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 20
1. “A Resorbable Antibiotic-Eluting Polymer Composite Bone Void Filler for Perioperative Infection Prevention 

in a Rabbit Radial Defect Model.” PLOS One. [In Press]. BD Brooks, KD Sinclair, DW Grainger, AE 

Brooks. 

2. “Therapeutic Strategies to Combat Antibiotic Resistance.” Advanced Drug Discovery Reviews. Volume 78, 

30 November 2014, Pages 14–27. BD Brooks, AE Brooks.

3. "Antimicrobial medical devices in preclinical development and clinical use." 2012 Springer Book Chapter in 

Biomaterial Associated Infection. BD Brooks, AE Brooks, DW Grainger, et al.

4. "Polymer-Controlled Release of Tobramycin from Bone Graft Void Filler.” Drug Delivery and Translational 

Research. [2013/02, 3(6): 518-530]. BD Brooks, SN Davidoff, DW Grainger, AE Brooks.

Nanoparticles

Nanoparticle research represents one of the fastest growing areas of translational research; however, major 

limitations exist in the understanding of the toxicity of these nanoparticles. As a postdoctoral fellow, I managed 

several projects that facilitated our understanding in the field of nanoparticle toxicity. 

1. Nanoparticle toxicity assessment using an in vitro 3-D kidney organoid culture model.” Biomaterials. 

[2014]. AI Astashkina, CF Jones, G Thiagarajan, K Kurtzeborn, H Ghandehari, BD Brooks, DW Grainger. 

2. “Cationic PAMAM dendrimers aggressively initiate blood clot formation.” ACS-Nano. [2012, 6 (11): 9900–

9910]. CF Jones, AS Weyrich, H Ghandahari, BD Brooks, AS Weyrich, DW Grainger, et al.

3. "Cationic dendritic nanoparticles disrupt key platelet functions." Molecular Pharmaceutics. [2012/06 

4;9(6):1599-611]. CF Jones, R Campbell, BD Brooks, H Ghandahari, AS Weyrich, DW Grainger.

4. “A Critical Comparison of Protein Microarray Fabrication Technologies.” Analyst. [2014, 139:303-1326]. V 

Romanov, SN Davidoff, AR Miles, DW Grainger, BK Gale, BD Brooks.

D. Research Support

Ongoing Research Support

 Fasttrack SBIR; NIH; NIGMS R44 4R44GM109738-02 Date 05/01/2014 – 04/30/2015

Title: “HT Label-Free Screening and Kinetic Analysis of Small Molecules and Biologics

Purpose: To develop an innovative SPR instrument with throughput and sensitivity for screening new drug 

candidates. Role: PI

 Innovations Grant/Convertible Debt. Pfizer-Rinat; Date 09/25/2014 – 09/25/2016

Title: “HT Label-Free Screening using Epitope Binning for Biologics”

Purpose: To develop a HT SPR instrument with throughput and sensitivity for epitope binning new drug 

candidates. Role: PI

Completed Research Support

 Phase I SBIR; NIH; NCI R43 1R43CA177146-01A1 Date 07/01/2014 – 06/30/2015

Title: “Multiplexed Ovarian Cancer Microfluidic Tissue Microarray”

Purpose: The purpose is to develop an instrument system for automated, multiplexed cell- and tissue-based 

experiments (i.e. tissue microarrays).The proposed instrument will be used to novel cell- and tissue-based 

assays in a highly parallel manner that are otherwise difficult to perform. Role: PI

 Phase I SBIR; NIH; NIGMS R43 1R43GM101859-01 (MPI) GRANT10940803 

Date: 9/01/2012 – 8/31/2014

 Title: “Submerged Printing of Lipid and Membrane Protein Arrays”

Purpose: This SBIR project developed a device for automated array printing of lipids and membrane proteins 

onto submerged microtiter plate surfaces in a way that maintains their activity and function. Role: PI

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 21
 Phase II STTR; NIH; NIMH R43 5R42MH084372-03 (MPI)

Date: 9/01/2011 – 8/31/2014 

 Title: “Multiplexed GPCR Characterization Using SPR”

Purpose: This Phase II STTR project developed a real-time label-free biosensor that can analyze 96 samples 

at a time, compared to the 6 samples possible with current technologies. Role: PI

Patents

Drug Release from a Polymer-Controlled Local Antibiotic Delivery System Using a Degradable Bone Graft

Inventors: Amanda Elaine Brooks, Benjamin Delbert Brooks, David W Grainger

Publication date: 2013/2/5 Application number: 13/759,904

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 22
OMB No. 0925-0001 and 0925-0002 (Rev. 10/15 Approved Through 10/31/2018)

BIOGRAPHICAL SKETCH

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

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

NAME: Bailey-Kellogg, Chris

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

POSITION TITLE: Professor

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

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

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Massachusetts Institute of Technology, Cambridge, 

MA

Ohio State University, Columbus, OH

Dartmouth, Hanover, NH

S.B., S.M.

Ph.D.

Postdoc

05/1993

3/1999

06/2001

Electrical Engineering & 

Computer Science

Computer & Information 

Science

Computational Biology

A. Personal Statement

Research in my laboratory focuses on the development and application of computational methods for data 

analysis and experiment optimization, in the context of protein sequence/structure/function studies. By tightly

integrating computation with experiment, we seek to maximize information gain while minimizing experimental 

complexity, and to rigorously evaluate the resulting confidence in the data interpretation. Recently my primary 

focus has been computational modeling and computationally-driven engineering of the immune response to 

foreign proteins, in the contexts of both desired responses in infection/vaccination and undesired responses to 

therapeutics.

Of particular relevance to the proposed project, we have developed methods to computationally model and 

design protein:protein interactions. Our methods integrate both biophysical modeling and experimental data 

in order to capture the amino acid determinants of specific recognition in a manner that enables prediction 

of binding as well as optimization of variants for improved or disrupted binding. We have recently begun 

putting this to use in the context of antibody:antigen recognition, and have demonstrated the utility of 

computationally-directed engineering in helping identify antibody epitopes. The proposed project will enable 

us to scale up our methods to the high-throughput, multiplexed context enabled by the Wasatch 

Microfluidics SPR, analyzing entire panels of antibodies and antigenic variants in parallel. It will also provide 

a compelling context for studying antibody:antigen recognition, in order to understand the impact of antibody 

and antigen variability in HSV.

B. Positions and Honors

Positions and Employment

1998 Research Intern, Xerox PARC.

1999-2000 Postdoctoral Research Associate, Computer Science, Dartmouth College.

2001 Research Assistant Professor, Computer Science, Dartmouth College.

2001-2004 Assistant Professor, Computer Sciences, Purdue University.

2004-2007 Assistant Professor, Computer Science, Dartmouth College.

2007-2013 Associate Professor (with tenure), Computer Science, Dartmouth College.

2013-present Professor, Computer Science, Dartmouth College.

Honors

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 23
1993 MIT chapters of Eta Kappa Nu, Sigma Xi, and Tau Beta Pi honor societies

1998 OSU chapter of Phi Kappa Phi honor society

2003 NSF CAREER award (awarded at Purdue University)

2004 Alfred P. Sloan Research Fellowship (awarded at Dartmouth College)

2007 Dartmouth Friedman Family Fellowship

2007 Dartmouth Karen E. Wetterhahn Award for Distinguished Creative or Scholarly Achievement

2014 Dartmouth Melville and Leila Straus 1960 Faculty Fellowship

C. Contributions to Science

1. Protein design. We have developed a portfolio of complementary computational methods to optimize the 

selection of mutations so as to reduce immunogenicity of foreign proteins (e.g., bacterial enzymes) while 

maintaining their therapeutic activity.

a. A.S. Parker, W. Zheng, K.E. Griswold, and C. Bailey-Kellogg, “Optimization algorithms for functional 

deimmunization of therapeutic proteins”, BMC Bioinf., 2010, 11:180. PMCID: PMC2873530.

b. L. He, A.M. Friedman, and C. Bailey-Kellogg, “A divide-and-conquer approach to determine the

Pareto frontier for optimization of protein engineering experiments”, Proteins, 2012, 80:790-806.

PMCID: PMC4939273.

c. R.S. Salvat, A.S. Parker, Y. Choi, C. Bailey-Kellogg, and K.E. Griswold, “Mapping the Pareto 

optimal design space for a functionally deimmunized biotherapeutic candidate”, PLoS Comput. 

Biol., 2015, 11:e1003988. PMCID: PMC4288714.

d. Y. Choi, C. Hua, C. Sentman, M.E. Ackerman, and C. Bailey-Kellogg, “Antibody humanization by 

structure-based computational protein design”, MAbs, 2015, 7:1-13. PMID: 26252731.

2. Computationally-driven protein engineering. We have applied computationally-driven protein 

engineering methods to successfully reengineer a number of therapeutic candidates for improved 

properties.

a. R.S. Salvat, Y. Choi, A. Bishop, C. Bailey-Kellogg, and K.E. Griswold, “Protein deimmunization via 

structure-based design enables efficient epitope deletion at high mutational loads”, Biotechnol. 

Bioeng., 2015, 112:1306-1318. PMCID: PMC4452428. 

b. K. Blazanovic, H. Zhao, Y. Choi, W. Li, R.S. Salvat, D.C. Osipovitch, J. Fields, L. Moise, B.L. 

Berwin, S.N. Fiering, C. Bailey-Kellogg, and K.E. Griswold, “Structure-based redesign of 

lysostaphin yields potent anti-staphylococcal enzymes that evade immune cell surveillance”, Mol. 

Ther. Methods Clin. Dev., 2015, 2:15021. PMCID: PMC4470366.

c. H. Zhao, D. Verma, W. Li, Y. Choi, S.N. Fiering, C. Bailey-Kellogg, and K.E. Griswold, “Depleting T 

cell epitopes in lysostaphin mitigates anti-drug antibody response and enhances antibacterial 

efficacy in vivo”, Chem. Biol., 2015, 22:629-639. PMCID: PMC4441767.

d. Y. Choi, C. Ndong, K.E. Griswold, and C. Bailey-Kellogg, “Computationally driven antibody 

engineering enables simultaneous humanization and thermostabilization”, Protein Eng. Des. Sel., 

2016, epub. PMID: 27334453.

3. Protein interaction modeling. We have developed data- and physics-driven approaches to model, in an 

interpretable and predictive fashion, the amino acid level determinants of protein interaction specificity.

a. J. Thomas, N. Ramakrishnan, and C. Bailey-Kellogg, “Graphical models of protein-protein 

interaction specificity from correlated mutations and interaction data”, Proteins, 2009, 76:911-929.

PMID: 19306342.

b. A.N. Kettenbach, T. Wang, B.K. Faherty, D.R. Madden, S. Knapp, C. Bailey-Kellogg, and S.A. 

Gerber, “Rapid determination of multiple linear kinase substrate motifs by mass spectrometry”, 

Chem. Biol., 2012, 19:608-618. PMCID: PMC3366114.

c. H. Kamisetty, A. Ramanathan, C. Bailey-Kellogg, and C.J. Langmead, “Accounting for 

conformational entropy in predicting binding free energies of protein-protein interactions”, Proteins, 

2011, 79:444-462. PMID: 21120864.

d. H. Kamisetty, B. Ghosh, C.J. Langmead, and C. Bailey-Kellogg, “Learning sequence determinants 

of protein:protein interaction specificity with sparse graphical models”, J. Comput. Biol., 2015, 

22:476-486. PMCID: PMC4449715.

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 24
4. High-dimensional immune profiling. We have developed a suite of data analysis methods to predictively 

model the antibody response to infection and vaccination, based on multiplexed data regarding antigen 

specificity, Fc properties, and effector functions, across subjects from different clinical classes and 

outcomes.

a. E.P. Brown, A.F. Licht, A.-S. Dugast, C. Bailey-Kellogg, G. Alter, and M.E. Ackerman, “Highthroughput, multiplexed IgG subclassing of antigen-specific antibodies from clinical samples”, J. 

Immunol. Methods, 2012, 386:117-123. PMCID: PMC3475184.

b. A.W. Chung, M. Ghebremichael, H. Robinson, E. Brown, I. Choi, et al., and C. Bailey-Kellogg, M.E. 

Ackerman, and G. Alter, “Polyfunctional Fc-Effector Profiles Mediated by IgG Subclass Selection 

Distinguish RV144 and VAX003 Vaccines”, Sci. Transl. Med., 2014, 6:228ra38. PMID: 24648341.

c. I. Choi, A.W. Chung, T.J. Suscovich, et al., and G. Alter, M.E. Ackerman, and C. Bailey-Kellogg, 

“Machine learning methods enable predictive modeling of antibody feature:function relationships in 

RV144 vaccinees”, PLoS Comput. Biol., 2015, 11:e1004185. PMCID: PMC4395155.

d. M.E. Ackerman, A. Mikhailova, E.P. Brown, K.G. Dowell, B.D. Walker, C. Bailey-Kellogg, T.J. 

Suscovich, and G. Alter, “Polyfunctional HIV-Specific Antibody Responses Are Associated with 

Spontaneous HIV Control”, PLOS Path., 2016, 2:e1005315. PMCID: PMC4706315.

5. Immunoinformatics. We have developed methods to predict the binding of peptides to specific MHC 

proteins as well as more generally characterize the potential for and implications of cross-reactive binding 

of T cell receptors to peptide-MHC complexes.

a. C. Bailey-Kellogg, A.H. Gutierrez, L. Moise, F. Terry, W.D. Martin, and A.S. De Groot, “CHOPPI: a 

web tool for the analysis of immunogenicity risk from host cell proteins in CHO-based protein 

production”, Biotechnol Bioeng. 2014, 111:2170-2182. PMCID: PMC4282101.

b. P.T. Losikoff, S. Mishra, F. Terry, A. Gutierrez, M.T. Ardito, L. Fast, M. Nevola, W.D. Martin, C. 

Bailey-Kellogg, A.S. De Groot, and S.H. Gregory, “HCV epitope, homologous to multiple human 

protein sequences, induces a regulatory T cell response in infected patients”, J. Hepatol., 2015, 

62:48-55. PMID: 25157982.

c. A.H. Gutierrez, W.D., Martin, C. Bailey-Kellogg, F. Terry, L. Moise, and A.S. De Groot, 

“Development and validation of an epitope prediction tool for swine (PigMatrix) based on the pocket 

profile method”, BMC Bioinf., 2015, 6:290. PMCID: PMC4570239

d. L. He, A.S. De Groot, and C. Bailey-Kellogg, “Hit-and-run, hit-and-stay, and commensal bacteria 

present different peptide content when viewed from the perspective of the T cell”, Vaccine, 2015, 

33:6922-6929. PMID: 26428457.

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/43195993/?sort=date&direction=ascending

D. Research Support

Ongoing Research Support

2R01GM098977 (Bailey-Kellogg) 07/01/11 – 06/30/20

NIH

Functional Deimmunization of Therapeutic Proteins

The goals of this project are the development of protein deimmunization algorithms and the application of 

these methods in the design and evaluation of functionally-deimmunized beta-lactamase enzyme variants.

Role: PI

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 25
STTR 1R41AI118133 (Bailey-Kellogg, Stealth Biologics, LLC) 02/15/15 – 01/31/17

NIH

Design and Development of Immunotolerant S. aureus Biotherapies

The goal of this project is advanced pre-clinical development of anti-S. aureus enzyme therapies that have 

been engineered for reduced immunogenicity. 

Role: PI

1R21AI119741 (Griswold) 07/01/16 – 06/30/18

NIH 

Engineer bifunctional antibacterial enzymes for treatment of S. aureus infections

The goal of this project is to develop a modular platform for construction of bifunctional anti-Staphylococcal

lysin therapies designed to possess intra-molecular synergy between complementary catalytic domains. 

Role: co-investigator

1P01AI120756 (Tomaras, Duke) 04/01/16 – 03/31/21

Analytical Core (Ackerman, Dartmouth)

NIH

Bridging Antibody Fc-mediated Antiviral Functions Across Humans and Non-human Primates

The program seeks to rigorously evaluate and improve the use of the non-human primate model for testing 

antibody-based interventions and vaccines.

Role: co-investigator

Recently Completed Research Support

NSF CCF-0915388 (Bailey-Kellogg) 09/01/09 – 08/31/12

Algorithmic Problems in Protein Structure Studies

The project entails designing and analyzing efficient algorithms for fundamental problems that arise in studies 

of the three-dimensional structures of proteins, with a particular focus on graph-theoretic approaches.

Role: PI

IIS-0905206 (Bailey-Kellogg, Friedman, Langmead, Ramakrishnan) 08/15/09 – 07/31/13

NSF

III-Medium: Collaborative Research: Integration, Prediction, and Generation of Mixed Mode Information using 

Graphical Models, with Applications to Protein-Protein Interactions

The project seeks to develop new approaches to integrating heterogeneous data, in order to produce models 

that are predictive and diagnostic of key physical properties, and can be used for design purposes.

Role: PI

IIS-1017231 (Bailey-Kellogg, Friedman) 09/15/10 – 08/31/14

NSF 

III: Small: Collaborative Research: Analysis of Multi-dimensional Protein Design Spaces with Pareto 

Optimization of Experimental Designs

The project aims to develop new methods to characterize complex protein design spaces and optimize highquality designs for experimental evaluation, balancing multiple complementary criteria assessing design quality 

in terms of sequence-structure-function relationships.

Role: PI

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 26
1R21AI098122-01 (Bailey-Kellogg, Griswold, De Groot) 02/01/12 – 01/31/15

NIH

Computationally optimized anti-staphylococcal biotherapeutics

The goal of this project is to engineer lysostaphin enzyme variants that efficiently kill drug-resistant 

Staphylococcus aureus while simultaneously exhibiting low level immunogenicity.

Role: PI

R01 AI102691 (Ackerman) 07/15/14 – 07/14/15

NIH

One year supplement to Applying High-Performance Protein Engineering Tools to HIV Immunogen Design

The goal of this supplement is to develop epitope probes that capture the variable loop topology and sequence 

diversity of the HIV envelope trimer, but in the context of a monomeric scaffold. 

Role: co-investigator

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 27
Contact PD/PI: Brooks, Benjamin Delbert

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

Gary H. Cohen, Ph.D.

POSITION TITLE

Professor of Microbiology

eRA COMMONS USER NAME (

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

Brooklyn College, Brooklyn, NY B.S. 1956 Biology

University of Vermont, Burlington, VT Ph.D. 1964 Microbiology

University of Pennsylvania, Philadelphia, PA PDF 1964-67 Virology

A. Personal Statement of research interests. 

The overall goal of my research is to understand the molecular events that mediate herpes simplex virus 

(HSV) entry into mammalian cells and promotes its pathogenesis in the human. Recently, I have 

broadened my efforts directed at understanding how the human responds to HSV entry glycoproteins after 

natural infection or vaccination. HSV entry requires binding of gD to a cell receptor, either HVEM or nectin1. This interaction triggers virus-cell fusion involving three additional virion glycoproteins, gB, and gH/gL. 

Our approach has been to relate the essential viral glycoproteins’ functional properties to their 3-D 

structures (all of which we solved in collaborations with crystallographers). Our working model for fusion 

posits that in a sequential fashion, binding of gD to receptor causes gD to undergo radical conformational 

changes that allow it to interact with gH/gL thereby activating it into a form that causes up-regulation of the 

prefusion form of gB into a functional fusion protein. Over the years we have developed extensive panels of 

monoclonal antibodies to gD, gH/gL, gB as well as to HVEM and nectin-1. We have expressed each 

protein in a baculovirus expression system and have become expert in preparing and studying purified 

proteins. We have also prepared various forms of each glycoprotein to understand more about how each 

protein functions. In addition we have also developed monoclonal antibodies to two other important 

glycoproteins, gC and gE, both of which have been implicated in evading the innate responses of the host 

to virus infection, and we have developed methods to purify each of these proteins as well. Our proteins 

have become valuable candidates for human vaccines and are being studied by multiple labs as potential 

vaccine candidates: gD2306t is considered a “Gold Standard” to compare efficacy of vaccine candidates. 

We have learned how to dissect the humoral response to HSV in naturally infected or vaccinated humans. 

These were small scale studies in which we employed prototypes of our extensive panels of monoclonal 

antibodies to gain an epitope profile for each specimen. We carried out competition studies between our 

monoclonals and IgGs purified from human sera on the BIAcore biosensor and these small scale studies 

will be expanded upon in this grant by taking advantage of the Wasatch Microfluidics (WM) technology for 

doing competition studies between sera and our panels of monoclonal antibodies. This will able us to 

examine a much larger cohort of naturally infected people (over 3000 samples). We will study the 

responses to sera from guinea pigs vaccinated with gD, or with gD in combination with gB or with a 

trivalent gD/gC/gE combination. Ultimately, we believe our studies will reveal the mechanism(s) of an 

effective host response to virus infection, thereby providing a solid intellectual basis for generating a unique 

“second” generation vaccine. 

B. Positions and Honors

Positions and Employment

1964 - 1967 Postdoctoral Fellow (NIAID), University of Pennsylvania

1967 - 1973 Assistant Professor of Microbiology, School of Dental Medicine, U. of PA 

1973 - 1980 Associate Professor of Microbiology, School of Dental Medicine, U. of PA

1976 - 1977 Visiting Scientist, Swiss Institute for Experimental Cancer Research, Lausanne

1980 -Present Professor of Microbiology, School of Dental Medicine, U. of PA

Biosketches Page 28
1985 -2012 Chair, Department of Microbiology, School of Dental Medicine, U. of PA

Other Experience and Professional Memberships

1990 Senior International Fellow, Fogarty International Center, NIH

1990 Invited Professor, Institute of Biochemistry of the Faculty of Medicine,

 University of Lausanne, Lausanne, Switzerland

1993-2003 Member, Cancer Center, U. of PA 

1969 - 1974 NIH Career Development Award (AI)

2003 Elected, Fellow of the American Academy of Microbiology

2004 Elected, Fellow of the American Association for the Advancement of Science

2005-2015 Merit Award Recipient National Institute of Allergy and Infectious Diseases

C. Selected Publications (Selected from over 200) 

Selected Publications on HSV

Hannah, BP, Cairns, TM, Bender FC, Whitbeck JC, Lou H, Eisenberg RJ and Cohen GH. 2009. Glycoprotein B 

of herpes simplex virus associates with target membranes via its fusion loops. J. Virol. 83(13):6825-6836. 

PMCID: PMC2698560

Wright CC, Wisner TW, Hannah BP, Eisenberg RJ, Cohen GH and Johnson DC. 2009. Fusion between 

perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB. 

J. Virol. 83(22):11847-11856. PMCID: PMC2772685

Atanasiu D, Whitbeck JC, Ponce de Leon M, Lou H, Hannah BP, Cohen GH and Eisenberg RJ. 2010. 

Bimolecular complementation defines functional regions of HSV gB that are involved with gH/gL as necessary 

steps leading to cell fusion. J. Virol. 84:3825-34. PMCID: PMC2849501

Atanasiu, D., W.T. Saw, G.H.Cohen and R.J. Eisenberg. 2010. The cascade of events governing cell-cell 

fusion induced by HSV glycoproteins gD, gH/gL and gB. J.Virol. 84: 12292-12299. PMCID: PMC2976417

Chowdary TL, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, and Heldwein EE. 2010. Crystal structure of 

the gH/gL complex – a conserved herpesvirus fusion regulator. Nat Struct Mol Biol. 2010:882-888. PMCID: 

PMC2849501

Stampfer SD, Lou H, Cohen GH, Eisenberg RJ and Heldwein EE. 2010. Structural basis of the local pHdependent conformational changes in fusion glycoprotein B from Herpes Simplex virus 1. J. Virol. 84: 12924-

12933. PMCID: PMC3004323

Stiles KM, Whitbeck, JC, Lou H, Gary H. Cohen GH, Eisenberg RJ and Krummenacher C. 2010. Herpes 

simplex virus glycoprotein D interferes with HVEM binding to its ligands through down-regulation and direct 

competition. J. Virol. 84:11646-11660. PMCID: PMC2977867

Bernstein DI, Earwood JD, Bravo FJ, Cohen GH, Eisenberg RJ, Clark JR, Fairman J, Cardin RD. 2011. Effects 

of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the 

guinea pig. Vaccine. 29:2071-8. PMCID: PMC3082315.

Cairns, TM, Whitbeck JC, Lou H, Heldwein EE, Chowdary TK, Eisenberg RJ, Cohen GH. 2011. Capturing the 

herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment. J.Virol. 85:6175-6184. PMCID: 

PMC3126480

Di Giovine P, Settembre EC, Bhargava AK, Luftig MA, Lou H, Cohen GH, Eisenberg RJ, Krummenacher C, 

Carfi A. Structure of HSV gD bound to the human receptor nectin- 1. PLoS Pathog. 2011 Sep;7(9):e1002277. 

Epub 2011 Sep 29. PMCID: PMC3182920

Lazear E, Whitbeck JC, Ponce-de-Leon M, Cairns TM, Willis SH, Zuo Y, Krummenacher C, Cohen GH, and 

Eisenberg RJ. 2012. Antibody-induced conformational changes in HSV gD reveal new targets for virus 

neutralization. J Virol. 86:1563-76. PMCID: PMC3264331

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 29
Contact PD/PI: Brooks, Benjamin Delbert

Atanasiu, D, Cairns TM, Whitbeck JC, Saw WT, Rao S, Eisenberg RJ, and Cohen GH: Regulation of Herpes 

Simplex Virus gB-Induced Cell-Cell Fusion by Mutant Forms of gH/gL in the Absence of gD and Cellular 

Receptors. mBio 4(2):(e00046-13), February 2013. PMCID: PMC3585445

Maurer UE, Zeev-Ben-Bordehai T, Pandurangan AP, Cairns TM, Hannah BP, Whitbeck JC, Eisenberg RJ, 

Cohen, GH, Topf M, Huiskonen JT and Gruenewald K. 2013. The Structure of Herpesvirus Fusion 

Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction.

Structure. 2013:1396-13405. PMCID: PMC3737472

Atanasiu D, Saw, WT, Gallagher JR, Hannah BP, Matsuda Z, Whitbecke JC, Cohen GH and Eisenberg, RJ. 

2013. A dual split protein-based fusion assay reveals that mutations to HSV glycoprotein gB alter the kinetics 

of cell-cell fusion induced by HSV entry glycoproteins. J. Virol.87:11332-11345. PMCID: PMC3807322

Gallagher JR, Saw WT, Atanasiu D, Lou H, Eisenberg RJ, Cohen GH. 2013. Displacement of the C terminus 

of herpes simplex virus gD is sufficient to expose the fusion-activating interfaces on gD. J Virol. 2013 

87(23):12656-12666. PMCID: PMC3838169

Lazear E, Whitbeck JC, Zuo Y, Carfi A, Cohen GH, Eisenberg RJ, Krummenacher, C. 2014 Virology. 448:185-

195. PMCID: PMC2224591

Cairns TM, Fontana J, Huang ZY, Whitbeck JC, Atanasiu D, Rao S, Shelly, SS, Lou H, de Leon, MP, Steven, 

AC, Eisenberg RJ, and Cohen GH. 2014. Mechanism of neturalization of HSV by antibodies directed at the 

fusion domain of glycoprotein B. J. Virol. 88(5): 52677-2689. PMCID: PMC3958082. 

Whitbeck JC, Huang ZY, Cairns TM, Gallagher JR, Lou H, Ponce-de-Leon M, Belshe RB, Eisenberg RJ, 

Cohen GH. 2014. Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes 

simplex virus 2 glycoprotein d. J Virol. 2014 88:7786-95. PMCID: PMC4097771

Gallagher JR, Atanasiu D, Saw WT, Paradisgarten MJ, Whitbeck JC, Eisenberg RJ, Cohen GH. 2014. 

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and 

after Execution of Membrane Fusion. PLoS Pathogens. 2014 Sep 18;10(9):e1004373. doi: 

10.1371/journal.ppat.1004373. eCollection 2014. PMCID: PMC4169481

Cairns TM, Huang ZY, Whitbeck JC, de Leon MP, Lou H, Wald A, Krummenacher C, Eisenberg RJ and Cohen 

GH. 2014. Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected 

humans. J. VIrol. 88(21):12612-12622. PMCID: PMC4248929

. 

D. Research Support 

Ongoing 

R37-AI-18289, Gary H. Cohen (PI); RJ Eisenberg, Co-Investigator Dates: 6/1/10-5/31/15 

NIH/NIAID 

Studies of Herpes Simplex Virus glycoproteins 

Biosketches Page 30
The major goal of this project is to understand the two roles of glycoprotein gD in binding to its receptors and 

activating the fusion machinery of HSV. Because it is the receptor binding proteins, we are developing new 

ways to identify small molecules that inhibit receptor binding. We are also dissecting the composition of 

polyclonal antibodies to gD using Biacore and our panel of monoclonal antibodies. This dissection of the 

immune response has direct bearing on development of subunit vaccines. 

2R01- AI-076231, Roselyn J. Eisenberg (PI) Dates: 7/1/97-7/31/14

NIH/NIAID 

Early events in herpes simplex virus entry into cells 

Title of renewal changed: Studies of the structure, function and regulation of gB, the fusion protein of 

HSV. This is the grant under review. 

The major focus of this grant is to define functional regions of gB using mutants and antibodies and develop 

new gB constructs that could be used to define pre-and post-fusion forms of this protein. We will explore new 

methods to study how gB functions as a fusion protein. No overlap (or less than 10% in science, none in 

budget)

R01-AI-056045, Roselyn J. Eisenberg (PI) Dates: 09/15/03-11/30/15

NIH/NIAID 

Uncovering the regulatory role of gH/gL in HSV fusion 

The major focus of this project is to understand the functional role of different regions of gH/gL in regulating cell 

fusion based on its newly solved structure. No overlap (or less than 10% in science, none in budget). 

Completed Research Support (past 3 years)

NIH 1 UC1 AI-062486. Richard Welch, P.I., G.H. Cohen (PI of Subproject) Dates: 09/01/05 – 8/31/08

NIH/NIAID Gary H. Cohen (PI) and Roselyn J. Eisenberg (Co-Investigator) 

Goal: To evaluate recombinant proteins of vaccine and smallpox developed by C-PERL using baculoviruses 

grown in insect larvae to produce proteins for a multiprotein subunit vaccine against poxvirus infections and to 

develop antisera to them. 

 

Evaluation of Control Measures Against Infectious Diseases Other Than AIDS. Dates: 12/1/11-10/31/13

This is a contract that originated at St. Louis University. 

Our subcontract has G.H. Cohen as PI and RJE as Co-Investigator. 

The purpose of our subcontract is to evaluate the composition of human sera from patients that had been 

immunized with a truncated form of gD by Glaxo Smith Kline as a test for an anti-HSV-2 vaccine. Because of 

the failure of the trial, one question posed to us was what constituted a neutralizing response in terms of the 

epitopes of gD in the vaccine. Could we dissect how people responded to immunization with gD? 

 

RCE-U54-AI57168, Gary H. Cohen (PI of Subproject) Dates: 03/01/09-02/28/14

NIH/NIAID 

Defense Against Biowarfare and Emerging Infectious Agents 

The major goal of this project was to develop a multiprotein subunit vaccine against smallpox and monkey pox 

viruses (consisting of the membrane proteins L1, A27, A33 and B5) and to better understand the role each 

protein in poxvirus infection. Current studies focus principally on L1 as the principal receptor binding protein of 

the MV form of vaccinia virus and a major contributor to fusion by the membrane fusion complex. No overlap 

with any herpes projects listed above.

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 31
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

Roselyn J. Eisenberg

POSITION TITLE

Professor of Microbiology

eRA COMMONS USER NAME (credential, e.g., 

agency login)

ROSELYN 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

Bryn Mawr College A.B. 1960 Biology

University of Pennsylvania, Philadelphia, PA Ph.D. 1965 Microbiology

Princeton University, Princeton NJ PDF 1966-68 Virology

A. Personal Statement of research interests specific to this proposal). 

The goal of my research is to understand the process by which the four essential glycoproteins of HSV 

cooperate to carry out membrane fusion, a process essential for productive virus infection and for cell-cell 

fusion. Together with my colleague, Dr. Gary H. Cohen, I have been interested in the basic biology of this 

process and in the humoral responses to those glycoproteins in cohorts of vaccinated people and in the course 

of natural infection and recurrences caused by HSV-1 and HSV-2. Over the course of many years, we have 

amassed a large collections of reagents, including antibodies (both monoclonal and polyclonal) to each of the 

proteins involved in the HSV entry process. In collaboration with several crystallographers, we have solved the 

structures of the four essential glycoproteins (gD. gH/gL and gB) as well as the two gD receptors, HVEM and 

nectin-1. Glycoproteins gB and gH/gL together comprise the core fusion machinery of all herpesviruses, so that 

what we learn about how they function is broadly applicable. Although we have learned a great deal about the 

cascade of events leading to fusion, many details are still lacking. Our current understanding is that this 

process involves key conformational changes in each protein in a well-regulated and coordinated fashion. 

Monoclonal antibodies in our collection are capable of blocking key steps. A hallmark of our research is to 

study the properties of purified glycoproteins and receptors using techniques such as Biosensor analysis, 

electron microscopy and other biochemical and immunological approaches to characterize wild type and 

mutant forms of each. Recently, we have used our knowledge of key epitopes of the glycoproteins to dissect 

the humoral responses of people who have been vaccinated with gD and of people who have naturally been 

infected by HSV-1 and/or HSV-2. These were small scale atudies in which we employed prototypes of our 

extensive panesl of monoclonal antibodies to gain an epitope profile for each specimen. We cararied out

competition studies between our monoclonals and IgGs purified from human sera on the BIAcore biosensor

and we also dissected the IgGs into gD and gB specific fractions. We found that the human antibodies 

competed with several of our mouse monoclonals while other epitopes we had characterized were not well 

represented. These initial studies did not examine epitope profiles of other important HSV glycoproteins. It is 

now our goal to expand these initial studies to different and larger cohorts and use all of our mouse antibodies 

to gain a more completed profile to match with such variables as shedding, recurrence and protection (the 

latter in the case of vaccinated animals and humans). We have panels of monoclonal antibodies to other key 

glycoproteins that we would like to incorporate into these new studies. We have >3000 samples of human sera 

from naturally infected people who have had genital HSV infections and we also have sera from people in two 

different vaccine trials as well as guinea pig sera from ongoing new vaccine trials to test. The Wasatch 

Microfluidics (WM) technology for doing SPR (in place of the BIAcore) will enable us to scale up our studies to 

larger sample numbers, take adavantage of our full panels of monocloanal antibodies and using sera rather 

than IgG for the competition studies. Ultimataly, our findings may impaact the design of future HSV vaccines. 

B. Positions and Employment

1966 - 1968 Postdoctoral Fellow (NIGMS), Princeton University

1968 - 1969 Research Assoc. Dept. of Microbiology, Sch. of Medicine, U of Pennsylvania (UPenn)

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 32
1969 - 1978 Assistant Professor of Microbiology, School of Dental Medicine, UPenn

1978 - 1982 Assistant Professor of Microbiology, School of Veterinary Medicine, UPenn

1982 - 1985 Associate Professor of Microbiology, School of Veterinary Medicine, UPenn

1989 - 1993 Chair, Program in Microbiology, Molecular Biology Graduate Group, UPenn

1989 - Head, Laboratory of Microbiology, School of Veterinary Medicine, UPenn 

1985 - Professor of Microbiology, School of Veterinary Medicine, UPenn 

Current Editorial Board Memberships: Journal of Virology, Virology 

A-hoc reviews: PNAS, Nature, Vaccine, J.Gen. Virol. PLoS Pathogens and a number of others

NIH Service

1994 - 1998 Member, Experimental Virology Study Section, NIH

1998 - Member, Center for AIDS Research, U. of PA.

2003 - 2006 Member, Virology A Study Section, NIH

2006 - present: Ad Hoc service on many special emphasis panels in Virology

Honors

1972 - 1977 NIH Career Development Award (NIDR), University of Pennsylvania

2003 Elected, Fellow of the American Academy of Microbiology

2004 Elected, Fellow of the American Association for the Advancement of Science

2007 to present ISI highly cited researcher

C. Selected peer–reviewed publications most relevant to the current application (from >170)

Hannah, BP, Cairns, TM, Bender FC, Whitbeck JC, Lou H, Eisenberg RJ and Cohen GH. 2009. Glycoprotein B 

of herpes simplex virus associates with target membranes via its fusion loops. J. Virol. 83(13):6825-6836. 

PMCID: PMC2698560

Wright CC, Wisner TW, Hannah BP, Eisenberg RJ, Cohen GH and Johnson DC. 2009. Fusion between 

Perinuclear Virions and the Outer Nuclear Membrane Requires the Fusogenic Activity of Herpes Simplex Virus 

gB. J. Virol. 83(22):11847-11856. PMCID: PMC2772685

Atanasiu D, Whitbeck JC, Ponce de Leon M, Lou H, Hannah BP, Cohen GH and Eisenberg RJ. 2010. 

Bimolecular complementation defines functional regions of HSV gB that are involved with gH/gL as necessary 

steps leading to cell fusion. J. Virol. 84:3825-34. PMCID: PMC2849501

Atanasiu, D., W.T. Saw, G.H.Cohen and R.J. Eisenberg. 2010. The cascade of events governing cell-cell 

fusion induced by HSV glycoproteins gD, gH/gL and gB. J.Virol. 84: 12292-12299. PMCID: PMC2976417

Chowdary TL, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, and Heldwein EE. 2010. Crystal structure of 

the gH/gL complex – a conserved herpesvirus fusion regulator. Nat Struct Mol Biol. 2010:882-888. PMCID: 

PMC2849501

Stampfer SD, Lou H, Cohen GH, Eisenberg RJ and Heldwein EE. 2010. Structural basis of the local pHdependent conformational changes in fusion glycoprotein B from Herpes Simplex virus 1. J. Virol. 84: 12924-

12933. PMCID: PMC3004323

Stiles KM, Whitbeck, JC, Lou H, Gary H. Cohen GH, Eisenberg RJ and Krummenacher C. 2010. Herpes 

simplex virus glycoprotein D interferes with HVEM binding to its ligands through down-regulation and direct 

competition. J. Virol. 84:11646-11660. PMCID: PMC2977867

Bernstein DI, Earwood JD, Bravo FJ, Cohen GH, Eisenberg RJ, Clark JR, Fairman J, Cardin RD. 2011. Effects 

of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the 

guinea pig. Vaccine. 29:2071-8. PMCID: PMC3082315.

Cairns, TM, Whitbeck JC, Lou H, Heldwein EE, Chowdary TK, Eisenberg RJ, Cohen GH. 2011. Capturing the 

herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment. J.Virol. 85:6175-6184. PMCID: 

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 33
Contact PD/PI: Brooks, Benjamin Delbert

PMC3126480

Di Giovine P, Settembre EC, Bhargava AK, Luftig MA, Lou H, Cohen GH, Eisenberg RJ, Krummenacher C, 

Carfi A. Structure of HSV gD bound to the human receptor nectin- 1. PLoS Pathog. 2011 Sep;7(9):e1002277. 

Epub 2011 Sep 29. PMCID: PMC3182920

Lazear E, Whitbeck JC, Ponce-de-Leon M, Cairns TM, Willis SH, Zuo Y, Krummenacher C, Cohen GH, and 

Eisenberg RJ. 2012. Antibody-induced conformational changes in HSV gD reveal new targets for virus 

neutralization. J Virol. 86:1563-76. PMCID: PMC3264331

Shelly SS, Cairns, TM, Whitbeck JC , Lou H, Gallagher J, Cohen GH and Eisenberg RJ. 2012. The membrane 

proximal region (MPR) of herpes simplex virus gB regulates association of the fusion loops with lipid 

membranes. mBio. 2012 Nov 20;3(6). doi:pii: e00429-12. 10.1128/mBio.00429-12. PMCID: PMC3585445

Atanasiu, D, Cairns TM, Whitbeck JC, Saw WT, Rao S, Eisenberg RJ, and Cohen GH: Regulation of Herpes 

Simplex Virus gB-Induced Cell-Cell Fusion by Mutant Forms of gH/gL in the Absence of gD and Cellular 

Receptors. mBio 4(2):(e00046-13), February 2013. PMCID: PMC3585445

Maurer UE, Zeev-Ben-Bordehai T, Pandurangan AP, Cairns TM, Hannah BP, Whitbeck JC, Eisenberg RJ, 

Cohen, GH, Topf M, Huiskonen JT and Gruenewald K. 2013. The Structure of Herpesvirus Fusion 

Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction.

Structure. 2013:1396-13405. PMCID: PMC3737472

Atanasiu D, Saw, WT, Gallagher JR, Hannah BP, Matsuda Z, Whitbecke JC, Cohen GH and Eisenberg, RJ. 

2013. A dual split protein-based fusion assay reveals that mutations to HSV glycoprotein gB alter the kinetics 

of cell-cell fusion induced by HSV entry glycoproteins. J. Virol.87:11332-11345. PMCID: PMC3807322

Gallagher JR, Saw WT, Atanasiu D, Lou H, Eisenberg RJ, Cohen GH. 2013. Displacement of the C terminus 

of herpes simplex virus gD is sufficient to expose the fusion-activating interfaces on gD. J Virol. 2013 

87(23):12656-12666. PMCID: PMC3838169

Lazear E, Whitbeck JC, Zuo Y, Carfi A, Cohen GH, Eisenberg RJ, Krummenacher, C. 2014 Virology. 448:185-

195. PMCID: PMC2224591

Cairns TM, Fontana J, Huang ZY, Whitbeck JC, Atanasiu D, Rao S, Shelly, SS, Lou H, de Leon, MP, Steven, 

AC, Eisenberg RJ, and Cohen GH. 2014. Mechanism of neturalization of HSV by antibodies directed at the 

fusion domain of glycoprotein B. J. Virol. 88(5): 52677-2689. PMCID: PMC3958082. 

Whitbeck JC, Huang ZY, Cairns TM, Gallagher JR, Lou H, Ponce-de-Leon M, Belshe RB, Eisenberg RJ, 

Cohen GH. 2014. Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes 

simplex virus 2 glycoprotein d. J Virol. 2014 88:7786-95. PMCID: PMC4097771

Gallagher JR, Atanasiui D, Saw WT, Paradisgarten MJ, Whitbeck JC, Eisenberg RJ, Cohen GH. 2014. 

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and 

after Execution of Membrane Fusion. PLoS Pathogens. 2014 Sep 18;10(9):e1004373. doi: 

10.1371/journal.ppat.1004373. eCollection 2014. PMCID: PMC4169481

Cairns TM, Huang ZY, Whitbeck JC, de Leon MP, Lou H, Wald A, Krummenacher C, Eisenberg RJ and Cohen 

GH. 2014. Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected 

humans. J. VIrol. 88(21):12612-12622. PMCID: PMC4248929

Biosketches Page 34
D. Research Support 

Ongoing 

R01-AI-056045, Roselyn J. Eisenberg (PI) Dates: 09/15/03-11/31/15

NIH/NIAID 

Uncovering the regulatory role of gH/gL in HSV fusion 

The major focus of this project is to understand the functional role of different regions of gH/gL in regulating cell 

fusion based on its newly solved structure. No overlap (or less than 10% in science, non in budget)

R37-AI-18289, Gary H. Cohen (PI); RJ Eisenberg, Co-Investigator Dates: 8/1/00-5/31/15 

NIH/NIAID 

Studies of Herpes Simplex Virus glycoproteins 

The major goal of this project is to understand the two roles of glycoprotein gD in binding to its receptors and 

activating the fusion machinery of HSV. Because it is the receptor binding proteins, we are developing new 

ways to identify small molecules that inhibit receptor binding. We are also dissecting the composition of 

polyclonal antibodies to gD using Biacore and our panel of monoclonal antibodies. This dissection of the 

immune response has direct bearing on development of subunit vaccines. 

Completed Research Support (past 3 years)

NIH/NIAID. Evaluation of Control Measures Against Infectious Diseases Other Than AIDS. This is a 

contract that originated at St. Louis University. Our subcontract has G.H. Cohen as PI and RJE as CoInvestigator. Dates: 12/111-6/30/12

The purpose of our subcontract is to evaluate the composition of human sera from patients that had been 

immunized with a truncated form of gD by Glaxo Smith Kline as a test for an anti-HSV-2 vaccine. Because of 

the failure of the trial, one question posed to us was what constituted a neutralizing response in terms of the 

epitopes of gD in the vaccine. Could we dissect how people responded to immunization with gD? 

 

RCE-U54-AI57168, Gary H. Cohen (PI of Subproject) Dates: 03/01/09-02/28/14

NIH/NIAID 

Defense Against Biowarfare and Emerging Infectious Agents 

The major goal of this project was to develop a multiprotein subunit vaccine against smallpox and monkey pox 

viruses (consisting of the membrane proteins L1, A27, A33 and B5) and to better understand the role each 

protein in poxvirus infection. Current studies focus principally on L1 as the principal receptor binding protein of 

the MV form of vaccinia virus and a major contributor to fusion by the membrane fusion complex. No overlap 

with any herpes projects listed above.

 

2R01- AI-076231 (formerly NS-36731), Roselyn J. Eisenberg (PI) Dates: 7/1/97-7/31/14

NIH/NIAID 

Early events in herpes simplex virus entry into cells 

Title of renewal changed: Studies of the structure, function and regulation of gB, the fusion protein of 

HSV. 

The major focus of this grant is to define functional regions of gB using mutants and antibodies and develop 

new gB constructs that could be used to define pre-and post-fusion forms of this protein. We will explore new 

methods to study how gB functions as a fusion protein. No overlap (or less than 10% in science, non in budget)

 

Contact PD/PI: Brooks, Benjamin Delbert

Biosketches Page 35
Contact PD/PI: Brooks, Benjamin Delbert

Principal Investigator: Brooks, Benjamin D 

For New and Renewal Applications (PHS 398) – DO NOT SUBMIT UNLESS REQUESTED For Noncompeting Progress Reports (PHS 2590) – Submit only Active Support for Key Personnel PHS 398/2590 

OTHER SUPPORT 

Brooks, Benjamin 

Active Support: 

1R44GM109738-01 (Brooks) “HT Label-Free Screening and Kinetic Analysis of Small Molecules and 

Biologics.” 4/1/2014 – 3/31/2017 ($ 3.0 Calendar 

Completed Support: 

1R43CA177146-01A1 (Brooks) “Multiplexed Ovarian Cancer Microfluidic Tissue Microarray.”

8/1/2014 – 7/31/2015 ($ 1.0 Calendar 

1R43GM101859-01 (Brooks) “Submerged Printing of Lipid and Membrane Protein Arrays “

9/1/2012 – 8/31/2014 ($ 2.0 Calendar 

5R42MH084372-03 (Brooks) “Multiplexed GPCR Characterization Using SPR “

9/1/2012 – 8/31/2014 ($ 2.0 Calendar 

Pending: 

NSF SBIR Phase I. “High-throughput SPR for Screening and Characterizing Vaccines “

10/1/2016 – 9/30/2017 ($ 1.0 Calendar 

 

NIH SBIR Direct to Phase II. “High-throughput SPR for Screening and Characterizing Vaccines “

10/1/2016 – 9/30/2018 ($ 2.0 Calendar 

NIH SBIR Phase II (Brooks) “Multiplexed Ovarian Cancer Microfluidic Tissue Microarray.”

8/1/2016 – 7/31/2018 ($ 2.0 Calendar 

Overlap: 

Not relating to this proposal. Overlap related to NIH and NSF proposals ““High-throughput SPR for 

Screening and Characterizing Vaccines.“

Current and Pending Support Page 36
Contact PD/PI: Brooks, Benjamin Delbert

Dartmouth Department of Computer Science

August 31, 2016

Dr. Ben Brooks

Wasatch Microfluidics

Dear Ben,

I’m writing to offer you my enthusiastic support for your NIH SBIR proposal, “High-throughput,

multiplexed characterization and modeling of antibody:antigen binding, with application to

HSV”. As you know, my lab has long been developing and applying computational methods

for modeling and designing protein:protein interactions. Recently, we have been focusing on

antibody:antigen interactions, and have developed new, integrated computational-experimental

techniques to identify the amino acids driving a particular interaction. This proposal provides

a chance to scale these methods to the high-throughput, multiplexed context enabled by your

technology, and to apply them to the interesting HSV system brought by Dr. Cohen. If the proposal is funded, I will work with both the Wasatch and Penn teams to research, design, develop,

and implement methods to analyze Wasatch SPR data in order to define antibody communities and localize antibody epitopes, and apply these methods to the study of antibody:antigen

binding in HSV. In short, I offer you my full support for this proposal, and I look forward to

building a strong collaboration, developing exciting new technology, and gaining interesting

new scientific insights.

Sincerely,

Chris Bailey-Kellogg

Professor

cbk@cs.dartmouth.edu http://www.cs.dartmouth.edu/∼cbk/

6211 Sudikoff Laboratory Hanover, NH 03755, USA

(603) 646-3385 Fax: (603) 646-1672

Current and Pending Support Page 37
Contact PD/PI: Brooks, Benjamin Delbert

OTHER SUPPORT –COHEN, GH

ACTIVE 

R01-AI-18289-34 (Cohen) 07/01/15-12/31/19 3.6 calendar months

NIH/NIAID $ 

Title: Functions of HSV glycoproteins in virus entry and the humoral immune response 

The major goals of this project are: 1) to use biochemical and immunological approaches to explore how 

conformational changes to each of the entry glycoproteins impact function, using monoclonal antibodies and 

protein structure; and 2) to use these monoclonal antibodies and purified glycoproteins to dissect the humoral 

response to vaccination against HSV as well as to natural HSV infections. This analysis will impact the design 

of future vaccine. 

OVERLAP: None 

1R44AI127039-01 (B. Brooks) 07/01/16-06/30/18 2.4 calendar months

NIH/NIAID $ 

Title: Improving vaccine development through high-throughput immunogenicity screening 

The major goals of this project are: 1) Develop the user interface and HT data analysissoftware. 2) 

Characterize the epitope profile of sera obtained from vaccinated guinea pigs and from naturally infected 

humans. 

PENDING 

None. 

 

7

Current and Pending Support Page 38
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date: 2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: WASATCH MICROFLUIDICS

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Benjamin Delbert Brooks PD/PI 2.0

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

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

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Computer Programmer 1.0

1 Application Sciences 1.0

2 Total Number Other Personnel Total Other Personnel

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

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

Contact PD/PI: Brooks, Benjamin Delbert

 Page 39
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: WASATCH MICROFLUIDICS

Start Date*: 07-01-2017 End Date*: 06-30-2018 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 0.00

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 0.00

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

 Page 40
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: WASATCH MICROFLUIDICS

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . MTA 40.0

Total Indirect Costs

Cognizant Federal Agency

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

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: WasatchBudgetJustificationPA-16-302.pdfMime Type: application/pdf

(Only attach one file.)

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

 Page 41
Contact PD/PI: Brooks, Benjamin Delbert

Budget Justification Wasatch Microfluidics 

Senior Personnel 

Two months support is requested for the PI, Ben Brooks. Ben will supervise all grant and research efforts and 

reagent/antibody generation and immunogenicity efforts through contract research organizations. 

Engineers and Technicians 

Funding for a senior programmer and application scientist are requested for 1 month to write updates for our 

epitope binning software and to perform experiments with Cohen/Eisenberg groups.

 

Fringe Benefits 

Fringe benefits are estimated at ~25% of salaries. These benefits include FICA for all personnel. 

Travel Funds are requested ($5,000) to allow the PI and/or application scientists to travel to or from UPenn and 

Dartmouth. 

Supplies- We request funds for supplies for work on the project ($ $ is requested for lab supplies 

and materials Including assorted chemicals, glassware, proteins and reagents, pumps, electrical supplies, fluid 

flow connections, and other test supplies. Most of these will only be used if necessary as wet lab experiments 

have already been performed. $ will be to conduct the X-Ray crystallography at the Pasteur and $ 

will be to generate mutants with Integral Molecular. $ is requested to clone and sequence mAbs used in 

computer algorithm

Indirect Costs 

40% is the indirect cost rate applied by the Wasatch Microfluidics to NIH Awards, which is charged on all items 

except equipment and subcontracts. 

Fee A fee of less than 7% is requested to cover other business costs, such as intellectual property costs.

Budget Justification Attachment Page 42
Budget Justification: Cohen and Eisenberg Lab

Personnel:

Gary H. Cohen, Ph.D., P.I., (1.8 calendar months) will be responsible for all scientific and budgetary aspects of

this grant. 

Roselyn J. Eisenberg, Ph.D., Co-Investigator, (1.8 calendar months) will collaborate with Dr. Cohen on all 

scientific aspects of the grant.

Tina Cairns, Ph.D., Research Specialist, (1.8 calendar months) She will carry out the monoclonal antibody 

blocking experiments using the ursing She is developing a high throughput method of dissecting human sera 

from vaccines (both human and animal) and naturally infected people to gain an epitope profile of large numbers 

of specimens using the WM Continuous Flow Microspotter (CFM) coupled to the Ibis MX96 imager. She also 

assembles and catalogues all of our monoclonal antibodies into Groups depending on their properties. As part 

of this, she will do antibody binning on known and thus far uncharacterized monoclonal antibodies using the WM 

Continuous Flow Microspotter (CFM) plus Ibis MX96 imager. She will collaborate with Dr. Atanasiu in this effort. 

Doina Atanasiu, Ph.D., Research Specialist, (1.8 calendar months) is a mainstay in the running of the laboratory, 

in manuscript preparation and formatting. She will help to characterizd specific monoclonals to the entry 

glycoproteins in terms of their functions in virus entry and cell-cell fusion. She is responsible for maintaining all 

stocks of cells and viruses used for this purpose. She will purify antibodies to specific glycoproteins from serum 

specimens using streptavidin coated magnetic beads to capture biotinylated proteins. She will also assist Dr. 

Cairns in carrying out biosensor studies. A number of MAbs have not been fully characterized, particularly ones 

to gE and gC and she will be in charge of fully elucidating their properties. She will assist Dr. Cairns in grouping 

of Mabs in terms of their ability to neutralize virus and to block fusion. 

Ms. Huan Lou, Research Specialist, (1.8 calendar months) is an accomplished insect culturist who grows large 

volumes of baculovirus and insect cells using the Celligen Bioreactor. She is a mainstay of our laboratory in its 

efforts to purify the large quantities of protein needed for all experiments involving purified proteins. She is skilled 

in the use of this equipment as well as equipment designed for smaller scale purification. She routinely handles 

3-5 liters of cell supernatant fluids and she purifies the proteins by various chromatography techniques and 

characterizes them by SDSPAGE, silver staining, Western blotting, and ELISA. 

Ms. Wan Ting Saw, Research Specialist, (1.8 calendar months) works closely with Dr. Atanasiu. She is highly 

skilled in all of the techniques used including cell culture molecular biology and virology. Her work is exceptional 

in quality. 

Supplies. We are requesting funds on an annual basis to cover the costs of reagents, hybridoma production, 

preparation of immunosorbent columns for additional protein purification, magnetic beads, tissue culture 

supplies, media serum, plasticware, etc for cell culture and virus production and neutralization assays. We are 

Contact PD/PI: Brooks, Benjamin Delbert

Budget Justification Attachment Page 43
also requesting funds for supplies related to the WM biosensor. We are also requesting funds to cover page 

charges on publications, service for the ultracentrifuges and gel filtration equipment. 

Travel. Funds are requested to offset travel to relevant scientific meetings such as the International Herpesvirus 

Workshop and others as appropriate. 

Budget Justification Biosensor Tools

David Myszka, PhD. 

Pi Myszka will test the new CFM/E-SPR at the end of Aim 2 for one calendar month per year. Rebecca Rich of 

Biosensor Tools will be employed as an application scientist for two weeks each year. 

Materials and Supplies.

Funds are requested to test the next-generation CFM/SPR at $4500 per year. 

Indirect Costs 25% is the indirect cost rate applied by Biosensor Tools for this grant. 

Contact PD/PI: Brooks, Benjamin Delbert

Budget Justification Attachment Page 44
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

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 0.00

Section D, Travel

1. Domestic

2. Foreign 0.00

Section E, Participant/Trainee Support

Costs

0.00

1. Tuition/Fees/Health Insurance 0.00

2. Stipends 0.00

3. Travel 0.00

4. Subsistence 0.00

5. Other 0.00

6. Number of Participants/Trainees 0

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs 0.00

3. Consultant Services 0.00

4. ADP/Computer Services 0.00

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

Fees

0.00

7. Alterations and Renovations 0.00

8. Other 1 0.00

9. Other 2 0.00

10. Other 3 0.00

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

 Page 45
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date: 2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ❍ Project ● Subaward/Consortium

Enter name of Organization: The Trustees of the University of Pennsylvania

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Dr. Gary H Cohen PD/PI 0.6

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

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

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

1 Research Scientist 0.6

1 Research Specialist 1.2

2 Total Number Other Personnel Total Other Personnel

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

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

Contact PD/PI: Brooks, Benjamin Delbert

 Page 46
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: The Trustees of the University of Pennsylvania

Start Date*: 07-01-2017 End Date*: 06-30-2018 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 0.00

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 0.00

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 0.00

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

Contact PD/PI: Brooks, Benjamin Delbert

 Page 47
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: The Trustees of the University of Pennsylvania

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research 61.0

Total Indirect Costs

Cognizant Federal Agency

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

DHHS, Louis Martillotti, (212) 264-2069

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:

Cohen_Lab_Budget_Justification_PA-16-302.pdf

Mime Type: application/pdf

(Only attach one file.)

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

 Page 48
Budget Justification: Cohen Lab 

Personnel: 

Gary H. Cohen, Ph.D., P.I., (0.6 calendar months) will be responsible for all scientific and budgetary aspects of 

this grant. 

Tina Cairns, Ph.D., Research Specialist, (0.6 calendar months) Dr. Cairns will carry out high throughput 

epitope dissection of different herpes proteins and antibodies using the WM Continuous Flow Microspotter 

(CFM) coupled to the Ibis MX96 imager. She will assemble, analyze and catalogue the novel proteins and 

antibodies generated in this study. She will collaborate with Dr. Atanasiu in this effort. 

Doina Atanasiu, Ph.D., Research Specialist, (0.6 calendar months) is a mainstay in the laboratory. She will be 

involved in the day-to-day effort to characterize the novel glycoproteins in terms of their altered function in virus 

entry and cell-cell fusion. She will assist Dr. Cairns in carrying out biosensor studies 

Ms. Huan Lou, Research Specialist, (0.6 calendar months) grows baculovirus and insect cells to produce and 

purify protein needed for experimentation. She purifies proteins by various chromatography techniques and 

characterizes them by SDS-PAGE, silver staining, Western blotting, and ELISA. 

Supplies: 

We are requesting funds to cover the costs of reagents, DNA sequencing, preparation of reagents, protein 

purification, tissue culture supplies; media, serum, plastic ware, etc. 

2

Contact PD/PI: Brooks, Benjamin Delbert

Budget Justification Attachment Page 49
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

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 0.00

Section D, Travel 0.00

1. Domestic 0.00

2. Foreign 0.00

Section E, Participant/Trainee Support

Costs

0.00

1. Tuition/Fees/Health Insurance 0.00

2. Stipends 0.00

3. Travel 0.00

4. Subsistence 0.00

5. Other 0.00

6. Number of Participants/Trainees 0

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs 0.00

3. Consultant Services 0.00

4. ADP/Computer Services 0.00

5. Subawards/Consortium/Contractual

Costs

0.00

6. Equipment or Facility Rental/User

Fees

0.00

7. Alterations and Renovations 0.00

8. Other 1 0.00

9. Other 2 0.00

10. Other 3 0.00

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee 0.00

 Page 50
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date: 2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ❍ Project ● Subaward/Consortium

Enter name of Organization: Dartmouth College

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Christopher Bailey-Kellogg PD/PI 0.5

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

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

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

1 Graduate Students 12.0

Undergraduate Students

Secretarial/Clerical

1 Total Number Other Personnel Total Other Personnel

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

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

Contact PD/PI: Brooks, Benjamin Delbert

 Page 51
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: Dartmouth College

Start Date*: 07-01-2017 End Date*: 06-30-2018 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 0.00

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 0.00

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

 Page 52
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: Dartmouth College

Start Date*: 07-01-2017 End Date*: 06-30-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs 0.00

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 . 62% of Dartmouth total direct - equip

Total Indirect Costs

Cognizant Federal Agency

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

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name:

Dartmouth_Personnel_Justification.pdf

Mime Type: application/pdf

(Only attach one file.)

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

 Page 53
Personnel Justification 

Prof. Chris Bailey-Kellogg (.25 Summer Months) will oversee all computational components of 

the project, advising the Dartmouth PhD student and working together with the Wasatch 

programmer. We will research, design, develop, and implement methods to analyze Wasatch 

SPR data in order to define antibody communities and localize antibody epitopes. We will work 

with the other investigators to apply the methods to the study of antibody:antigen binding in 

HSV. 

Dartmouth PhD student (TBD, 9 Calendar Months) will lead the research effort into new 

methods for analyzing Wasatch SPR data for antibody:antigen binding analysis. 

Contact PD/PI: Brooks, Benjamin Delbert

Budget Justification Attachment Page 54
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 1

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment 0.00

Section D, Travel

1. Domestic

2. Foreign 0.00

Section E, Participant/Trainee Support

Costs

0.00

1. Tuition/Fees/Health Insurance 0.00

2. Stipends 0.00

3. Travel 0.00

4. Subsistence 0.00

5. Other 0.00

6. Number of Participants/Trainees 0

Section F, Other Direct Costs 0.00

1. Materials and Supplies 0.00

2. Publication Costs 0.00

3. Consultant Services 0.00

4. ADP/Computer Services 0.00

5. Subawards/Consortium/Contractual

Costs

0.00

6. Equipment or Facility Rental/User

Fees

0.00

7. Alterations and Renovations 0.00

8. Other 1 0.00

9. Other 2 0.00

10. Other 3 0.00

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee 0.00

Contact PD/PI: Brooks, Benjamin Delbert

 Page 55
Contact PD/PI: Brooks, Benjamin Delbert

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

 

Total Direct Costs less Consortium F&A

 

NIH policy (NOT-OD-05-004) allows applicants to exclude consortium/contractual F&A costs when determining if an application falls at or beneath any applicable direct cost limit. When a direct

cost limit is specified in an FOA, the following table can be used to determine if your application falls within that limit. 

Category Budget

Period 1

Budget

Period 2

Budget

Period 3

Budget

Period 4

Budget

Period 5

TOTALS

Total Direct Costs less Consortium F&A 0 0 0 0

 Page 56
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

Program Type (select only one)*

● SBIR ❍ STTR ❍ Both  (See agency-specific instructions to determine whether a particular agency allows a single submission for

both SBIR and STTR)

SBIR/STTR Type (select only one)*

● Phase I ❍ Phase II ❍ Fast-Track (See agency-specific instructions to determine whether a particular agency participates in FastTrack)

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

1a. Do you certify that at the time of award your organization will meet the eligibility criteria for a

small business as defined in the funding opportunity announcement?*

● Yes ❍ No

1b. Anticipated Number of personnel to be employed at your organization at the time of award.* 15

2. Does this application include subcontracts with Federal laboratories or any other Federal

Government agencies?*

❍ Yes ● No

If yes, insert the names of the Federal laboratories/agencies:*

3. Are you located in a HUBZone? To find out if your business is in a HUBZone, use the mapping

utility provided by the Small Business Administration at its web site: http://www.sba.gov *

❍ Yes ● No

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

States?*

❍ Yes ● No

If no, provide an explanation in an attached file. Explanation:* Pasteur_Letter_-_PA-16-302.pdf

5. Has the applicant and/or Program Director/Principal Investigator submitted proposals for

essentially equivalent work under other Federal program solicitations or received other Federal

awards for essentially equivalent work?*

❍ Yes ● No

If yes, insert the names of the other Federal agencies:*

6. Disclosure Permission Statement: If this application does not result in an award, is the

Government permitted to disclose the title of your proposed project, and the name, address,

telephone number and e-mail address of the official signing for the applicant organization, to

organizations that may be interested in contacting you for further information (e.g., possible

collaborations, investment)?*

❍ Yes ● No

7. Commercialization Plan: If you are submitting a Phase II or Phase I/Phase II Fast-Track Application, include a Commercialization

Plan in accordance with the agency announcement and/or agency-specific instructions.*

Attach File:*

Contact PD/PI: Brooks, Benjamin Delbert

 Page 57
Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

SBIR-Specific Questions:

Questions 8 and 9 apply only to SBIR applications. If you are submitting ONLY an STTR application, leave questions 8 and 9 blank and

proceed to question 10.

8. Have you received SBIR Phase II awards from the Federal Government? If yes, provide a

company commercialization history in accordance with agency-specific instructions using this

attachment.*

● Yes ❍ No

Attach File:* Wasatch_Microfluidics_Commercialization_History.pdf

9. Will the Project Director/Principal Investigator have his/her primary employment with the small

business at the time of award?*

● Yes ❍ No

STTR-Specific Questions:

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

blank.

10. Please indicate whether the answer to BOTH of the following questions is TRUE:*

(1) Does the Project Director/Principal Investigator have a formal appointment or commitment either with the small business directly

(as an employee or a contractor) OR as an employee of the Research Institution, which in turn has made a commitment to the small

business through the STTR application process; AND

(2) Will the Project Director/Principal Investigator devote at least 10% effort to the proposed project?

❍ Yes ❍ No

11. In the joint research and development proposed in this project, does the small business

perform at least 40% of the work and the research institution named in the application perform at

least 30% of the work?*

❍ Yes ❍ No

Contact PD/PI: Brooks, Benjamin Delbert

 Page 58
Professor	Felix	A.	Rey

Unité	de	Virologie	Structurale

Institut	Pasteur	-	CNRS	UMR	3965

25	rue	du	Docteur	Roux

Paris	75015	France

Tel	:	+33	1	45	68	85	63

e-mail	rey@pasteur.fr

Paris,	August	18th 2016

OBJECT:	 Collaboration	 letter	 for	 NIH	 small	 business	 grant	"Predicting	 Epitopes	 in	

Vaccine	and	Therapeutic	Antibody	Research”		

To: Gary	Cohen	and	Roselyn	Eisenberg,	Benjamin	Brooks,	Chris	Bailey-Kellogg	

The	Structural	Virology	Unit of Institute	Pasteur	in	Paris studies viruses	 of	global	

public	health	and/or	of	veterinary	concern by	using	structural	biology	techniques,	

primarily	 X-ray	 crystallography.	 The	 knowledge	 gained	 can	 be	 used	 for	

translational	structure-based	design	of	preventive	or	curative	antiviral	agents.	One	

of	 our	 scientific	 goals	 is	 to	 provide	 a	 structural	 basis	 for	 understanding	 the	

molecular	mechanisms	of	membrane	 fusion	used	by	enveloped	viruses	 to	enter	a	

target	 cell.	 Herpesviruses	 are	 important	 human	 pathogens	 and	 they	 present	 an	

interesting	and challenging	model	for	studying	this	process.	We	have been	using an	

animal	 herpesvirus	 called	 pseudorabies,	as	well	as	 human	 herpesviruses	 such	as	

cytomegalovirus	and	human	herpesvirus-8	as	model	systems	in	our	lab.		

Herpes	simplex	viruses	(HSV)	have	been	the	most	examined	and	best	described	of	

all	human	herpesviruses,	and	it	is	our pleasure	to	have	established	a collaboration	

with	 Gary	 Cohen	 and	 Roz	 Eisenberg on	 co-crystallization	 of	 HSV	 envelope	

glycoproteins	in	complex	with	key	Fab	molecules,	such	as DL11	Fab	bound	to	HSV2	gD.	We	think	that	solving	the	structures	of	these	complexes	will	yield	important	

data	 regarding	 the	 interface	 between	 virus–neutralizing	 antibodies	 and	 their	

epitopes.	The extensive	studies	done	in	the	Cohen	and	Eisenberg	labs,	on	epitope	

mapping	 using	 biochemical	 techniques	 and	 cryo-EM,	 will	 augment	 what	 we	 will	

discover	 from	 the	 structures,	 and	 of	 course	 what	 we	 learn	 will	 be	 of	 value	 to	

understanding	 the	 roles	 of	these	glycoproteins	in	HSV	entry.	As	 our	experiments	

develop,	 a	 deeper	 insight	 into	 how	 antibodies	 block	 important	 glycoprotein	

functions	involved	in	viral	entry	will	be	gained.

We	 thank	 our	 collaborators	for	 continuing	 to	 send	 purified	 recombinant	 gD	 and	

and	Fab	proteins.	As	you	know,	Marija	Backovic	in	the	lab	has	obtained	crystals	of	

HSV-2	gD	bound	 to	 Fab	DL11,	an	important	neutralizing	antibody	against	HSV-2.	

Efforts	to	improve	those	crystals	are	underway.	We	have	open	access	to	the	robotic	

facilities	 for	 protein	 crystallization	 at	 the crystallogenesis	 core	 facility	 of the	

Institute,	 together	 with	 fluent	 synchrotron	 X-ray	 beam	 time	 allocation.	 We	 thus	

Contact PD/PI: Brooks, Benjamin Delbert

Research & Development Outside the U.S. Page 59
Contact PD/PI: Brooks, Benjamin Delbert

believe	 that	 we	 have	 all	 the	 elements	 required	 for	 the	 execution	 of	 our	

collaborative	 project	 of	 co-crystallization	 and	 successful	 structure	 determination	

of	HSV-2	gD-DL11	Fab	complex.	

We	look	forward	to	a	fruitful	collaboration.			

Professor	Felix	A.	Rey	

	 	

Research & Development Outside the U.S. Page 60
Wasatch Microfluidics Commercialization History. 

Wasatch Microfluidics has received 4 Phase II award (less than the 15 required for documentation). 

The NSF Phase II grant had a start date of September 1, 2009. The grant was for the Continuous 

Flow Microspotter which has been successfully commercialized and is currently being sold. 

Wasatch is currently in Phase III for the NIH Phase II that was completed in June of 2013 entitled 

“Multiplexed GPCR Characterization Using SPR Multiplexed GPCR Characterization Using SPR” 

(5R42MH084372-03). Release of this product is scheduled for Q3 of 2017.

Wasatch is in Phase II of a Fasttrack for the third awarded Phase II grant entitled “HT Label-Free 

Screening and Kinetic Analysis of Small Molecules and Biologics” (1R44GM109738-01).

Wasatch just began working on a Phase II of a Direct to Phase II award entitled “Improving vaccine 

development through high-throughput immunogenicity screening“ . 1R44AI127039-01

(GRANT12062711).

Contact PD/PI: Brooks, Benjamin Delbert

Prior SBIR Phase II Awards Page 61
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ❍ Yes ● No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

❍ Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Brooks, Benjamin Delbert

 Page 62
PHS 398 Cover Page Supplement

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302 . Received Date:

2016-09-06T17:27:06.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

❏ Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ● No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Brooks, Benjamin Delbert

 Page 63
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12244575 Funding Opportunity Number: PA-16-302. Received Date:

2016-09-06T17:27:06.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

Research Plan Section

2. Specific Aims aims_PA-16-302.pdf

3. Research Strategy* Research_Plan-v4.5_PA-16-302.pdf

4. Progress Report Publication List

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals

10. Select Agent Research

11. Multiple PD/PI Leadership Plan MultiPI_Leadership_Plan_PA-16-302.pdf

12. Consortium/Contractual Arrangements  

13. Letters of Support Letters_of_Support_2016_NIH_-_PA-16-302.pdf

14. Resource Sharing Plan(s)

15. Authentication of Key Biological and/or

Chemical Resources

Appendix

16. Appendix

Contact PD/PI: Brooks, Benjamin Delbert

 Page 64
Specific Aims

Antibodies are central to modern biomedicine, with their discovery, characterization, and engineering 

experiencing explosive growth, yielding powerful new treatments, and enabling breakthroughs in both 

biotherapeutic and vaccine development. Understanding how antibodies interact with their antigens is critical to 

defining and distinguishing mechanisms of action and even developing improved versions of therapeutic 

antibodies as well as the antigen components of vaccines. While structure determination by x-ray 

crystallography or cryo-EM can define antibody:antigen interactions at atomic resolution, these techniques 

(and other related and even less detailed methodologies) are too expensive and time consuming to support

studies with large sets of antibodies from polyclonal samples or engineered libraries, or likewise large sets of 

antigen variants from diverse populations. At the same time, more experimentally tractable methods, such as 

alanine scanning and pairwise antibody blocking, do not provide nearly as rich or robust information.

In order to scale detailed characterization of antibody:antigen binding to handle entire panels of antibody and 

antigen variants, we seek here to integrate two complementary high-throughput approaches: the experimental 

measurement of binding via multiplexed Wasatch Microfluidics Surface Plasmon Resonance (SPR) and the 

computational modeling and design of interactions. Glycoprotein D (gD) from herpes simplex virus (HSV) 

provides an ideal focus for development, testing, and application of the new approaches, due to the availability 

of a wide variety of antibody and antigen variants and extensive prior low-throughput data for assessing results 

from the new methods. GD also still poses interesting biological questions suitable for study with the new

methods, regarding variation in two HSV serotypes that resulted in failure of a vaccine trial.

The proposed methodologies will address two distinct levels of characterization: 

Aim 1. Define communities of antibodies with similar antigen binding patterns. 

Here, we seek broad strokes across a wide range of antibodies, not being too 

sensitive to small differences, and requiring limited experimental effort. By analyzing

patterns of antibody blocking with a set of antigen variants, our approach will identify 

functionally-related antibodies to infer the general binding regions on the antigen. 

Aim 2. Localize antibody epitopes. Here, we seek to tease apart key contributors 

that can explain and predict subtle but significant impacts on interaction, requiring 

relatively more experimental effort to gain this level of detail. By analyzing binding 

between a panel of antibodies and a panel of natural and computationally designed 

antigen variants, our approach will identify hot-spot residues mediating binding. 

The methods will be tested retrospectively against existing low-throughput data, and applied prospectively to 

predict binding of new antibodies and binding modes to be confirmed by x-ray crystallography.

Strength of the Premise: Other experimental techniques either do not scale or do not robustly provide the 

desired richness of information required to address these aims. Computational techniques are improving but 

are not yet by themselves able to reliably map interactions. The Wasatch SPR instrument provides a wealth of 

data and scales to large panels, but the panels need to be appropriately defined and analyzed. By combining 

computational modeling with Wasatch multiplexed SPR experimental measurement, this proposal thus builds 

on solid technologies and promises to hurdle limitations of existing techniques.

Proposed Innovation: The project will chart as-yet unexplored territory in analyzing data across large panels of 

antibodies and antigens, both carefully defining general binding patterns and specifically localizing binding 

regions. It will integrate computational and experimental methods to rationally design antigenic variants 

(beyond simple alanine scans and natural variants) so as to improve resulting experimental information. 

Unmet Clinical Need and Potential Health Impact: The methods will be broadly applicable in the development 

of vaccines and antibody therapeutics. The specific application to HSV will provide deeper insights into vaccine 

studies and neutralizing antibodies that may be effective against different serotypes.

Team and Outlook: The project brings together investigators with the necessary complementary expertise in 

the instrument (Brooks), the experimental system (Cohen), and the computational methods (Bailey-Kellogg), 

along with collaborators to generate variants (Integral Molecular) and to structurally validate models (Felix Ray, 

Pasteur Institute), see Letters of Support. The successful completion of Phase I will lay the foundation for 

application to additional antigens from HSV and other targets, scale up and engineering of the analysis 

platform for commercial distribution, and incorporation of both more detailed kinetics data and even broader 

antibody and antigen sequence data from next-generation sequencing. 

Positions on HSV gD targeted 

by a few different antibodies.

Contact PD/PI: Brooks, Benjamin Delbert

Specific Aims Page 65
Research Strategy

A. Significance

Due to their inherent capacity to be developed against wide-ranging antigens at high affinity and specificity, 

antibodies are driving much of the therapeutic market1 and provide correlates of protection for most vaccines2

. 

Characterizing antibody:antigen binding, including the localization of epitopes on an antigen that are central to 

recognition by different antibodies, provides important insights into functionality and mechanisms of action3–5. 

Such information is particularly valuable when faced with significant antigenic diversity (e.g., from rapidly 

evolving pathogens) or a large set of lead candidates (e.g., from a polyclonal sample or engineered library)6

. 

Thus the characterization would ideally be performed early in the development of a drug or vaccine, so as to fill 

the pipeline with epitopically-diverse candidates, increasing the number of different shots on goal and reducing 

the risk of exceedingly costly late-stage failure 7

. Unfortunately, while techniques such as x-ray crystallography, 

NMR spectroscopy, and cryo-EM characterize antibody:antigen recognition at atomic resolution, these 

techniques are laborious, expensive, and time-consuming 5,6,8,9, and thus not suitable for studying large sets of 

antibodies and antigenic variants. They are typically employed too late in a development pipeline, and on too 

limited a sample, to identify and help overcome a lack of epitope diversity 7,10–12. 

In order to enable large-scale but still detailed characterization of antibody:antigen recognition, we pursue here 

a novel approach based on surface plasmon resonance (SPR), a mature biosensing technology that provides 

high-resolution data regarding the kinetics and affinity of two (or more) binding partners7

. Wasatch Microfluidics

has developed commercial products with previous SBIR funding, including the commercially-available 

Continuous Flow Microspotter (CFM) and the soon-to-be-released, high-throughput SPR instrument for 

screening antibody candidates. Plans also exist to improve the SPR instrument to possess small-molecule 

sensitivity and 384 spots that allows for >80X increase in throughput versus current SPR systems. 

Of course, kinetics and affinity data for an individual antibody:antigen pair characterize how well they bind, not 

how they bind. However, we hypothesize that the high-throughput multiplexed binding studies supported by the 

Wasatch SPR instrument, over entire arrays of antibodies and antigens, will provide sufficiently rich information 

to enable computational analysis methods to infer and model details of recognition6,7. In particular, by 

characterizing differences in binding across antibody and antigen variants, the methods will group functionally 

similar antibodies and associated antigenic binding regions, as well as more finely localize epitopes of 

particular antibodies7

. Thus this integrated computational-experimental analysis will enable the epitope profile 

of an antigen to be mapped much earlier and in more detail than previously possible, supporting better and 

faster prioritization, reducing failure rates, lowering drug/vaccine costs, and saving lives6,7. The opportunity to 

develop this approach is unique as the extent and quality of binding data is only available with a highthroughput SPR instrument such as the

Wasatch SPR instrument. 

The herpes simplex viruses (HSVs) 

serve as an excellent system for 

developing and applying our new 

methodology, with extensive availability 

of suitable antibodies and antigenic 

variants for experimental evaluation, 

sufficient “ground truth” data by which to 

evaluate the new methods, and 

compelling biological questions to be 

answered with the methods13. As 

enveloped viruses, HSVs must fuse 

their membranes with a host membrane 

for replication to occur. Entry is a 

coordinated process requiring four 

glycoproteins: gD, gB, a heterodimer of 

gH/gL, and either of two cell receptors—

herpes virus entry mediator (HVEM) or 

nectin-1 14. Absence of any single 

protein abolishes entry and cell-cell 

Figure A.1. (A) Previously established relationships of the gD mAbs used in 

these studies. ( ) = regions of gD involved in binding. Magenta = neutralizing 

mAbs. (B) Representation of extracellular domain of gD used in these studies. 

Nectin-1 disrupting mutations are in red. 

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 66
fusion. Each step in this cascade of events leading to fusion is subject to blocking by epitope-specific virusneutralizing mAbs. The Cohen-Eisenberg labs have crystallized and solved the structures of each of the entry 

proteins and the cell receptors, so that epitopes may be mapped onto the 3-D structures of the proteins. In this 

phase I project, we will focus on the gD glycoprotein, for which the Cohen-Eisenberg labs have developed a 

large library of antibodies against both type I and type II HSV (Figure A.1). In addition to generally gaining a 

broader and deeper understanding of the binding patterns in these antibodies and other variants as much still 

remains uncharacterized in the library (especially specific epitopes), we will study an intriguing question about 

antigen diversity raised by a recent vaccine trial. GlaxoSmithKline developed a vaccine using gD from type II 

HSV; unfortunately the vaccine only provided protection against Type II HSV for ~35% of the study 

participants, but surprisingly it provided over 80% protection against Type I HSV15. Studying the large-scale 

impacts of antigenic variability on antibody recognition will be made possible by the proposed approaches. 

Moreover, this work stands uniquely at the intersection between basic studies of recognition and immune 

responses to glycoproteins responsible for virus entry. 

Strength of the premise. The importance of characterizing antibody:antigen binding, particularly localizing 

epitopes, has been well established 5,7,11. Due to the time and expense of structure determination discussed 

above6,12, a variety of other methods have been developed, but they do not provide both the throughput and 

resolution of the proposed approach. For example H-D exchange mass spectrometry is still relatively low 

throughput6,12, while peptide arrays do not use native antigenic structures6,12. In addition, alanine scanning is 

laborious and of a low hit rate16, while at the other extreme shotgun mutagenesis provides a lot of data but not 

of the same resolution and quality as SPR and due to random variation so literally hit-or-miss while not 

capturing impacts due to natural variation17. Purely computational approaches for antibody epitope prediction 

are not yet of sufficient accuracy to be generally useful18,19, though the steady improvement in antibody 

modeling and docking20–27 provides some hope particularly for antibody-specific approaches, especially when 

integrated with experimental data 28 as we propose to do here. In particular, the proposed approach builds 

upon the well-established Wasatch SPR platform, for multiplexed evaluation of a matrix of either 

antibody:antigen binding or antibody:antibody competition for a single antigen. The latter approach is 

commonly termed “binning”, as antibodies possessing similar blocking profiles are placed in the same “bin” 7

. It 

is now commonly being conducted at the earliest possible stages in drug discovery 7,10–12; e.g., Yasmina 

Abidche recently detailed how epitope binning using instrument packages from Wasatch and ForteBio is

improving Pfizer-Rinat’s drug discovery processes29. Thus our proposed method builds upon this strong 

technological platform in order to address a significant gap in large-scale, rapid, detailed characterization of 

antibody:antigen binding. 

B. Innovation

Researchers from both the vaccine community and the antibody therapeutic community need better, faster, 

and more scalable techniques to characterize antibody epitopes, across entire arrays of antibodies and 

antigenic variants. Since neither purely computational nor purely experimental techniques meet these goals, 

our innovative integrated methodology promises to combine the best characteristics of each side. The highthroughput, multiplexed analysis of antibody:antigen binding promises to fill pipelines with epitopically diverse 

candidates, inherently leading to more functional diversity and increased odds of success at the clinical stage.

7

Specific innovations of our proposed approach include the following: 

• Integration of computational docking with large-scale antibody:antigen binding studies. 

Computational methods alone are not yet sufficient to localize epitopes and binding mechanisms, and 

neither are binding studies alone, but the integration of these techniques promises to provide 

complementary insights enabling modeling of the binding.

• Computational design of sets of mutations to test specific binding hypotheses. In contrast to 

alanine scanning, single point mutation, and site saturation, our methods will design mutations based 

on prior knowledge and modeling, so as to best (most efficiently and confidently) evaluate putative 

binding modes.

• Exploitation of natural and artificial antigenic variability. In order to localize epitopes, our methods 

will analyze the relationship between sequence differences and binding differences, inferring which sets 

of residues are likely to play key roles.

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 67
• Systematic analysis of antibody competition assays across multiple antigens. While antibody 

competition with respect to a single antigen has become a standard approach, our approach will 

combine competition results across 

multiple variants to better characterize 

communities.

• Development of reference panels. Our 

computational methods will select among

natural variants and design new ones, so 

as to best support both coarse and fine 

grained epitope localization.

• Computational analysis methods for 

large-scale antibody competition and 

binding studies. Our platform breaks new 

ground in characterizing antibody:antigen 

binding by integrated analysis of the largescale data provided by the Wasatch SPR.

C. Approach 

We will pursue two complementary approaches for 

the systematic characterization of antibody:antigen

binding, requiring different experimental effort and 

yielding representations of different generality and 

specificity (Fig. C.1). 

An antibody vs. antibody binning experiment (Fig. 

C.1, top) evaluates competition between 

antibodies in binding an antigen, and can thereby 

reveal which antibodies are likely to be recognizing 

overlapping regions on the antigen. Thus 

competition results from a large binning 

experiment, as Wasatch SPR can produce, are 

naturally represented in terms of "communities" of 

antibodies that tend to compete with each other. In 

our preliminary results below, we showed that 

binning identified communities consistent with 

previous low-throughput experiments conducted 

over many years, and that binning across

antigenic variants resulted provided important 

information about the epitopic landscape of the protein, has 

been traditionally been much more difficult to obtain. In our 

proposed work, we will extend from using binning only to 

identify antibody communities to also simultaneously 

characterize their rough "footprints" on the antigen. Consider a 

hypothetical scenario where two antigenic variants differ by only 

one amino acid, and the antibodies in a community bind one 

variant but not the other. Intuitively, we can infer that the 

variable amino acid is likely to be important for the binding of 

those antibodies. This intuition also drives the popular but 

limited alanine scanning approach to epitope16, more focused 

site-directed mutagenesis methods17,30 as well literally hit-ormiss approaches like 'shotgun mutagenesis'16. Our proposed 

approach will exploit this insight in the more natural scenario, 

which is also generally more helpful, where there are multiple 

antigen variants and each pair of antigens differs at multiple 

positions (Fig. C.2). 

An antibody vs. antigen binding experiment (Fig. C.1, bottom) 

Figure C.1. Cartoon intuition for the approaches. 

(top, aim 1) Binning-based inference of antibody communities and 

general epitope localization. Each matrix captures binning results 

between each pair of antibodies (rows and columns) competing to 

bind a single antigen; red indicates high competition, yellow medium, 

green low, and gray indeterminate. The data enable the inference of 

antibody communities from mutual competition, as well as general 

regions on the antigen recognized by each community from 

competition differences with respect to antigenic sequence variation.

(bottom, aim 2) Binding-based inference of fine epitope localization. 

The single matrix represents binding between each antibody:antigen

pair (antibodies as rows, antigenic variants as columns), with a 

quantitative binding measurement indicated in grayscale. Once 

again, the impact of antigenic sequence variation on the 

experimental results enables localization of antibody binding, but 

here at a finer resolution due to the larger extent of antigenic 

variability and the incorporation of additional antibody and antigen 

structural modeling.

Figure C.2. Superposition of gD1 structure and gD2 

homology model, with backbones in ribbon mode and side 

chains shown only for positions with different amino acids.

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 68
evaluates the strength of a particular interaction; Wasatch SPR can do this in parallel for a whole matrix of 

pairs of antibodies and antigens. In our preliminary results below, we show that the SPR instrument readily 

enables identification of linear epitopes via antibody:peptide binding. In our proposed work, we will more 

precisely localize the "hotspots" mediating antibody:antigen recognition, with respect to native antigen 

conformations rather than individual peptides, and we will scale up to whole panels of antibodies. As described 

intuitively above, impacts of antigenic variation on antibody binding enables inference of residues important for 

recognition. Here, in characterizing the epitope of a particular antibody, we will have a richer set of variants 

enabling finer grained localization. Our proposed approach will leverage this data with and without reference to 

predictions of putative binding modes by computational docking methods. Furthermore, we will assess the 

augmentation of preexisting natural antigenic variants with new ones that have been computationally designed 

to be informative in revealing epitopes.

Overall, the contrast in the two approaches is that our binning method will generalize across antibodies, 

providing coarse localization information such that, assuming we have a sufficiently comprehensive panel, new 

antibodies are likely to fall into the same communities with the same interaction patterns. On the other hand, 

our mapping method will finely localize a specific interaction in a way that captures details of particular 

antibody:antigen pairs. In both cases, we leverage and assess the impact of antigenic variability, though using 

more extensive variability (at consequently more expense) for binding than for binning. In the binding case, we 

also consider use of antibody sequence (again, at more expense) and computationally hypothesized binding 

modes, and target the design of artificial antigenic variants to provide further information.

Aim 1. Define communities of antibodies with similar antigen binding patterns. 

Preliminary Data. A preliminary epitope binning study was performed by Wasatch and the Cohen/Eisenberg 

teams to evaluate the effectiveness of the high-throughput system for characterizing epitopes of a library of 

mAbs with multiple variants of an antigen. The results set the stage for this Phase I SBIR project. The study

assessed four different gD constructs (truncations 

of length 285 and 306, for both type I and type 2

Figure C.3. Antibody community maps from four gD variants. A 

community plot is a network plot (e.g. social networks) generated 

from the epitope binning heat maps. The different colors represent 4 

Wasatch derived unique communities formed by the binning 

characteristics of 40 different gD monoclonal antibodies obtained 

from 6 different laboratories. Circles represent binding in both 

directions (surface attached and in solution) whereas squares 

represent binding only in one direct. Chord length indicates degree 

of relatedness s defined by relative completion in the binning assay. 

Dashed chord indicate that there is asymmetry in the relationship 

(i.e. blocking does not reciprocate).

Figure C.4. Structural localization of antibody epitopes for 

antibodies of different specificities. The colors on the 3-D 

structures represent the presumed location of two major 

epitopes IA and IB (pink and red) derived by genetic and 

mutagenic analysis. The C-terminus shown in black, lies along 

the intersection of IA and IB and also intersects the Nectin-1 

receptor binding site. The N-terminus (shown in yellow) is 

presented to show the complexity of the Nectin-1 binding face. 

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 69
HSV). The classical sandwich binning assay format was used, with antibody competition used to identify pairs 

of antibodies with overlapping HSV epitopes via Wasatch’s CFM and Ibis’ MX96 surface plasmon resonance 

imager (SPRi) biosensor. Communities of co-competing antibodies were then identified, separately for each

antigen (Figure C.3) which shows how the four general binding communities are related for each mutant. While 

the communities are generally the same between type 1 and 2 for gD 285, important structural differences in 

the proteins are exposed in gD 306 between type 1 and 2 two as shown in the differences between the 

community maps. Importantly, these differences in communities relate to important structural and epitope 

details that had been identified by previous low-throughput methods (Figure C.4). 

Proposed Work: Experimental. The Cohen/Eisenberg Labs at the University of Pennsylvania has available 

multiple gD variants and multiple antibodies (Fig. A,1). These constructs as well as constructs obtained from 

Integral Molecular (see letters of support) will be used to conduct a set of epitope binning experiments for up 

to 60 selected antibodies across at least 10 gD variants, to feed into the computational analysis. 

Proposed Work: Computational. Our goal is to find communities of related antibodies and corresponding 

regions on the antigen that mediate their binding. We will focus on the case where antibody communities are 

already defined by binning as in our preliminary results, since we have shown that that method yields 

communities consistent with external characterizations of related antibodies. But now we will have 

communities identified for multiple different antigenic variants, with amino acid differences in the antigen 

leading to perturbations in binding (see again Fig. C.3) and thereby the competition results and identified 

antibody clusters. Under the assumption that antibodies in a community recognize nearby residues in the 

antigen's structure (since they compete for binding to the antigen), those residues should be spatially proximal 

on the antigenic surface. Thus the approach is to cluster, with respect to the antigenic surface, the variable 

residues that perturb a community. We will implement and assess standard clustering techniques (hierarchical, 

k-means, etc.), with custom metrics that account for a combination of residue proximity on the surface and 

expected vs. observed perturbation due to mutation. Robustness of the identified epitope clusters will be 

assessed by bootstrapping-like techniques, where subsets of antibodies and/or antigens are selected for 

clustering, and consistency of the results characterized (e.g., by region overlap). Accuracy will be evaluated in 

retrospective tests by comparison to known epitope localization obtained from other experimental studies. 

Pitfalls and Alternatives. Given our extensive experience with analysis of binning experiments, and our 

preliminary analysis of gD antigenic variants, we see no significant hurdles to successfully analyzing 

perturbations across multiple antigens and localizing binding regions. If we do not find sufficient local variation 

in the antigens, we will leverage artificial alanine variants, or even computationally designed variants (see aim 

2). On the other hand, if it is necessary to reduce the set of antigens to test, we will employ computational 

sequence analysis to select a relatively diverse pool. Finally, if we find too little consistency of perturbation 

effects within a community, we will move from staged clustering (first antibodies then epitopes) to co-clustering 

(simultaneously).

Aim 2. Localize antibody epitopes. 

Preliminary work: Experimental. A preliminary epitope mapping study was performed by Wasatch and the 

Cohen/Eisenberg teams to evaluate the effectiveness of a high-throughput system for characterizing epitopes 

of a library of mAbs. The 306 truncation of gD1 was represented by an overlapping library of 33 biotinylated 

peptides . All of the known linear epitopes (mainly the brown community in Fig. C.4) were identified. The logical 

extension of this study is to create a larger panel with conformational mutated epitopes to identify 

conformational epitopes.

Preliminary work: Computationally-driven design of disruptive mutations. Building upon their extensive 

track record in developing and applying computational protein design methods to redesign therapeutic proteins 

so as to remove T cell epitopes, the Bailey-Kellogg lab has recently begun developing methods to optimize

variants so as to disrupt protein:protein interactions (e.g., to eliminate allosteric hotspots, antibody binding, or 

aggregation). In the context of disrupting antibody:antigen interactions, an initial cross-validation assessment 

found that a statistical sequence potential like that of Pons et al., trained on a dataset of 250 mutations from 11 

Ab:Ag pairs, yielded ~70% accuracy in predicting disruptive mutations31. 

Preliminary work: Computationally-driven epitope mapping. While computational docking methods are not 

yet sufficient to always correctly identify how an antibody binds an antigen, recent studies have shown that at 

least one near-native conformation is usually found among the sampled poses20. Thus we treat the docking 

poses as hypotheses to be experimentally tested, so as to ferret out the correct one. Building upon the

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 70
disruption design method described in the preceding paragraph, the Bailey-Kellogg lab has developed a 

computational protein design method to optimize, for each such docking hypothesis, a variant whose antibody 

binding should be ablated if the pose is correct. Thus experimental assessment of binding would confirm/reject 

the various docking poses. In order to gauge the feasibility of this approach, we performed an initial 

retrospective analysis of 20 diverse antibody:antigen pairs from SAbDab 32, with server-generated models for 

both antigen33 and antibody26 based on moderate identity templates, combined into server-generated docking 

models 20. Sets of triple mutants were then optimized for each docking model, predicted to disrupt binding. 

Strikingly, at least one of the known epitope residues was targeted for mutation within the top 3 designs for 

80% of the targets, and within the top 5 designs for all targets.

Proposed Work: Computational. Our core approach uses natural antigenic variants and arbitrary antibodies, 

exploiting the same intuition underlying our binning analysis regarding impacts of sequence variation on 

possible binding, but now on a per-antibody basis and with a much larger set of antigenic variants. (The scale 

up is due to the fact that a single binding experiment generates a matrix of antibodies vs. antigens, whereas a 

single binning experiment generates a matrix of antibodies vs. antibodies for a single antigen.) Again, 

clustering methods and metrics will be tested retrospectively with respect to known epitope localization. We will 

then evaluate the utility of computational docking in further refining the analysis. Note that this requires an 

antibody sequence from which to model the antibody structure25–27 and generate antibody:antigen docking 

poses20,22. These poses can then either be integrated with the clustering of perturbed residues (i.e., preferring 

clusters supported by docking) or potentially even supplant them (i.e., directly assessing clusters of docking 

poses for enrichment in perturbed residues). After localizing epitopes as much as possible with natural 

variants, we will use our computational antibody:antigen disruption design to design additional informative 

variants. The antibody and antigen will be re-docked to focus on the epitope regions hypothesized from the 

initial analysis, and mutational variants designed to confirm/reject the docking hypotheses as described in 

preliminary results.

Proposed Work: Experimental. Seven to ten mAbs, including DL1134–36, and at least ten gD variants will be 

selected based on an initial computational analysis of existing data. Antibodies will be sequenced to enable 

computational modeling. An SPR binding experiment will then be conducted across all these antibody:antigen 

pairs, to feed into the computational analysis approach described. Subsequent to this analysis, a follow-up set 

of at least ten antigenic variants will be selected or computationally designed to further disambiguate and 

localize epitopes 

To verify the model, the structure of DL11 will be characterized using X-Ray crystallography in the Felix Rey 

group at the Pasteur Institute as a subcontractor. In addition, virus neutralizations and functional binding 

assays will also be performed to evaluate with the goal of the functionality of the epitopes. In past studies, the 

Cohen/Eisenberg labs have studied these gD mutants against a panel of characterized murine monoclonal 

antibodies (selected from amongst those tested for this grant) possessing virus-neutralizing activity. 

Cohen/Eisenberg groups identified multiple epitopes that elicit strong humoral responses that provided new 

insight into the human response to known neutralizing gD2 epitopes 14,15. 

Pitfalls and Alternatives. Again, given our extensive experience with the computational and experimental 

methods, and our extensive set of available antibodies and antigens, we are confident that this aim will be 

successfully accomplished. As in aim 1, if necessary, computational methods can be used to preselect 

antigens, and alanine variants can also be incorporated.

Phase II Look Ahead. Upon Phase I completion, the team will demonstrate the ability to integrate multiplexed 

experimental binding studies with sophisticated computational analysis methods to characterize 

antibody:antigen binding more broadly and more deeply than previously possible at this throughput.. In Phase 

II, Wasatch will integrate the analysis and visualization tools into its custom epitope binning and mapping 

software that is sold currently with our Wasatch SPR instrument packages, enabling facile analysis of the 

results by scientists. In addition, additional targets within the HSV system will be used to further apply the 

algorithms, with selective validation (as prioritized by the analysis) through our collaboration with the X-Ray 

crystallography group at the Pasteur Institute directed by Felix Rey (see letters of support). Also, the algorithms 

will be expanded to leverage kinetics and quantitative affinity data. Lastly, the approach will be applied at an 

even larger scale in the context of next-generation sequencing (NGS) of antibody libraries and natural 

repertoires. Wasatch can then commercialize this approach as a structural characterization tool to can 

fundamentally shift the focus of vaccine and drug development to leverage this information more extensively 

and earlier in the process. 

Contact PD/PI: Brooks, Benjamin Delbert

Research Strategy Page 71
Multiple PI Leadership plan:

Benjamin Brooks, Gary Cohen, Roselyn Eisenberg and Chris Bailey-Kellogg: Multiple PD/PI 

leadership plan

This application has a Primary Investigators (B. Brooks with Wasatch Microfluidics) with three additional 

consulting investigators, (Chris Bailey-Kellogg with Dartmouth College and Dr. Gary Cohen/Dr. Roselyn 

Eisenberg for the University of Pennsylvania). PI Brooks will provide the overall management of the project. 

Dr. Bailey-Kellogg will provide the management and computer programming. Drs. Cohen and Eisenberg will 

provide HSV glycoproteins, monoclonal antibodies to those proteins and human as well as guinea pig sera 

obtained from collaborations with Drs. Friedman, Bernstein and Cardin (see letters). 

PI Ben Brooks provides expertise in assay development, microfluidic printing, and SPR Imaging design. PI 

Gary Cohen and his colleague Roselyn Eisenberg provide vaccine development management expertise. PI 

Bailey Kellogg provides extensive expertise in computer algorithm for predicting protein-protein interaction. 

Each will be responsible for corresponding components of the project. 

PIs Brooks, Bailey-Kellogg and Cohen will converse regularly including monthly group meetings, while they 

each meet with employees and students assigned to their portions of projects weekly, or more often as 

needed. In cases of disagreement or discussion, we will defer to the PI with the most expertise in the 

particular area. The entire team will meet monthly as noted, and overarching project aims will be discussed 

and progress assessed. Adjustments will be made as needed by forming a consensus within the group. 

Meeting organization and reports will be the responsibility of PI B. Brooks. 

As needed, either PI Brooks will fly to the other’s facility for face to face management meeting, for 

troubleshooting, and for cross-training. Currently, a senior application scientist from Wasatch

(Noah Ditto) and Wasatch equipment (CFM and SPRi) are collocated within the Cohen lab to 

facilitate communication and workflow. 

Contact PD/PI: Brooks, Benjamin Delbert

Multiple PD/PI Leadership Plan Page 72
References

1. (Rob) Aggarwal, S. What’s fueling the biotech engine—2012 to 2013. Nat. Biotechnol. 32, 32–39 (2014).

2. Plotkin, S. A. Correlates of Protection Induced by Vaccination. Clin. Vaccine Immunol. 17, 1055–1065 

(2010).

3. Ladner, R. C. Mapping the epitopes of antibodies. Biotechnol. Genet. Eng. Rev. 24, 1–30 (2007).

4. Liljeroos, L., Malito, E., Ferlenghi, I. & Bottomley, M. J. Structural and Computational Biology in the Design 

of Immunogenic Vaccine Antigens. J. Immunol. Res. 2015, (2015).

5. Abbott, W. M., Damschroder, M. M. & Lowe, D. C. Current approaches to fine mapping of antigen-antibody 

interactions. Immunology 142, 526–535 (2014).

6. Noah Ditto & Benjamin Brooks. The emerging role of biosensor-based epitope binning and mapping in 

antibody-based drug discovery. Expert Opin. Drug Deliv. (2016). 

doi:http://dx.doi.org/10.1080/17460441.2016.1229295

7. Abdiche, Y. N. et al. High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can 

Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with 

Functional Activity. PLoS ONE 9, e92451 (2014).

8. Garman, E. F. Developments in x-ray crystallographic structure determination of biological 

macromolecules. Science 343, 1102–1108 (2014).

9. Zheng, H. et al. X-ray crystallography over the past decade for novel drug discovery–where are we 

heading next? Expert Opin. Drug Discov. 10, 975–989 (2015).

10. Benjamin D. Brooks. The Importance of Epitope Binning for Biological Drug Discovery. Curr. Drug Discov. 

Technol. 14, (2014).

11. Brooks, B. D., Miles, A. & Abdiche, Y. High-throughput epitope binning of therapeutic monoclonal 

antibodies: why you need to bin the fridge. Drug Discov. Today doi:10.1016/j.drudis.2014.05.011

12. Davidoff, S. N., Ditto, N. T., Brooks, A. E., Eckman, J. & Brooks, B. D. Surface Plasmon Resonance for 

Therapeutic Antibody Characterization. Label-Free Biosens. Methods Drug Discov. 35–76 (2015).

13. Eisenberg, R. J. et al. Herpes Virus Fusion and Entry: A Story with Many Characters. Viruses 4, 800–832 

(2012).

14. Eisenberg, R. J. et al. Herpes virus fusion and entry: a story with many characters. Viruses 4, 800–832 

(2012).

15. Awasthi, S., Shaw, C. & Friedman, H. Improving immunogenicity and efficacy of vaccines for genital 

herpes containing herpes simplex virus glycoprotein D. Expert Rev. Vaccines 13, 1475–1488 (2014).

16. Cunningham, B. C. & Wells, J. A. High-resolution epitope mapping of hGH-receptor interactions by 

alanine-scanning mutagenesis. Science 244, 1081–1085 (1989).

17. Davidson, E. & Doranz, B. J. A high-throughput shotgun mutagenesis approach to mapping B-cell antibody 

epitopes. Immunology 143, 13–20 (2014).

18. Kringelum, J. V., Lundegaard, C., Lund, O. & Nielsen, M. Reliable B Cell Epitope Predictions: Impacts of 

Method Development and Improved Benchmarking. PLOS Comput Biol 8, e1002829 (2012).

19. Ponomarenko, J. V. & Bourne, P. E. Antibody-protein interactions: benchmark datasets and prediction 

tools evaluation. BMC Struct. Biol. 7, 64 (2007).

20. Brenke, R. et al. Application of asymmetric statistical potentials to antibody–protein docking. Bioinformatics

28, 2608–2614 (2012).

21. Krawczyk, K., Liu, X., Baker, T., Shi, J. & Deane, C. M. Improving B-cell epitope prediction and its 

application to global antibody-antigen docking. Bioinforma. Oxf. Engl. 30, 2288–2294 (2014).

22. Sircar, A. & Gray, J. J. SnugDock: paratope structural optimization during antibody-antigen docking 

compensates for errors in antibody homology models. PLoS Comput. Biol. 6, e1000644 (2010).

23. Lensink, M. F. & Wodak, S. J. Docking, scoring, and affinity prediction in CAPRI. Proteins 81, 2082–2095 

(2013).

24. Kozakov, D. et al. How good is automated protein docking? Proteins 81, 2159–2166 (2013).

25. Sivasubramanian, A., Sircar, A., Chaudhury, S. & Gray, J. J. Toward high-resolution homology modeling of 

antibody Fv regions and application to antibody-antigen docking. Proteins 74, 497–514 (2009).

26. Marcatili, P., Rosi, A. & Tramontano, A. PIGS: automatic prediction of antibody structures. Bioinformatics

24, 1953–1954 (2008).

27. Marze, N. A., Lyskov, S. & Gray, J. J. Improved prediction of antibody VL-VH orientation. Protein Eng. Des. 

Sel. PEDS (2016). doi:10.1093/protein/gzw013

Contact PD/PI: Brooks, Benjamin Delbert

References Cited Page 73
28. Sela-Culang, I., Ofran, Y. & Peters, B. Antibody specific epitope prediction-emergence of a new paradigm. 

Curr. Opin. Virol. 11, 98–102 (2015).

29. Miles, Adam & Abdiche, Yasmina. High-throughput epitope binning assays on label-free array-based 

biosensors can yield exquisite epitope discrimination that facilitates the selection of clones with functional 

activity. PLoS ONE

30. Benjamin, null & Perdue, null. Site-Directed Mutagenesis in Epitope Mapping. Methods San Diego Calif 9,

508–515 (1996).

31. Pons, C., Talavera, D., de la Cruz, X., Orozco, M. & Fernandez-Recio, J. Scoring by Intermolecular 

Pairwise Propensities of Exposed Residues (SIPPER): A New Efficient Potential for Protein−Protein 

Docking. J. Chem. Inf. Model. 51, 370–377 (2011).

32. Dunbar, J. et al. SAbDab: the structural antibody database. Nucleic Acids Res. 42, D1140–D1146 (2014).

33. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment 

for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).

34. Whitbeck, J. C. et al. Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes 

simplex virus 2 glycoprotein D. J. Virol. 88, 7786–7795 (2014).

35. Chen, K.-J. A thermoresponsive bubble-generating liposomal system for triggering localized extracellular 

drug delivery. ACS Nano 7, 438–446 (2013).

36. Whitbeck, J. C. et al. The major neutralizing antigenic site on herpes simplex virus glycoprotein D overlaps 

a receptor-binding domain. J. Virol. 73, 9879–9890 (1999).

Contact PD/PI: Brooks, Benjamin Delbert

References Cited Page 74
Contact PD/PI: Brooks, Benjamin Delbert

Letters of Support Page 75
Contact PD/PI: Brooks, Benjamin Delbert

Director, Botswana-UPenn Partnership

Harvey M. Friedman, M.D.

Professor of Medicine

522E Johnson Pavilion 

Philadelphia, PA 19104-6073 

Tel: 215.573.8432 

Fax: 215.349.5111 

hfriedma@mail.med.upenn.edu

website: http://www.upenn.edu/botswana/

August 4, 2016 

Gary H. Cohen 

School of Dental Medicine 

University of Pennsylvania 

Philadelphia PA 19104 

Dear Gary: 

I am pleased to join you in collaborating with Wasatch Microfluidics who have existing technology 

that improves the throughput over more traditional biosensor instruments such as the 

BIAcore. The Wasatch approach allows for analysis of many more samples in much less time. I am 

pleased to be included as a collaborator with you and Wasatch in the submission of an SBIR 

application to evaluate the performance of a second-generation instrument that combines two current 

technologies for printing and imaging into a single unit. Thus, I anticipate that we will be in a position 

to evaluate the array of antibody epitopes present in the sera of immunized animals that we have 

available and currently are in the process of obtaining. These samples are from guinea pigs immunized 

with HSV-2 gD2 subunit glycoproteins. The new instrument that you are evaluating will be an 

invaluable addition to a full understanding of the immune responses in our animal experiments. We 

look forward to a more thorough epitope analysis and will be pleased to collaborate with you on this 

project. 

Sincerely, 

Harvey Friedman 

Letters of Support Page 76
Contact PD/PI: Brooks, Benjamin Delbert

Letters of Support Page 77
02032011

Pfizer Inc

235 East 42nd Street

New York, NY 10017

 Worldwide Research & Development

Tuesday, September 6, 2016

825 North 300 West, C309

Salt Lake City, UT 84103

Reference: Non-Binding Letter of Support for Wasatch Microfluidics

Dear Dr. Brooks

Thank you for contacting us on a non-confidential basis seeking a letter from us to give non-binding and

general support of the grant that you are seeking from the SBIR. Within my role as leader of the 

bioanalytical group at Rinat-Pfizer, I have been collaborating with the Wasatch team for the past three years 

and have acquired first-hand, in-depth working knowledge of their platform in the context of monoclonal 

antibody characterization. The Wasatch system is helping to fill a gap between the capacity to generate 

antibodies and our industry’s ability to characterize them.

This area of research may be of potential interest to Pfizer in the future and as you continue to develop your 

technology related to this field, we would be interested in maintaining an open dialogue with you on a nonconfidential basis (unless we agree otherwise in writing) regarding such technology and any potential related 

emerging needs at Pfizer. Any such potential future interest by Pfizer will be predicated on several factors, 

including Pfizer’s strategic needs, agreement upon business terms, and Pfizer’s completion of an appropriate 

due diligence process. However, for clarity, neither Pfizer nor you is in any way obliged to negotiate or enter 

into any agreement in relation to this work or any research performed in relation to this work or otherwise. 

We wish you every success in attempting to address this important area of research through your pursuit of 

the grant from the SBIR. 

Sincerely,

Yasmina Abdiche

Contact PD/PI: Brooks, Benjamin Delbert

Letters of Support Page 78
Professor	Felix	A.	Rey

Unité	de	Virologie	Structurale

Institut	Pasteur	-	CNRS	UMR	3965

25	rue	du	Docteur	Roux

Paris	75015	France

Tel	:	+33	1	45	68	85	63

e-mail	rey@pasteur.fr

Paris,	August	18th 2016

OBJECT:	 Collaboration	 letter	 for	 NIH	 small	 business	 grant	"Predicting	 Epitopes	 in	

Vaccine	and	Therapeutic	Antibody	Research”		

To: Gary	Cohen	and	Roselyn	Eisenberg,	Benjamin	Brooks,	Chris	Bailey-Kellogg	

The	Structural	Virology	Unit of Institute	Pasteur	in	Paris studies viruses	 of	global	

public	health	and/or	of	veterinary	concern by	using	structural	biology	techniques,	

primarily	 X-ray	 crystallography.	 The	 knowledge	 gained	 can	 be	 used	 for	

translational	structure-based	design	of	preventive	or	curative	antiviral	agents.	One	

of	 our	 scientific	 goals	 is	 to	 provide	 a	 structural	 basis	 for	 understanding	 the	

molecular	mechanisms	of	membrane	 fusion	used	by	enveloped	viruses	 to	enter	a	

target	 cell.	 Herpesviruses	 are	 important	 human	 pathogens	 and	 they	 present	 an	

interesting	and challenging	model	for	studying	this	process.	We	have been	using an	

animal	 herpesvirus	 called	 pseudorabies,	as	well	as	 human	 herpesviruses	 such	as	

cytomegalovirus	and	human	herpesvirus-8	as	model	systems	in	our	lab.		

Herpes	simplex	viruses	(HSV)	have	been	the	most	examined	and	best	described	of	

all	human	herpesviruses,	and	it	is	our pleasure	to	have	established	a collaboration	

with	 Gary	 Cohen	 and	 Roz	 Eisenberg on	 co-crystallization	 of	 HSV	 envelope	

glycoproteins	in	complex	with	key	Fab	molecules,	such	as DL11	Fab	bound	to	HSV2	gD.	We	think	that	solving	the	structures	of	these	complexes	will	yield	important	

data	 regarding	 the	 interface	 between	 virus–neutralizing	 antibodies	 and	 their	

epitopes.	The extensive	studies	done	in	the	Cohen	and	Eisenberg	labs,	on	epitope	

mapping	 using	 biochemical	 techniques	 and	 cryo-EM,	 will	 augment	 what	 we	 will	

discover	 from	 the	 structures,	 and	 of	 course	 what	 we	 learn	 will	 be	 of	 value	 to	

understanding	 the	 roles	 of	these	glycoproteins	in	HSV	entry.	As	 our	experiments	

develop,	 a	 deeper	 insight	 into	 how	 antibodies	 block	 important	 glycoprotein	

functions	involved	in	viral	entry	will	be	gained.

We	 thank	 our	 collaborators	for	 continuing	 to	 send	 purified	 recombinant	 gD	 and	

and	Fab	proteins.	As	you	know,	Marija	Backovic	in	the	lab	has	obtained	crystals	of	

HSV-2	gD	bound	 to	 Fab	DL11,	an	important	neutralizing	antibody	against	HSV-2.	

Efforts	to	improve	those	crystals	are	underway.	We	have	open	access	to	the	robotic	

facilities	 for	 protein	 crystallization	 at	 the crystallogenesis	 core	 facility	 of the	

Institute,	 together	 with	 fluent	 synchrotron	 X-ray	 beam	 time	 allocation.	 We	 thus	

Contact PD/PI: Brooks, Benjamin Delbert

Letters of Support Page 79
Contact PD/PI: Brooks, Benjamin Delbert

believe	 that	 we	 have	 all	 the	 elements	 required	 for	 the	 execution	 of	 our	

collaborative	 project	 of	 co-crystallization	 and	 successful	 structure	 determination	

of	HSV-2	gD-DL11	Fab	complex.	

We	look	forward	to	a	fruitful	collaboration.			

Professor	Felix	A.	Rey	

	 	

Letters of Support Page 80