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<div class="ocr">Confidential Business Information-Deleted Risk Analysis
Consisting of
The Summary Information Format & Risk Assessment
for

Foot and Mouth Disease Virus Vaccine, Live Adenovirus Vector

PerOS USA Inc. (under contract with GenVec, Inc.)

U.S. Veterinary Establishment No. 419

</div>

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<div class="page"><p></p>
<div class="ocr">Category III
Summary Information Format (SIF)

Foot and Mouth Disease Virus Vaccine,

Serotype | Live Adenovirus Vectored

UPDATED

Version Date: 03Apr09

PerOs USA Inc. (under contract with GenVec, Inc.)

U. S. Veterinary Establishment No. 419

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April 3, 2009

SIGNATURES:

Page 2 of 27

GenVec, Inc.

GenVee, Inc.

GenVec, Inc.

Date

Date

Date
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April 3, 2009

TABLE OF CONTENTS
I. INTRODUCTION 0... ccceeceeseceeenseesensecaceecenseseecesenseenaeeeseseaecsaeseaeseseesneeeenecsssessonsoneeneneeseaes 6
A. ODJOCtIVE oe eeecccccsteceetcessseeesneceseeesseeeeeseceesnesnereeeseeeeeeeecceceeseesneasesessntenseestensssnsessesses 6
B. Proposal... eeeesceeeceseeseeceseesseeeseeeeseessesaessecssessaessesseeceeasnssccaseseesnsesssassssssesseesssassaseasenseeaes 7
Il. DESCRIPTION OF THE REGULATED BIOLOGICAL AGENT CONSTRUCTION ......... 8
A. The Backbone Biological Agent... ce eeesecseeceeceeeseeeessecneusenesseessesseseesessessaeenesseseas 8
B. Donor Biological Agents and Donor DNA of Gee .........:ccceecceseeeceteceeeeteneceeereesserenersees 12
C. Construction and Characterization of the Regulated Biological Agent... ee 14
Ill. BIOLOGICAL PROPERTIES OR VIRULENCE FOR THE REGULATED BIOLOGICAL
AGENT USED FOR MASTER SEED.\.u... ec cccceceneeeeeeecneeneeeeesaeseeeeaeeaeeeeseeesesseseenetneaeseneee 20
A. What are the known phenotypic characteristics or effects of the Regulated Biological
Agent identified as Master Seed? oo... ccceceesccseeseceneeeeereseseaeceseeeaeenaecaeeaseateaeeaeeeaeeeneaees 20
B. What are the virulence characteristics of the Regulated Biological Agent used for the
Master Seed? oo... eeesesscsseesseseeceeeneceeeseesseesseesecsaecaesseeeaeseceeeseeeaesnaeeneseeseaesserasesaesesenees 2]
C. Is the Regulated Biological Agent used for the Master Seed virulent for target animals,
NONn-target ANiMals? ....... ce cccceseesscesceenscsscesssceseccsssecessessaeecensecuesesseecesesessceseceaeseneenseeeees 21
D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to
survive in the target animals, non-target animals?............ccccccsseceeeeeeeeeeseeeeeceneetaeeneeeeeens 21
E. Discuss the potential for horizontal gene transfer or recombination of the Regulated
Biological Agent. 0.0 ..cccccccscccctscssscssecssccseesseeeeeecsnecceseecssseseusecesueceseeceseseasseaeteneseneeeresseeed 23
F. Describe the Shed/Spread Capabilities of the Regulated Biological Agent... 24
G. Discuss the expected environmental impact or survivability of the Regulated
Biological Agent, and provide available scientific evidence. ..........:ecceseecceceesetseteeeeeeenee 26
FIGURES

Figure 1: Construction of the 7 base plasmid (The Backbone Biological Agent)

Figure 2: Nucleotide sequence of the
Figure 3: Schematic of

Figure 4: Construction of the Regulated Biological Agent
Figure 5A: Donor Biological Agent (Foot and Mouth Disease Virus Serotype

and [J deletion junctions (GenVec Research

Report , March 2007)

. Numbers correspond
to nucleotide position in

Figure 5B: Donor Biological Agent (Foot and Mouth Disease Virus Serotype f

Figure 6: The Regulated Biological Agent [I
Figure 7: Genetic Structural Identity assay by PCR
Figure 8: Western blot of the RBA vector expressing [ protein

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April 3, 2009

APPENDICES

Appendix A Part 1. Adenovirus Containment Level, NIH Office of Biotechnology Activities
Part 2. The SACGM Compendium of Guidance of Adenovirus BL containment
requirements (section 2.7)

Appendix B

Appendix C

Appendix D

Appendix E_ Electronic DNA Sequence File, RBA transgene region

Appendix F Part 1. Mayr et. al; 1999. Virology 263:496-506
Part 2. Mayr et. al.; 2001. Vaccine 19:2152-2162

Appendix G Mouse Safety Test

Appendix H 10X Master Seed Calf Safety Test

AppendixI Master Seed Backpassage Study of Foot and Mouth Disease Virus Vaccine,

Serotype 7 Live Adenovirus Vector Product Code 1FM1.R0, Master Seed

Appendix J Master Seed Shed and Spread Study of Foot and Mouth Disease Virus Vaccine,

Serotype 7 Live Adenovirus Vector Product Code 1FM1.R0, Master Seed

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GLOSSARY
: A replication-defective adenovirus 5 encoding FMDV fF

: An adenovirus encoding the FMDV Po

Ad.Null: An adenovirus vector based upon the BBA with no donor transgene insertion, for
use as a vector control.

APHIS: Animal and Plant Health Inspection Service. An agency under the USDA that is
responsible for protecting and promoting U.S. agricultural health, administering the Animal
Welfare Act, and carrying out wildlife damage management activities

ARS: Agricultural Research Service

BBA: Backbone Biological Agent

BL: Biosafety Level

CAR: Coxsackie-Ad Receptor

CDC: US Centers for Disease Control

DIVA: Differentiating infected from vaccinated animals

DHS: US Department of Homeland Security

CMV: Cytomegalovirus

DOI: Duration of immunity

FDA: US Food and Drug Administration

FFU: Focus Forming Units

FMDV: Foot and Mouth Disease Virus

FRR: Final Research Report

GSI: Genetic Structural Identity

HSPG: Heparin Sulfate Proteoglycans

IM: Intramuscular

IVP: Investigational Veterinary Product

MSV: Master Seed Virus

NIH: National Institutes of Health

OIE: World Organization for Animal Health; also known as Office International des
Epizooties. Recommends sanitary regulations for trade in animals and animal products and
establishes appropriate testing procedures.

OOI: Onset of immunity

PLS: Prelicense Serial

PU: Particle Unit

RBA: Regulated Biological Agent

RCA: Replication Competent Adenovirus
REA: Recombinant El Adenovirus Assay
RNA: Ribonucleic acid

SV40: Simian Virus 40

TetR: Tetracycline Repressor

TIS: Transcriptionally Inert Spacer
USDA: US Department of Agriculture:
UTR: Untranslated Region

VSS: Vector Seed Stock (pre-master seed)

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April 3, 2009

J. INTRODUCTION

Foot-and-mouth disease (FMD) is a highly contagious viral disease of livestock. Although FMD
is not currently present in North America, it is endemic in over 70 countries in South America,
Africa, Middle East, Asia and the Pacific Rim. The causative agent, foot-and-mouth disease
virus (FMDV) is a single, positive stranded RNA virus belonging to the Picornaviridae family.
FMDV spreads quickly between animals, and herds, resulting in devastating epizootics and
significant economic hardship primarily due to OIE imposed trade barriers. While outbreaks are
normally controlled by slaughter and vaccination with chemically inactivated virus, concerns
with the effectiveness and onset of immunity (OOD of current vaccines and the fact that Federal
law prohibits their production in the United States (US) creates a significant need for new
genetically engineered (molecular), marker FMD vaccines as alternatives. A US government
interagency blue ribbon panel has identified FMD as the highest priority threat to the US
livestock industry. The objective is to discover and develop FMD molecular vaccines that have
been specifically designed for the control and eradication of FMDV to protect our Nation against
intentional or accidental outbreaks of this disease. This Summary Information Format (SIF)
describes the discovery and development of an experimental FMD lead vaccine candidate against
FMDV (vaccine specific for FMDV serotype i). This vaccine candidate will serve as the
roadmap for the future development of FMD molecular vaccines specific for other FMDV
serotypes and subtypes (e.g. a. The strategic use of these products will be
determined by USDA APHIS Emergency Programs, including possible deployment of vaccine
from the National Veterinary Stockpile in the event of an FMD outbreak. FMD molecular
vaccine products will be manufactured in the US, provide adequate OOI and duration of
immunity (DOD), prevent the shed and spread of FMDV infection, and will be compatible with
differentiating infected from vaccinated animals (DIVA) diagnostic tests currently in
development by USDA APHIS.

A. Objective

1. Identify where the Regulated Biological Agent was constructed and where the product
will be made, tested, and manufactured. Address the available level of containment.

The Regulated Biological Agent (RBA), adenovirus vector [I is a

replication defective Human Adenovirus serotype 5-vectored FMD vaccine that was constructed

at GenVec, Inc. (Gaithersburg, MD). The adenovirus vector, , was
constructed by insertion of the structural capsid gene of FMDV serotype and the
nonstructural protease gene of FMDV serotype |. The vector

seed stock (VSS), was prepared and tested at the GenVec research laboratories in Maryland,
under NIH-defined conditions for Biosafety Level 2 (BL-2) laboratories.

Efficacy testing in cattle was performed at the
. The PIADC facilities are collaboratively managed by , and are operated at

Biosafety Level 3 (BL-3).

The US location for RBA manufacturing will be [M. The RBA will be manufactured
and tested within the PerOs USA Inc., facility (Est. No. 419) located at

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The PerOs USA Inc. establishment is managed on behalf of PerOs USA Inc. by Benchmark
Biolabs personnel. Both locations have been inspected by USDA APHIS CVB-IC for
manufacturing and testing the RBA. These facilities include BL1 and BL2 areas designated for
use in veterinary biologics production and testing in compliance with USDA APHIS CVB
regulation.

2. Provide a brief (one sentence) description of the Regulated Biological Agent.

is a replication deficient, recombinant human adenovirus serotype 5
(Ad5) viral vector carrying the inserted structural capsid gene [I and nonstructural
protease gene [MY of FMDV, which when used as a cattle vaccine, induces an immune
response against FMDV serotype im while allowing differentiation of vaccinates from
naturally infected animals.
B. Proposal
1. What is the intended use of the product?

The product will first be developed for use in efforts aimed at the control and eradication of
intentional or accidental outbreaks of FMDV in cattle which might occur in the United States.

the target animal for this vaccine is cattle, the intended market is the United
States national defense stockpiling for emergency vaccination, and any future claim expansions
fall outside the scope of this document.

a. Species: Bovine.
b. Proposed claim: Recommended as an aid in the prevention of FMDV, a. in cattle.
c. Geographic area: The US (in connection with a USDA emergency program and/or veterinary
stockpile) and in countries where FMD is endemic.
d. Route of administration: Intramuscular (IM).
e. Brief description of the expected safety profile: Human adenoviruses cause mild cold-like
symptoms in humans but are not considered pathogens of cattle or other animals. The human
adenovirus vector used to produce this vaccine has been further attenuated by rendering it
replication defective by complete deletions of the || and || genes from the virus. Only 2 genes
from the FMD virus are inserted into this replication defective vector, and neither of the two
inserted FMD genes (the structural capsid gene fF | and the nonstructural protease gene

is associated with FMDV virulence nor are any of the FADV genomic sequences used
for virus specific replication included in the construct. This vector has been shown to be
incapable of establishing itself in injected cattle in a backpassage/reversion to virulence study
(Appendix I). The vector is also not expected to be shed from vaccinates, as has been
demonstrated in a shed/spread study (Appendix J). The resulting regulated biological agent
(RBA), a replication-deficient FMD-expressing human adenovirus serotype 5, is expected to be
safe for use as a parenterally administered vaccine in cattle, and is also expected to be safe for
the environment.

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April 3, 2009

II. DESCRIPTION OF THE REGULATED BIOLOGICAL AGENT
CONSTRUCTION

A. The Backbone Biological Agent

1. What organism was used for the Backbone Biological Agent? Are there any known
virulence features associated with the Backbone Biological Agent? What happens in the
target species?

The Backbone Biological Agent (BBA) for I consists of a Human
Adenovirus C, serotype 5 vector (adenovector). Wild-type human adenovirus serotypes cause

common cold symptoms in humans. Since adenovirus infection is species-specific, human

adenovirus serotypes are incapable of infecting the target species (bovine) and hence avirulent in
cattle.

The Human Adenovirus C, serotype 5 (Ad5) genome with deletions in the i. and

regions is replication deficient in target host cells. The || region deletion, including the

and ij promoters and open reading frames, renders the adenovector replication deficient. The
region is not essential for growth of Ad5 in tissue culture. The [ff deletion eliminates. _

essential elements for Ad5 replication through removal of the || open reading frames. There is

also a two base deletion in the [ region that interrupts a minor alternative transcription

start site. The multiple-deletion adenovector backbone is termed |. The

adenovector containing partial deletion of the [J domain and complete deletions of the [Jf and

| domains is incapable of replicating in normal mammalian cells (see Figure 1).

Using GenVec’s proprietary manufacturing technology, the | | adenovirus backbone was
chosen for FMDV vaccine vector generation to reduce the risk of replication-competent
adenovirus (RCA) generation during vaccine production and testing, and during later field usage
of the product. The backbone contains deletions of two regions essential for adenovirus
replication, [fj and ff, as well as a IJ deletion. The generation of an RCA would
require two independent recombination events in a single adenovirus genome. Although the rate
of RCA generation has not been determined, it is predicted to be extremely rare. Two events
would be required and any recombination event in [J would be of the infrequent non-
homologous type. The RCA rate in a first-generation Ad5 system, which measures one
homologous recombination event, was recently reported to be 5.6 x 107° to 6.7 x 107! RCA per
infectious unit (Duigou, G.J.; et. al., 2005. J. Virol. 79(9), 5437-5444). Thus, the frequency of
two events would be <10”° RCA per particle unit. The specific backbone used for this
construction, “ P, contains a sequence in the
deleted region.

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April 3, 2009

a. What is the previous safe use of the Backbone Biological Agent?

As described in the previous section, GenVec’s proprietary platform technology uses replication-
deficient human adenovectors for both therapeutic and vaccine applications in humans. This
technology was also recently used in proof of concept studies involving cattle and swine.

(i) If available, provide history of previous safe use, using published literature or internal
documents. Include the recommended CDC/NIH biosafety level for use of the Backbone
Biological Agent.

Wild-type human adenoviruses have been classified by CDC/NIH for handling as BL-2 agents.
Modification of the adenovirus classification for replication deficient vectors such as the |
BBA has been suggested as a possibility by NIH when such reclassification is supported by
appropriate risk assessment (RA) documentation. (Refer to Appendix A, part 1). Recent
guidance from the Science Advisory Committee on Genetic Modification (SACGM) also
suggests reclassification of [MM-deleted adenovirus vectors could be expected to occur as
the result of proper risk assessment documentation. (Refer to Appendix A, part 2).
Accordingly, the proprietary GenVec Ad5 i Backbone has been handled as a BL-2 agent,
and will continue to be until it has been formally re-classified consistent with use as a live
vaccine vector as a result of review and approval of documents such as this SIF and
accompanying RA by the relevant government agencies.

The GenVec Adenovirus Type 5 I backbone is considered to be fully attenuated for humans
and, in fact, is presently being studied for use as a vector for medical products.

GenVec and USDA/ARS collaborated in a proof of concept study using replication deficient
Human Adenovirus C, serotype 5 (Ad5) vectored type I Interferon biologicals in
swine. Twenty (20) pigs
, at

Final Research Report

were administered the investigational veterinary product (IVP),

concluded that neither the adenovirus nor the
appeared to have deleterious effects in swine. contain
the same deletions as described in this document for the BBA (Figure 1).

GenVec replication deficient Human Adenovirus C, serotype 5 (Ad5) vectored FMDV subunit
vaccines have undergone proof of concept studies for IM administration in cattle under
GenVec/USDA/DHS collaborations. These studies were conducted at MJ under BL-3Ag
conditions since these studies involved live FMDV challenge. All studies used the same gene
deletions in the Human Adenovirus C, serotype 5 (Ad5) as described for the BBA (Figure 1).
Three completed studies are discussed below.

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GenVec FRR study number Si
BE <v aluated the safety of two replication defective Ad5-vectored test vaccines

produced in complementing cell lines ( ). The results
showed that vaccine candidate is safe in cattle following a single immunization

IM. Eighteen (18) cattle were administered [VP during this study. No untoward systemic or
local reactions were observed during or immediately following administration of the test
vaccines, and no injection site swellings were observed in any of the eighteen (18) vaccinates.
Safety evidence for lack of | vectored vaccine shed was supported by the absence
of seroconversion to Ad5 in the ten (10) non-vaccinated controls co-mingled with vaccinates for
a period of three weeks post injection

GenVec FRR study number

evaluating the antigenic, safety, and efficacy of a Human Adenovirus C,
Ad5 FMDV subunit vaccine AdS | observed that there were no untoward systemic or local
reactions observed immediately following administration of Adil in five (5) cattle. There was
no injection site swelling in any of the five (5) vaccinates during the observation period (24 — 73
hours post-immunization). Safety evidence for lack of Ad5 im vectored vaccine shed was
supported by the absence of seroconversion to Ad5 in five (5) naive cattle co-mingled with
vaccinates during the three-week study. The study demonstrated that the IVP was not shed from
vaccinates in sufficient amounts to cause production of Ad5-specific vaccine vector antibody in
contact exposed cattle (see Section III.F. regarding shed/spread)

GenVec FRR study
evaluated the antigenic and safety properties of a replication deficient

Human Adenovirus C, Ad5 fF vectored FMDV subunit vaccine (| produced in
EE ells. The report concluded that the vaccine,

administered by the IM route was safe in cattle. This was based on the absence of detectable
local or systemic reactions following a single dose immunization in five (5) cattle. No injection
site swelling or febrile response was observed in any vaccinates during the observation period.
Similar to previous studies, safety evidence for lack of Ad5 vectored vaccine shed
was supported by the absence of seroconversion to Ad5 in five (5) naive cattle co-mingled with
vaccinates during the study.

Replication defective Human Adenovirus type 5 expressing capsid proteins of FMDV strain
has been shown to be safe in cattle (target species) (Pacheo, J.M.; et. al., 2005. Virology 337:
205-209) and swine (Moraes, M.P.; et. al., 2002. Vaccine 20:1631-1639).

The master seed itself TE was recently tested ina

backpassage and reversion to virulence test as well as a shed spread study conducted in cattle in
support of licensure of the RBA by USDA APHIS CVB. The back passage study is described in

detail in the official report es ee “Master Seed Backpassage Study of Foot and
Mouth Disease Virus Vaccine, Serotype , Live Adenovirus Vector Product Code 1FM1.R0,
Master Seed ns which was submitted to USDA APHIS CVB on =
. The shed spread study is described in detail in the official report
entitled, “Master Seed Shed and Spread Study of Foot and Mouth Disease Virus Vaccine,
Serotype a. Live Adenovirus Vector Product Code 1FM1 i
and was submitted to USDA APHIS CVB on . The back

passage study showed that inoculation of Master Seed virus into 10 animals showed no reversion

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April 3, 2009

to virulence. Only virus vector inoculum containing the RBA could be isolated from nasal swabs
from four of the animals after intranasal inoculation. Similar findings have been reported for use
of these types of replication deficient virus vectors in humans (See Section II.A.1.a.i). The shed
and spread study indicated that virus vector could not be isolated from animals after
administration of the inoculum by IM route, which has shown to be efficacious against
challenge. This supports earlier studies in that animals commingled with vaccinated animals do
not seroconvert whereas animals inoculated by IM do seroconvert. A copy of both reports is
provided in Appendices I and J.

2. Physical Characteristics of the Backbone Agent.

a. Provide a flow diagram or explanation of the process of how the Backbone Biological
Agent was constructed.

Figure 1 summarizes the process by which the Backbone Biological Agent, the | |
plasmid, was constructed.

The BBA consists of the Human Adenovirus C, serotype 5 (Ad5) plasmid, with deletions in the
aa. and {J regions, termed the | backbone agent plasmid. All Ad5 coordinates
provided in the schematic are based on a wild-type adenovirus genome size of 35,934 base pairs.
An | region deletion Pe total base pairs renders the adenovector replication deficient. The
deletion includes bases through [lf resulting in deletion of the [J and HB promoters
and open reading frames. The JJM region is not essential for the growth of Ad5 in tissue culture
and the deletion spans the bases
deletion is denoted as [ff The
removes all the [J open reading frames, therefore eliminating essential elements for
Ad5 replication (¢ . The

nucleotide sequences adjacent to the deletion junctions are depicted in Figure 2.

Production of I, and

spacer within the former

deficient adenoviral vectors is maximized by incorporation of a
region.

A Transcriptionally Inert Spacer (TIS1) was developed in

order to maintain maximum production of an adenoviral vector deficient in BE, and Bj and
assure that no expression occurs from the | region. The TIS1 element is flanked by sequences
that prevent transcription from the inserted region. The TIS1 element has been tested in similar
backbone vectors and shown to be inactive for transcription

(see Figure 3.).

(i) Describe the proposed site for Donor DNA insertion.

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The donor gene insertion site is between the [J promoter and PF of an AdS
expression cassette, and the expression cassette is inserted into the BBA at the || region (see
Figure 1 and Figure 4).

(ii) Do the flanking regions of the proposed insertion site Backbone Biological Agent have
any known regulatory elements that could moderate the expression of the inserted Donor
DNA?

The adenovirus based molecular vaccine is designed to avoid regulatory elements that could
moderate the expression of the inserted Donor DNA and lead to possible expression issues.

The [if expression cassette, located at the || region deletion junction, reads right-to-left with
respect to the viral genome RNA transcription. There are no known regulatory signals in the
flanking nucleotide sequence of the insertion cassette. The [jf enhancer/promoter controls the
initiation of transcription. Within this sequence is the viral enhancer

The IJ sequences are followed by an artificial Pe containing a splice

donor sequence. The open reading frame of the gene to be expressed follows the 3’ splice site
cr

(iii) Identify unique restriction endonucleases (not more than five) that will give identifiable
digestion patterns useful for characterizing the final Backbone DNA.

Restriction enzymes HindIII and Spel give an identifiable digestion pattern, (see Figure 1).
B. Donor Biological Agents and Donor DNA or Genes

1. What are the Donor Biological Agents used as the source of each Donor DNA sequence
inserted into the Backbone Biological Agent?

Sourcing of the FMDV cDNA sequences used as donor DNA is schematically presented in
Figures 5A and 5B. Source of all donor DNA is summarized in Table 1, below.

Table 1: Donor Biological Agents inserted into the Ad5 (BBA).

| Insertedin | Function
Ad5 If region Promoter
Ad5 [ll region Transcription
g terminator

Ad5 Ff region Donor gene

Stuffer to optimize
vector yield

Ad5 J region

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a. Has there been safe use of the Donor Sequences or Donor Genes as well as Safe use of the
Donor Biological Agent?

Yes; the GenVec J backbone, which includes the JJ promoter, I, and ||
has been used in animal biologic Investigational Veterinary Products (IVPs) in the US and also
in humans as an

The donor sequences from this expression cassette are also used extensively by GenVec in

animal biologic [VPs; for example, replication defective, recombinant Ad5 vaccines co-
expressing the and of different FMDV serotypes
. The safe use of

these [VPs were discussed previously in Section II.A.1.a.(i).

The FMDV gene has also been used in other safety studies.

studied one dose of an Ad5-vectored vaccine expressing the Jj coding
region of an FMDV field strain, , and evaluated the ability to protect cattle
and swine
vaccines were administered IM. Five (5) cattle and twelve (12) swine
receiving Ad5 failed to develop generalized disease signs. The twelve (12) swine received
identical clinical scores (0) and fever results (negative) as the eight (8) swine receiving the
commercial vaccine ( , lable 1).

The RBA itself PO was tested for calf safety (Appendix

H), in the previously mentioned backpassage and reversion to virulence study (refer to
Appendix J), and in the previously mentioned shed spread study (Appendix J). In these direct
studies the vector carrying the inserted genes was proven safe in cattle even during overdose
studies and in attempted forced backpassage. In addition, the virus vector poses no threat to the
environment or to other animals in that no virus or virus vector could be isolated in animals from
a forced shed and spread study. All cattle tested remained clinically normal following exposure
to the RBA within the MSV samples.

(i) Provide relevant references for safe use.

Refer to Appendix B for the

Refer to Appendix C for

b. Are there specific parts of the Donor Gene(s) or Sequences that were used for insertion?
Show pertinent sequences or restriction endonuclease sites.

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The FMDV capsid coding sequence of and the coding
sequence of I were introduced into the shuttle plasmid, by

scientists at and were supplied to GenVec research and development, in the form

The specific donor FMDVgenes which were used as donor DNA are described below:

C. Construction and Characterization of the Regulated Biological Agent.

1. Provide a flow diagram on the construction of the Regulated Biological Agent.

The beginning shuttle plasmid,
a. This plasmid is described by
is attached as Appendix D

, was constructed and provided by
researchers in a previously published report which

The AdFAST procedure for adenovirus vector construction (Brough DE, Lizonova A, Hsu C, et
al. J Virology 70:6497-6501, 1996) was then followed at the GenVec research and development
laboratories. The starting plasmid received from [J and was transformed into E. coli

. The plasmid product was digested to isolate the gene and subcloned in
between the [J promoter and . The resulting plasmid
was called .

The AdFAST plasmid was generated by recombining [I and the BBA
plasmid . (see Figure 4, flow diagram of RBA construction.)

The vector was identified using a PCR based assay, Genetic Structural
Identity (GSI) assay, and confirmed using protein expression by western blot technique (Figures
7 and 8).

a. Final Backbone Biological Agent.

See Figures 1 and 2.

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b. Donor DNA or gene
See Figure 5A and 5B
c. All shuttle vectors

See Figure 4, where the use of shuttle vectors is diagrammatically presented and to Appendix D
where J construction of the plasmid EE is described.

d. Host cell lines used

e. Selection techniques and methods used to construct the final Regulated Biological Agent

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2. Describe the laboratory methods or criteria used to evaluate the Regulated Biological
Agent.

The RBA, was evaluated by the Po
a PCR-based technique, to confirm the vector’s identity. The

expression cassette DNA was confirmed by sequencing, and the function of the vector was
confirmed protein expression confirmation in Western blot.

Results for the testing of the RBA, Pe are in Table 2

below.

The following are brief descriptions of the assays performed:

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3. Physical characterization of the Regulated Biological Agent

a. Characterize the physical map, using unique Donor DNA and Backbone Biological
Agent restriction endonuclease sites and describe resulting restriction fragments and
digestion patterns.

Restriction size and digest patterns are shown in Figure 6.
b. Devise a PCR or restriction endonuclease test based on the Backbone Biological Agent
sequence and the Donor Sequence that will identify and characterize the Donor

DNA/Backbone Biological Agent construct.

Primer pairs were designed for the RBA Genetic Structural Identity (GSI) assay.

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c. What will be the criteria for stability and purity of the Regulated Biological Agent
Master Seed n and n+5?

GenVec has tested the Po Master Seed Po in

accordance with the relevant regulatory guidelines, demonstrating the expression of the FMD
|| transgene, and confirming the genotypic and phenotypic analysis of the viral vector. Also,
absence of bacteria, fungi, mycoplasma, and a range of potential adventitious agents, including
viruses originating from bovine, and porcine sources have been demonstrated.

Recently the required in vivo calf and mouse safety tests of the Master Seed have also been
performed (Appendices G and H) and the master seed was tested in a very conventionally
designed live vaccine backpassage and reversion to virulence study (see Appendix I).

The master seed was found to be safe in both calves and mice, following the procedures defined
in 9CFR 113.33 (Appendix G) and 9CFR113.41 (Appendix H), respectively. In the mouse
safety test, eight of eight mice which were administered 0.5 ml of the master seed by
intraperitoneal injection remained healthy throughout the seven-day post-inoculation observation
period. Likewise, in the calf safety study, both of the two specified test calves which were
administered 10 vaccine doses of the MSV (EM) of the master seed by
intramuscular injection remained healthy throughout the 21-day post-inoculation observation
period.

For licensure as live vaccines, master seeds are routinely required by USDA APHIS CVB to be
tested in backpassage and reversion to virulence studies. A study designed to fulfill this
requirement was recently completed in BL2 containment facilities in J, Nebraska.
Results confirmed this master seed is incapable of establishing itself in cattle, as evidenced in a
backpassage procedure conducted in susceptible calves, even under conditions where the RBA
was administered in high titer by the oronasal route, considered to be the route most likely to
permit adenovirus infection in mammals. In this experiment, the RBA was recovered only after
the first 24 hours post inoculation and from only 4 of the 10 animals to which it had been directly
administered. Attempts to further pass the recovered virus collected in the nasal secretions of
was not performed since by genetic identification testing only RBA inoculum was detected..
All of the calves ate and drank normally and appeared healthy and normal during the entire study
term. One animal (calf | y died eight days into the study from a non-vaccine related cause.
The cause of mortality was diagnosed as chronic interlobular pneumonia with an enlarged
tracheobronchial lymph node causing bloating due to pressure against the esophagus (see report

. The remaining 9 animals did not show any signs of clinical disease attributable
to the administration of the master seed, even under conditions designed specifically to promote
backpassage of the virus and to reveal any increase in virulence which might be associated with
passage of the RBA in live cattle. On the contrary the virus was not able to sustain itself in any
of the original animals to which it was inoculated at the beginning of the protocol.

The master seed + 5 has been sequenced in the modified regions of the genome

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d. Provide the genetic sequence in electronic format for any new or altered genetic
sequences, including insertion and flanking sequence.

ees: cassette DNA sequence, of the RBA Po including

the promoter, has been verified in GenVec Research. Sequencing data analysis confirmed
the nucleotide sequence identity between the plasmid map and the DNA extracted from

ff viral particles. The DNA sequence of the | expression cassette
(including promoter, I, and was confirmed on both strands except
Hl nucleotides in the WM transgene from and [ff nucleotides in the 8,

A single strand sequence for this was matched with the expected plasmid sequence.

4. What is the recommended NIH/CDC biosafety level for the Regulated Biological Agent?

Biosafety level 2 (BL-2) (NIH Guidelines for Research Involving Recombinant DNA Molecules,
April 2002) has been recommended up to the present time. It is proposed the biosafety level
should now be modified to BL-1 based on review and acceptance of this SIF and accompanying
risk assessment, and consistent with use of the RBA as a live vaccine, subject to USDA APHIS
CVB approval of a pending product license application.

5. Provide a short summary or description of genetic motifs that may have resulted as a
consequence of the genetic recombination (II.C.3.d). Are there any known motifs that may
promote homologous recombination, DNA insertion, or gene expression of existing or new
open reading frames?

pg However, it is expected that the generation of a replication competent adenovirus

(RCA), requiring two independent recombination events in a single adenovirus genome, is
expected to be a rare occurrence (see Section IJ.A.1. for a detailed explanation).

It is known that | vectors grown in MM cells, the parental precursor to the fF

line, have the potential to undergo a recombination event between the vector and cell line that
will produce a replication incompetent process impurity within the vector stock. The
recombination event can occur between the homologous regions flanking the Hl deletion in the
| and the a complementation region contained within the cellular genome of

cells. The transgene expression cassette contained within the BM region of the |
vector would be removed as a result. The resulting recombinant is a replication incompetent
adenovirus that is Pe and is termed Recombinant [jj
Adenovirus (REA). GenVec has developed methods to detect REA in stocks of | vectors.

In preliminary studies using the final , was
serially passaged on the || cell line and the genetic integrity of assessed by the GSI

assay. Results show that after 10 passages, there was no scientific evidence of homologous
recombination, DNA insertion, changes in gene expression or evidence of new open reading
frames.

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III. BIOLOGICAL PROPERTIES OR VIRULENCE FOR THE REGULATED BIOLOGICAL
AGENT USED FOR MASTER SEED

The RSE) PIM Master Seed was prepared by expansion of a Vector Seed
Stock (VSS) in cells derived from an official Master Cell Stock. The MSV and its
preparation and testing are the subject of a report and report addendums submitted to USDA
APHIS CVB in support of licensure. The labeling of the Master Seed is as follows:

The biological properties of the
was used to produce the Master Seed have been tested in cattle (

peeerereerecmern Se IM administration of the Ad5 at vaccine
doses up to approximately total particle units in cattle did not cause any febrile

responses, site reactions, systemic reactions, clinical disease, or mortality. Similar results using a
highly similar human adenovirus serotype 5 replication defective viral-vectored FMDV
experimental vaccine have been previously published in swine (

: and cattle (
|). Based on its genetic properties, the

vector replication in the mammalian host. The FMDV
proteins expressed in vivo by the
deleterious or virulent effect on the host.

that

is incapable of

vaccine do not elicit any

A. What are the known phenotypic characteristics or effects of the Regulated Biological
Agent identified as Master Seed?

The Human Adenovirus C, serotype 5 (Ad5) genome with deletions in a. and i regions is
replication deficient in target host cells. The || region deletion, including the and
promoters and open reading frames, renders the adenovector replication deficient. The

deletion eliminates essential elements for Ad5 replication through removal of the Bl open
reading frames. The multiple-deletion adenovector backbone is incapable of replicating in non-
complementing cells, and the RBA, therefore, has been shown to share this characteristic
replication deficiency.

(VSS i) was confirmed to express {J protein in vitro in
cells by a western blot assay, yielding a reactive protein that migrated to the position of

approximately | KDa (see Figure 8). The antibody used for detection in the Western Blot
assay is a monoclonal antibody provided by BRM. The molecular weight of the BBB protein
expressed from the RBA was indistinguishable from that observed following transfection of the
positive control plasmid into mm cells. (Refer to Figure 8).

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B. What are the virulence characteristics of the Regulated Biological Agent used for the
Master Seed?

The master seed is considered avirulent for man and animals. The FMDV donor structural
capsid i) and nonstructural protease i) genes have no known virulence properties
or sequences associated with host virulence. The structural proteins encoded by the FMDV ||
Bl capsid gene do not possess any toxin, immune suppression, immune evasion, or other
properties associated with host virulence. The nonstructural protease enzyme encoded by the
FMDV | protease gene possesses enzymatic activity that is restricted to and specific for
the FMDV capsid protein, and does not have any known properties associated with host
virulence (Refer to Appendix F).

The backbone biological agent itself is considered avirulent for man and animals due to its |
deletions which produce an adenovector with no known virulence or
oncogenic properties in mammalian cells.

The RBA, therefore, is a multiple- deletion vector containing non-virulence associated FMDV
genes, which does not replicate in any known target host cells in nature.

C. Is the Regulated Biological Agent used for the Master Seed virulent for target animals,
non-target animals?

Based on several studies in cattle performed to date at J, the Po

RBA used to produce the Master Seed is not virulent for target animals (domestic cloven-hoofed
livestock) or anticipated to be virulent for non-target animals (e.g., wildlife, domestic companion
animals, birds, etc.). In cattle administered the RBA, no adverse reactions, morbidity or
mortality has been observed. Based on studies to date in cattle, the host immune response to the
adenovirus components of the RBA and those of the BBA is indistinguishable (based on
generation of anti-Ad5 serum neutralization antibodies) following administration.

The virulence of the Master Seed has been directly investigated in mouse and calf studies

described in Appendices G and H, respectively. The master seed was found completely avirulent
in these studies.

The master seed of the RBA is considered avirulent for man and animals.

D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to
survive in the target animals, non-target animals?

No. Virulence and survivability of the Master Seed in any host is predicated on the ability of the
Master Seed vector to achieve full replication in vivo. The genomic deletions in the vector
backbone render the Master Seed replication deficient. The FMDV DNA donor sequences do
not enhance the virulence or the ability of the RBA to survive in the
target animal, e.g., bovine. These DNA donor sequences do not convey any replication
competency properties. Results from cattle studies using Po RBA show
that the ee vector displays a lack of virulence in cattle. There is no

scientific basis to believe that the donor DNA sequences provide any growth advantage or confer

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any virulence attributes on the BBA agent for cattle for any other mammalian or non-mammalian
host. This conclusion is supported by the aforementioned backpassage and shed/spread studies
(Appendices I and J).

1. What is the tissue tropism of the Regulated Biological Agent in target and non-target
animals?

Since the master seed virus is replication deficient, its tissue tropism is very difficult to
determine and may be of little practical relevance. Since the master seed virus is incapable of
completing a full replication cycle within the host cells, it is unlikely to disseminate beyond the

immediate injection site. Accordingly, GenVec has not designed, nor executed tropism studies
to date.

The genetic recombination which resulted in construction of the replication incompetent master
seed would not be expected to alter the underlying tissue tropism of the wild-type adenovirus.
Current scientific literature indicates that target cell infection of wild-type adenoviruses is
mediated by binding of the fiber knob region to the receptor of the target cell, which is the
coxsackie-Ad receptor (CAR) for most serotypes, including Human Adenovirus serotype 5.
Thus, the tissue tropism of wild type viruses is predicted to be restricted to any cell types which
express the CAR receptor, including muscle cells and dendritic cells. However, recent reports
provide evidence that secondary cell receptors can be used for wild type adenovirus binding, in
particular heparin sulfate proteoglycans (HSPGs), so it is possible that cells in tissues expressing
HSPGs can also serve as targets for adenovirus infection. Yet, cells expressing CAR or HSPG
are anticipated to be potential targets for adenovirus attachment and are not associated sites of
pathology for FMDV, nor will the virus vector replicate in cells with either receptor in nature.

2. Provide direct scientific evidence, including any relevant scientific publications, if
available.

There are publications by EE scientists using an adeno-based vector
(replication defective adenovirus serotype 5) similar to the vector used at GenVec. A Human
Adenovirus serotype 5, replication defective viral-vectored FMDV | experimental vaccine
was tested in swine and cattle. For example:

i [BBE scientists vaccinated five (5) cattle with Ad5 FMDV subunit vaccine and two
(2) control cattle with Ad5S Vector containing glycoprotein gene of vesicular
stomatitis virus

ii [EB scientists constructed a replication defective Ad5 containing the BB coding
region of FMDV [i and the I coding region of and evaluated its ability to
induce neutralizing antibodies in swine (

These studies obtained results similar to results described in GenVec’s

. In

this study IM administration of the in cattle did not cause febrile
responses, site reactions, systemic reactions, clinical disease, or mortality.

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The Master Seed Virus was recently tested for safety in eight mice (per 9 CFR 113.33) and in
two calves (per 9CFR 113.41). As expected, the MSV was found safe. The backpassage and
reversion to virulence study confirmed safety of the MSV in cattle, as well. In addition, the
Shed/Spread study indicates that there is no virus shedding to contact animals or to the
environment (Appendices I and J)

E. Discuss the potential for horizontal gene transfer or recombination of the Regulated
Biological Agent.

1. Is there any reason to believe that the potential for horizontal gene transfer or
recombination is different in target and non-target animals?

No. The potential for horizontal gene transfer or recombination is predicted to be extremely low
due to the nature of the RBA, which is non-integrating/non-replicating, and this effect is
independent of the species of host cell to which the MSV would be exposed. Further supporting
this expectation is the observation made during the proof of concept studies conducted at the

| (as described previously in Section II.A.1.a.(i)). There was no seroconversion seen
among the co-mingled cattle.

a. Include contributions from the Backbone Biological Agent and Donor DNA Sequence in
the discussion of the horizontal gene transfer and recombination potential.

The RBA is expected not to have increased potential for horizontal gene transfer or
recombination for several reasons relating to its BBA and the type and method by which the
Donor DNA sequences were inserted.

i Unlike wild-type Human Adenovirus serotype 5 strains, the RBA is incapable of
completing a full round of viral replication within normal host cells, due to lack of
essential ORF within the [JJ and [J regions of the RBA genome. As a result, any
horizontal gene transfer would be required to occur within the severely limited viral
expression process which occurs upon introduction of the RBA into a non-
complementing cell.

ii The RBA is human serotype 5 adenovirus, which are not known to readily infect cattle.
The probability the replication deficient human adenovirus RBA could co-infect a cattle
cell which happened to be harboring another replication competent adenovirus is very
small. That these viruses could then recombine within the aborted time period of the
transcription cycle of the RBA within that cell is not a credible possibility, especially
since deletion of the | | region removes important DNA replication enzymes necessary to
melt DNA during replication and pre-requisite of homologous recombination exchange.

iii The donor DNA reads right to left relative to the remainder of the RBA’s adenoviral
DNA.

iv The inserted FMDV genetic material does not contain sequences that are specific to any
FMDV’s replicative enzymes.

b. Reference any relevant scientific publications.

A list of relevant references to the scientific literature are supplied at the conclusion of this SIF.

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F. Describe the Shed/Spread Capabilities of the Regulated Biological Agent
1. Include shed/spread potential in non-target animal species.

A replication-defective Human Adenovirus C, serotype 5 vector has the ability to attach and
internalize into cells from various species; however, the virus is only capable of replication in
vitro in specialized cell lines (MM). Humans are exposed to virulent adenovirus
serotypes at an early age, so the presence of a replication-defective human viral vector is not
expected to pose a threat to humans.

The shed/spread potential of the [NB BA in non-target animal species is
predicted to be virtually nonexistent based on the lack of replication competency in the target
animal species. A cattle study specifically designed to detect or measure the shed/spread
potential of the a RBA in non-target animal species indicates that no
virus can be isolated from the animal by direct sampling or by contact exposure in commingled
and housed animals (see Appendix I). The indirect evidence strongly supports the conclusion
that there is minimal to no risk of shed/spread to non-target animal species. The indirect
evidence is that cattle co-mingled in confined quarters for up to 42 days following IM
administration of [I 2.3 A failed to seroconvert to the Ad5 vector
backbone (e.g., produce serum neutralizing antibodies to Ad5). In all cattle studies performed to
date, all co-mingled study cohorts that did not receive Ad5 vectored test articles failed to

seroconvert to Ad5, suggesting that cattle that directly received Ad5 vectored test articles did not
shed Ad5 vectored test articles into the environment.

Direct evidence of that there is minimal risk of shed/spread to non-target species or risk to the
environment was demonstrated by inoculating the test material intranasally into 10 healthy
commercial 5-6 month-old cross-bred calves to determine whether any clinical signs of disease
would occur and also to determine whether the Master Seed could be recovered from the animals
subsequent to inoculation. In the event of virus recovery, the study design allowed for
subsequent backpassages and observations to determine whether the Master Seed increased in
virulence upon sequential passage in the vaccine’s target animal. Swab samples of nasal and oral
secretions were collected at Days 0, 1, 2, 3, 7, and 14 post-inoculation, and virus isolation was
attempted on each sample. Nine of ten test subjects remained healthy and normal throughout the
observation period. One animal died due to bloat (a cause unrelated to the study) on Day 8 post-
inoculation. Adenovirus vector was recovered from nasal swabs collected from 4 of 10 cattle at
the 24-hour sampling time point; however, all the oral swabs of all ten animals at this time point
were negative, indicating the virus vector recovered from the 4 positive calves was not dispersed
beyond its original (nasal) inoculation site. All virus isolation tests done on all sample types at
all time points later than 24 hours post-inoculation were negative (Appendix I).

The lack of any later isolations of virus from any test samples was considered consistent with the
known history and makeup of the Master Seed Virus. PCR testing confirmed the virus recovered
from the nasal swab samples at the initial (24-hour) sampling was genotypically
indistinguishable from Master Seed. This virus was undoubtedly a residual of the intranasal
Master Seed Po inoculum collected from the site of inoculation. Detection
of residual inoculum at the earliest samples taken from the inoculation site was not unexpected,
and its recovery serves to confirm the laboratory virus isolation methods used were effective. No

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later isolations were made, confirming that Master Seed NE did not

achieve successful replication in these calves, nor did it permanently establish itself. The PCR
testing of the recovered virus indicated its genotype was not altered as a result of bovine passage.

Based on these data and the highly defined, genetic make-up of the Po
RBA that confers a replication defective genotype, it is reasonable to assume that the shed/spread

potential of the Po RBA in non-target animal species is virtually
nonexistent.

2. Include in the discussion any contributions to shed/spread capabilities that might be
associated with particular attributes of the Backbone Biological Agent and Donor DNA
sequence.

GenVec FRR study number

the safety of A produced in the cell line.

The replication defective, recombinant adenovirus 5 vectored vaccine co-expressing the
structural capsid gene of FMDV serotype [J and the non structural gene of
FMDV serotype ma were constructed and recombinant viruses produced in the

cell line. No untoward systemic or local reactions were observed during or immediately
following administration of Ada IVP in five (5) cattle. No injection site swellings were
observed in any of the five (5) vaccinates during the observation period (see Section
II.A.1.a.(i)). The report concluded that Ada did not shed from vaccinates in a sufficient
amount to induce the production of Ad5-specific vaccine vector antibody in naive cattle co-
mingled during the three-week study. Data show that there were no detectable AdS SVN

antibody titers in the five (5) sham immunized cattle co-mingled with the five (5) Ad
vaccinates.

EE scientists constructed a vaccine using replication-defective adenovirus
serotype 5 containing the structural capsid of FMDV serotype | and the nonstructural
protease coding regions of FMDV serotype im virus and vaccinated swine ({

. Two groups of swine (three (3) in each group) were injected IM with
either the vaccine or phosphate buffered saline (PBS). The vaccination protocol used in this
study also gave a booster injection at 4 weeks post initial injection. The swine were co-mingled
to demonstrate the inability of the replication-defective virus to spread from the inoculated group
to the uninoculated control group. The results showed that the three (3) control swine did not
seroconvert to FMDV after vaccinating and boosting three (3) co-mingled trial swine. The
control swine did not develop neutralizing antibodies against adenovirus, proving that the
vaccine did not spread from the vaccinated swine. (Refer to Appendix F, part 1)

In 2001, EE scientists Wes pesemmerens vaccinated
swine IM and boosted at four weeks with Ad5- (a replication-defective adenovirus
5 encoding FMDV serotype i structural capsid and serotype [J protease coding regions)
and injected swine and boosted with Ad5- (an adenovirus encoding the FMDV
capsid coding region and inactive form of the . Six to eight-week old gilts were
injected using three (3) animals per group. A control group injected with PBS was co-mingled
with swine vaccinated and injected with the constructs being tested in three experiments. In the
first experiment, six (6) swine were co-housed (three (3) vaccinated with AdS a. and
three (3) injected with PBS). In the second experiment, six (6) swine were co-mingled (three (3)

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vaccinated and boosted with Ad5- and three (3) injected with PBS). In the third
experiment, nine (9) swine were co-mingled three (3) vaccinated and boosted with Ad5-

, three (3) injected and boosted with Ad5-EEEE, and three (3) injected
with PBS). None of the PBS injected swine developed neutralizing antibodies against Ad5.
Proving that the Ad5 vectors did not spread to naive animals in the same room. (Refer to
Appendix F, part 2)

The conclusion drawn from this work is that the Master Seed will be, based on the characteristics
of its BBA and of its inserted donor DNA, and the evidence gathered in studies of similarly
constructed vectors, both non-shedding and non-spreading in all animal species.

These expectations of innocuousness were confirmed when a backpassage and reversion to
virulence study conducted using a high dose of the MSV revealed its live titer rapidly declined
within the nasal secretions of the test cattle, and ultimately the MSV was not recoverable from
inoculated cattle after 48 hours. Nine of ten cattle remained free of any clinical signs of disease
during the entire post-inoculation period of the study (see III.F.1) One animal died at day 8 of
the study to a cause unrelated to the inoculation. This is considered direct evidence of the safety
of the MSV and its lack of shed/spread capability. These phenotypic characteristics of the MSV
are consistent with its known genotype.

3. Reference relevant scientific publications
A list of relevant references to the scientific literature is supplied at the conclusion of this SIF.

G. Discuss the expected environmental impact or survivability of the Regulated Biological
Agent, and provide available scientific evidence.

Specific data have not been generated to date to examine the survivability of this RBA in the
environment. However, based on the nature of I specifically that itis a
human adenovirus being injected into cattle and that it is replication incompetent, and the lack of
sentinel control seroconversion in the cattle studies completed to date, both the environmental
impact and survivability of the RBA is predicted to be extremely low in the environment.

The backpassage and reversion to virulence study conducted using the MSV revealed the live
virus vector titer recovered from nasal swabs of directly oronasally inoculated test cattle declined
rapidly from the initial level (| total PU inoculated) to undetectable levels (sd
within 48 hours in ten study animals. At 24 hours post-inoculation, RBA was detected at very
low levels (ED in only 4 of 10 animals. This lends support to the expectation that the RBA
is not replication competent, and is thus incapable of exerting any effect on the environment
other than an immunizing effect on the animal to which it is directly parenterally administered.
This study also confirms the RBA is not able to infect cattle productively to make a self-
sustaining population as would be required to support ongoing environmental survival of the
RBA in nature. A shed/spread study conducted with a similar titer of MSV as the back passage
revealed that the live virus vector could not be detected in any of the 10 animals inoculated by
IM, which is the recommended method of vaccine administration.

1. Do the Donor DNA sequences or gene sequences associated with the Backbone Biological
Agent enhance the ability to survive in the environment or increase resistance to
therapeutic agents?

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No. Although specific tests have not been designed, nor has data been generated to date to
directly examine the ability of donor DNA sequences or gene sequences associated with the
Backbone Biological Agent to survive in the environment or increase resistance to therapeutic
agents, the nature of the genetic changes give no reason for concern of this nature.

More specifically, using a designed study to examine the back passage and reversion to virulence
demonstrates, as predicted by the literature for similar adenovector backbone agents used in
human application, that the virus vector cannot be reisolated from any preferred sites for
adenovirus replication except that from the nasal orifice within the first 24 hours post-inoculation
in 4 of 10 animals. Furthermore, the recovered virus vector presented equivalent genetic identity
with the RBA inoculum, indicating the recovered virus was residual inoculum at the
administration site. The virus titer was reduced by approximately 10 logs from the applied
inoculum to the residual level still detectable after 24 hours. At 48 hours, no virus was
recoverable from oral or nasal samples. Thus, by a preferred site of exposure to the environment
and a preferred site for entry of adenovirus into any mammalian population the virus is not
capable of replicating. (see Appendix 1)

When the virus was inoculated by the specified IM method for efficacy in a designed
shed/spread study, no virus could be isolated from direct sampling from the animal by preferred
oronasal route of transmission. (see Appendix J)

Based on the known genotypic and phenotypic characteristics of both the BBA and RBA and
direct evidence from these two designed experiments it is very confidently predicted the altered
sequences will not enhance survival in the environment nor increase resistance to therapeutic
agents.

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<div class="ocr">Risk Assessment

Foot and Mouth Disease Virus Vaccine,

Serotype , Live Adenovirus Vector

UPDATED

Version Date: 14 Sep 09

PerOs USA Inc. (under contract with GenVec, Inc.)

U. S. Veterinary Establishment No. 419

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September 14, 2009

SIGNATURES:

GenVec, Inc.

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Date

Date

Date

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Table of Content
TABLE OF CONTENT 3
I OBJECTIVE/PROPOSAL 4
II SUMMARY OF INFORMATION FORMAT (SIF) PROVIDING CHARACTERIZATION
FOR THE VACCINE MICROORGANISM 4
A. MICROBIOLOGICAL PROPERTIES ........ccsscccosseeseseecossaceceesenencnsnaneversnaneerensnaneessesasesensnesesenanssaseesseteeneess 4
I. Master Seed Virtus (MSV) ......cccccccccccecceceteceeneenevieeneeeecnee teenie saeenee tn eeneenaetessneneeceernesesteessesenesieenees 4
2. Master Cell Stock (MCS). ....ccccccccccccscesccescesecececceecsecisesenecacecaeeeneeeeesuneseesseseseseseerieesseseseseenneneerges 5
3. Prodhuct Seriqls....ccccccecccsscsesecsesssenecseveneseeeeeneeecneeeecnsesaeenesaecesenseeessessaecaeeseesseceeeseesesassasnesannecaaas B)
B. BIOLOGICAL PROPERTIES ......ceccsccccssseeesscecseseessseesenessecsecneneesenenesersnanseresersaeesessearesssatesnarereneesesneeesas 5
Ill RISK ASSESSMENT 6
A. HAZARD IDENTIFICATION .......ccssccsescseceessrecscesneceeeeseeeesaeeseneensaeecsarerseaeeeensateeneessteeseeuessgessateseneeneessas 6
I. Potential Hazards of the Product to animals .....c.ccceccccceceecessecsereeeeneeceeneenenaeeetseevaceesnenensrneessnsies 6
2. Public Health Safety... ccccccscccccccscccssctssnecsene seen snes cee ene ee eet ene cbe serene ene cnt ne eee EE SESE Sd ee ea ene caenene es 18
3. Exavirormental Safety... cc ccccecccecscseesssecesseeeceeenesceneneeeeeeeenenneeteeeteesneseeseeeenerienernenenieseseeessees 19
B. RELEASE ASSESSMENT ......cscecesessscesscssesseeceecseeeeseeenecseeseuseeeecesensecnaeeesaesnecraesenersnesientissiedseeeesssenvags 19
LT. — Location of Test Sites ....eccccsccccccscsesscsevesseveceececenceeecneeensnseseeneceeeeseseeeneetecneevaenecenevisenssensnsesensenss 19
2. Characteristics of the test Sites ....ccccccccccscscsccsssertseeceueenessevecnetnereseceeenecescie tens enenseneneessaeeseescaes 20
3. Personnel at the test Sites... ccccccsessceseresisseetsesensenseeeeeeneenseeene ties nteien ene sernennenernetegeceeseeenenees 20
4. Experimental design... ccccccccccsercseccseveenesecne sects ene ct ene ene eee re shen ne een c nH Ere S nH ELE SDE DE LE SE PASE g tee eee catia 21
5. Potential for escape and dispersal from the identified test Sites ........ccccccscssscesetsetsetsetereeeeees 21
6. Potential for establishment in the environment of the test Site ........cccccscscsete tsetse reeseneeneneees al
7. Monitoring in and around the test Site 0. cccccecsecscsssesseseneessvceseuseeceeneeeenecaeseeneeseaesaceeraeereneneses 21
8 Contingency plans in case of AAVErSE EVENT... cece scensene ces eeeneetisensetecescareserscesesensecsesnensevee 22
C. RISK CHARACTERIZATION ......cccsccecessceceseeeececnecesueeeessnaeeeeseneeeecseaeecessaeaeeneceetaneerersnaresensaasesaeeseseereenees 23
IV. RISK MANAGEMENT 23
A. CONTAINED RELEASE.......cccssccecesseceessceeeeseaceceuseecssaceeesenaeeseneaeeeeseneneetensenaeenseseeadaseseesesssusesaneesenesenies 23
B. ENVIRONMENTAL RELEASE.......ccesccccesseceessneeesseeecseneeensnneeessanaeeeccnneeeesensnsareerenseneeesesesesusiauessneesienneneee 23
Vv RISK COMMUNICATION 23

APPENDIX 1: FIELD SAFETY STUDY PROTOCOL . 26

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1 Objective/Proposal

It is proposed that two pre-license serials of Foot and Mouth Disease Virus Vaccine,
Serotype m7. Live Adenovirus Vector, Product Code 1FM1.RO0 be released by the
United States Department of Agriculture Animal and Plant Health Inspection Service
Center for Veterinary Biologics (USDA-APHIS-CVB) for use in field safety testing.
This multi-site field safety test, to be conducted under normal husbandry conditions using
healthy cattle is being proposed by GenVec as partial fulfillment of the USDA’s
conditional product licensing requirements for this product. This proposal describes
analysis of the risk of release of the vaccine into the environment, first for use in the field
safety testing, and assuming a favorable outcome of that work, its release in a possible
future deployment in a federally directed Foot and Mouth Disease emergency
management program by APHIS officials. Availability of a conditionally licensed
FMDV veterinary vaccine bank is a part of a critical U.S. Department of Homeland
Security (US DHS) and USDA preparedness initiative.

II Summary of Information Format (SIF) providing characterization for the
vaccine microorganism

A preliminary SIF describing the MSV and MCS was first submitted to USDA APHIS
CVB on 15 Jun 07 in support of the product license of Foot and Mouth Disease Virus
Vaccine, Serotype a. Live Adenovirus Vector, Product Code 1FM1.R0., Est. no. 419.
Updated SIF documents dated 04 Aug 08 and 03 Apr 09 were subsequently submitted. A
further updated Risk Assessment dated 14 Sep 09 is submitted herewith.

A. Microbiological Properties

1. Master Seed Virus (MSV)

The Master Seed Virus, I, is a human

adenovirus C, serotype 5, which has undergone complete deletions in the | | and

| regions of the viral genome, and a partial deletion of the Mf region. In
addition, the virus has undergone a [J deletion in the region
that interrupts a minor alternative transcription start site. A

has been inserted in the deleted [JJ region of the virus, to optimize
virus production in the companion MCS. The resulting biological backbone agent
(BBA), identified as the [J base plasmid is an adenoviral plasmid, which,
when linearized and transfected into a complementing cell line such as
produces complete adenovirus particles which are themselves incapable of
replication in normal mammalian cells but which replicate to high titer in cells
derived from the complementing MCS. These complementing cells have been
genetically altered to supply the essential [J and [J proteins necessary for
production of complete virus particles when transfected with the BBA or

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derivatives of the BBA, such as the Master Seed Virus, Po

Into the BBA have been inserted the structural capsid gene ( of Foot and
Mouth Disease Virus (FMDV) Serotype (strain B. cattle
isolate) and the nonstructural protease gene , strain
119 (strain from repeated laboratory passage in guinea pigs). Both donor FMDV

sequences were supplied as plasmid DNA to GenVec, Inc., by

on December 4, 2004.

The resulting recombinant Master Seed Virus is a unique replication-deficient
human adenovirus serotype 5 which contains DNA coding for the structural
HMM gene of FMDV serotype [J and the nonstructural BE cence of
FMDV serotype i.

2. Master Cell Stock (MCS)

The I MCS ME is a proprietary cell line created by GenVec,

Inc. specifically for the purpose of contained in vitro production of GenVec’s
various replication-deficient adenoviruses, many of which are derived of the BBA
described above. The stable human cell line from which this MCS was prepared
has undergone insertion of genetic sequences which complement for the deleted
p regions of the adenovirus BBA. The MCS therefore complements for the

deletions in the MSV, and serves as the key unique cell substrate required
for its full viral replication. The || cell line is used in vaccine production.

3. Product Serials

Serials of the vaccine for use in cattle are prepared using the above-described
MSV and MCS under the master seed/cell principle, in compliance with USDA-
APHIS-CVB regulations in the licensed biologics production and testing
establishment of PerOs USA, Inc.,

Each serial is produced and tested for purity, safety and potency in accordance
with the detailed procedures specified in the product’s filed Outline of Production.

B. Biological Properties

The biological properties of the MSV when grown in normal mammalian cells are
consistent with its nature as a replication deficient adenovirus. Like its BBA, the
MSV is capable of penetrating certain normal mammalian cells, and once inside
the cells it is able to display its genetic material (including the inserted donor
DNA) but due to its lack of complete [I gene products it is
incapable of expressing all proteins necessary to complete a fully functional

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complete virus particle, and thus the BBA and the MSV are not able to complete
the repeated replication cycle necessary to achieve infection in normal
mammalian host cells nor dissemination inside the host animal and shedding to
the environment. As a result the adenovirus present in the MSV, which in its
original unaltered form in nature is believed to have a host range limited in all but
a few rare reported cases to man, in its altered MSV form is replication deficient
in normal mammalian cells.

In contrast, the MSV, when grown in the MCS, is fully infectious to these
complementary cells in vitro, because the MCS cells complement for the [J and
| deletions present in the MSV genome. Therefore, while in normal
mammalian cells the MSV is fully attenuated (because it can succeed only ina
single aborted round of replication), when the MSV is introduced into the MCS it
is fully infectious for the MCS cells. This results in multiple complete rounds of
viral particle production, including production of the non-genetically modified
adenovirus proteins and also production of the inserted FMDV structural capsid
protein and the nonstructural FMDV protease protein. Expression of the
structural FMDV capsid protein results in production of FMDV serotype ma.
capsid protein within the MSV-infected MCS cells in culture. Furthermore, when
the complete virus particles thus produced are injected into cattle, an active
immune response occurs within the cattle, characterized by the development of
FMDV serotype BE -specific antibody titer. Because the MSV is incapable of
establishing a full infection cycle in normal mammalian cells, the vaccinated
cattle exhibit no signs of clinical disease.

The serials of product are made using only the approved MSV and approved MCS
under the USDA-APHIS CVB-mandated production and testing procedure,

ensuring pure, safe potent and efficacious serials of uniform character are offered
for release.

Ill Risk Assessment
A. Hazard identification

1. Potential Hazards of the Product to animals

The proposal is for field safety testing and eventual conditional licensure of Foot
and Mouth Disease Virus Vaccine, Serotype [I], Live Adenovirus Vector,
Product Code 1FM1.RO0.

Cattle, which are non-permissive hosts for the MSV are the primary target for
vaccination to which the product will be directly administered. Other mammalian
species also within the same cattle testing facilities might include rodents, rabbits
or other wild animals which might enter the testing facilities. In ranch and farm

settings the possible exposure of canine, feline equine, ovine and/or swine might
also occur.

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a. Risk of FMD due to introduction of complete FMDV into animals
i Discussion of risk

FMD is considered exotic to the United States. A release of FMD
virus into the US environment would be an animal health emergency
of the gravest concern. In an environmental FMDV outbreak, animal
producers’ direct losses due to FMD itself would doubtlessly be
economically severe. Beyond these direct costs lie broader
consequences, since the presence of FMD within a nation’s animal
population is effectively a bar on trade in animals and animal products
with many countries worldwide.

The proposed release is of an adenovirus vector containing only
limited and well characterized sections of the FMD genome. Vaccine
was fully characterized and purity tested by PerOs USA Inc. and
confirmed by USDA APHIS CVB Laboratory. Under this proposal
no release of FMDV into the environment is possible.

Direct experiments with high doses of the master seed in cattle and
mice as well as extensive laboratory evidence have shown the master
seed used for production is avirulent and safe and incapable of
releasing replicating FMDV.

ii Consequence rating (and degree of certainty rating) of risk of
FMD disease in animals inside this proposal

The consequence rating for FMD disease, should it occur, would be
moderate because the study is to be done under direct veterinary
supervision and the majority of the animals in the test groups are
expected to develop protective vaccine immunity to FMD as a result of
vaccination. In the very unlikely event an FMD event would occur,
therefore, it would need to occur in a seronegative individual and its
spread to pen mates would be unlikely, as great majority of all the
vaccinates will be seropositive as a result of vaccination. Any FMD
infection would almost certainly be limited to a single animal.
Immediate sacrifice of any symptomatic animal followed by
quarantine and complete decontamination of the animal facility would
limit the consequence of the event to the premises of the test itself
only. The consequence rating can be made with a high degree of
certainty, based on direct evidence.

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iii Likelihood rating (and degree of certainty) of FMD disease in
animals

To ensure there could be no possible release of complete FMDV into
any animal, only two genes of the FMDV genome were inserted into
the BBA to form the RBA, and thus only these two FMDV proteins
are present within the MSV. In addition, because none of the
replication competent gene signals of FMD virus exist in the RBA
construct, it is impossible that a complete FMDV virus could be
created upon direct administration of any amount of the MSV, MCS
and/or product into any cell or into any test animal. Due to the
incomplete FMDV protein expression profile, only a characteristically
incomplete (but protective) antibody response occurs when animals are
exposed to the MSV, consisting of production of FMDV [ij protein -
antibody. Thus animals exposed to the MSV are readily differentiated
from animals which have been wild-type FMDV infected and as a
result have developed a full complement of antibodies specific to all
the proteins present on complete infectious FMDV particles.

Neither of the FMDV proteins present in the MSV, the structural

capsid protein gene [J nor the nonstructural capsid gene a. is
associated with virulence in FMDV.

GenVec has conducted studies in collaboration with J scientists,
using cattle, swine and mice. These studies give direct evidence that
only an [ij antibody response, rather than a complete FMDV
antibody response, occurs in RBA-exposed animals.

GenVec has also conducted Master Seed Virus testing confirming the
genotype of the vaccine virus, backpassage and reversion to virulence
testing in cattle, shed/spread testing in cattle and swine, plus, mouse
and calf safety tests. The testing of the master seed for purity and
identity was confirmed at the USDA APHIS CVB-L. All these studies
lend direct evidentiary support to the expectation that FMDV virus
cannot be produced as a consequence of vaccine administration, but
that animals can become immunized as a consequence of an
incomplete yet effective production of key FMDV proteins by the
virus vector.

The conclusion based on a combination of indirect scientific evidence
and direct evidence is that the proposal’s likelihood of risk of FMD
disease in animals, in all species, is extremely low, with an extremely
high level of certainty.

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b. Risk of Adenovirus Disease
i Discussion of Risk

Adenoviruses are double-stranded DNA, non-enveloped, viruses. The
Adenoviridae family, genus Mastadenoviridae, only infects mammals
and contains among others, 51 human adenovirus (HAd; 1-51), 4
bovine adenovirus (BAV; 1-3, 10) and 7 porcine adenovirus (PAV; 1I-
7) serotypes (Davison A.J., et. al., 2003. J. Gen. Virol. 84:2895—908).
HAd serotypes 2, 5 and 35 have been most often used and reported in
the literature as adenovector backbones for the construction of
recombinant replication-deficient adenoviral-vectors for vaccine
product applications.

Host cell infection by human adenovirus serotype 5 (HAdS) is initiated
by virus capsid binding to a 42-kDa glycoprotein receptor termed the
Coxsackievirus and Ad receptor (CAR) (Howitt J., et. al. 2003. Curr
Top Microbiol Immuno. 272:331-364). CAR is a ubiquitous integral
membrane glycoprotein of the immunoglobulin superfamily which
also plays a role in cell adhesion. Following fiber knob binding, the
capsid penton base interacts with a,83 and ayfs cell surface integrins
which triggers clathrin-mediated endocytosis of the HAdS virus. It has
also been shown that heparan sulfate poteoglycans expressed on CAR-
negative cell surfaces are involved in the binding and infection of
HAdS5 (Dechecchi M.C., et. al. 2001. J Virol. 75:8772-8780).

In its native state, HAd5 is associated only with relatively mild upper
respiratory disease of man. Although HAdS replication was initially
believed to be restricted to human cells, emerging evidence
demonstrates that some cell lines from other species can support wild-
type HAd5 replication. Porcine primary kidney cells and the
established porcine cell line PK15 support HAdS replication to similar
levels seen in the highly permissive human A549 cell line JJlogler, C.,
et al., 2006. J. Virol. 80(7):3549-3558). These in vitro results are
consistent with the reported in vivo infectivity of wild-type HAd5 in
swine (Torres, J.M. et. al. 1995. Virology. 213:503-516), (Jogler, C., et
al., 2006. J. Virol. 80(7):3549-3558). In contrast, wild-type HAd5S
inoculation doses at least 1,000-fold higher are needed for mouse,
cotton rat, rabbit, guinea pig and woodchuck primary cell lines to
produce comparable amounts of infectious wild-type HAd5 virus
generated in human A549 cells (Jogler, C., et al., 2006. J. Virol.
80(7):3549-3558). Consistent with these observations, mice injected
with wild-type HAdS failed to provide clear evidence for virus
replication (Jogler, C., et al., 2006. J. Virol. 80(7):3549-3558).

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Importantly, HAd5 belongs to a virus species for which bovines are
not the natural host and there are no known published reports on the
wild-type HAd5 tropism and infection of cattle. Although wild-type
HAdS5 can replicate in the bovine MDBK cell line (Prevec, L., et. al.
1989. J. Gen Virol. 70:429-434), viral titers were reported to be
approximately 1 log lower compared to the standard reference human
293 cell line (van Olphen, A.L, et. al. 2002. J. Virol. 76(12):5882-
5892).

There are only two known reports of replication-competent,
recombinant HAd5 experimental vaccine inoculation in larger animals.
In the first report replication-competent HAdS vector carrying a
Vesicular Stomatitis Virus (VSV) gene was inoculated into 4-month
old calves, 8-week old piglets and > 6 mo. old dogs by either the
subcutaneous or intranasal routes (Prevec, L., et. al. 1989. J. Gen
Virol. 70:429-434). The authors did not directly comment on vaccine
safety or mention any clinical signs of adenovirus-induced disease in
these species. In the second report replication-competent wild-type
HAd5 or a replication-competent HAd5 vector expressing the FMDV
precursor polypeptide capsid gene (HAd5-P1) was tested in nine, 12-
18 month old cattle (Sanz-Parra, A. et. al. 1999. J. Gen Virol. 80:671-
679). Cattle were immunized twice by the subcutaneous and/or
intranasal routes. The authors did not directly comment on vaccine
safety or mention any clinical signs of adenovirus-induced disease in
these species. Cattle immunized with replication-competent wild-type
HAdS5 or replication-competent HAd5-P1 developed similar primary
systemic anti-Ad5 antibody responses, but failed to develop any
detectable systemic ant-FMDV antibody response. Since no post-
vaccination mucosal anti-FMDV or anti-HAd5 IgA or IgG titers were
detected, the authors concluded that the replication-competent HAd5-
P1 vaccine did not replicate well at mucosal sites, and further
speculated that the limited immune responses observed in cattle was
due to the low infectivity of HAd5 in cattle.

The MSV was derived from a BBA which originated by genetic
manipulation of a human adenovirus serotype 5 strain and the potential
hazard to animals associated with exposure to the MSV, and/or
product has been further reduced by GenVec’s genetic manipulation of
the Human Adenovirus serotype 5 strain prior to use in preparation of
the MSV. The MSV has been rendered replication deficient in normal
permissive mammalian cells because it was constructed, not by
insertion of FMDV genes into the wild type human adenovirus, but
rather by insertion of the FMDV genes into GenVec’s proprietary
human adenovirus serotype 5 vector, |, which served as a
replication deficient BBA. This BBA was previously genetically
altered by GenVec so as to render the BBA replication deficient in

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normal permissive mammalian cells by deleting the genetic material
coding for complete [jf and J, and [I adenovirus proteins.
(refer to SIF for full description of the BBA and GenVec’s
construction preparation and testing of the MSV). Since the MSV is
incapable of achieving a full round of the viral replication and
infection in normal permissive mammalian cells, the risk of adenovirus
infection to animals is significantly reduced to the risk posed by a
single aborted round of virus replication after cell penetration and
incomplete cycle of virus specific DNA replication or protein
expression.

Replication-deficient HAd5 vectors do not replicate in CAR’, oyB3"
MDBK cells (van Olphen, A.L, et. al. 2002. J. Virol. 76(12):5882-
5892; Bangari, D.S., et. al. 2005. Biochem. Biophys. Res. Comm.
331:1478-1484). Moreover, in MDBK cells the transduction
efficiency of replication-deficient HAdS is 58% compared to an 85%
efficiency using a replication-deficient bovine Ad5 (BAd5)(Bangari,
D.S., et. al. 2005. Biochem. Biophys. Res. Comm. 331:1478-1484). The
relative transduction efficiency of replication-deficient HAdS vs.
replication-deficient BAd5 in bovine turbinate (BT-5705) and bovine
embyryonic (EBL) cell lines were also compared. Both cell lines were
poorly transduced by replication-deficient HAd5 but were efficiently
transduced by replication-deficient BAd5 (Bangari, D.S., et. al. 2005.
Biochem. Biophys. Res. Comm. 331:1478-1484). Collectively, these
results suggest that replication-deficient HAd5 vectors will have a
lower transfection efficiency in the bovine host compared to
replication-deficient BAd5 vectors.

Replication-deficient HAd5 vaccine vectors expressing the bovine
herpesvirus type 1 glycoprotein gC or gD gene were delivered by the
intranasal route to calves (Gogey, S. et. al. 2002. Vaccine. 20 (9-
10):1451-1465). Calves were injected intransally twice 3 weeks apart
with 1 x 10'° CCIDspo (cell culture infectious dose). The presence of
HAdS5 vaccine virus in nasal swabs was determined by daily collection
for 1 week post-primary immunization and booster immunization and
virus isolation on the replication-permissive 293 cell line. Results
showed very low amounts of vaccine virus shedding (~ 1.6 x 10°
CCIDs59) in 3/15 calves on days 1 and 2 following the 1° immunization
and 2/15 calves on the first day only following the 2" vaccine dose (<
10? CCIDs 0). It is likely that the vaccine virus detected was from the
vaccine inoculation itself, and the authors concluded that since the
vaccine vector carried a E1 deletion and dysfunctional E3 gene, that
the vaccine vectors could not replicate in infected bovine host cells.

No evidence of phenotypic complementation in vivo of E1 deleted
HAdS5 was seen upon superinfection by wild-type replication

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competent HAdS5 in the cotton rat, which is a species known to be
permissive to wild-type HAd5 replication (Oualikene, W. et al. 1995.
J. Virol. 69:6518-6524). These data support extension to cattle
immunized with El deleted HAd5 vaccine virus that may be
superinfected with bovine adenovirus. Similarly, no interference
between HAd5 and BAd 3, such as recombination of DNA or cross-
activation of virus replication was reported following up to 5 passages
in double-infected human cells (Rasmussen, U.B. et. al. 1999. Hum
Gene Ther. 10:2587-2599).

Replication-deficient HAd5 vectors do not replicate in the porcine PK-
15 cell line JJogler, C., et al., 2006. J. Virol. 80(7):3549-3558). These
results directly correlated to in vivo results in which i.v. inoculation of
a replication-deficient HAd5 vector into a sow resulted in no
detectable virus replication and liver GLDH levels remained in the
normal range throughout the study. Furthermore, direct
intramyocardial or intracoronary injection of a replication-deficient
HAd5 vector into mini-pigs showed no echocardiogram evidence of
either segmental or global left ventricular dysfunction (Muhlhauser, J.
et. al. 1996. Gene Therapy. 3(2):145-153). The authors concluded that
such vectors are safe for short-term gene transfer into the heart of large
animals.

In conclusion, the replication-deficient HAd5 published data in cattle
and other species suggest that replication-deficient HAd5 vectors are
environmentally safe.

Recently, GenVec has completed mouse safety testing per
9CFR113.33 in which there was no evidence any of the mice suffered
from adenovirus-induced signs of disease post inoculation. Likewise,
GenVec’s recently completed calf safety test per 9CFR 113.41 resulted
in no signs of disease in the test animals.

Finally, GenVec recently completed USDA-CVB mandated back-
passage and reversion to virulence studies in cattle wherein J
he MSV was inoculated directly onto the nasal mucosa of
test cattle. All the cattle remained well, and the virus vector was
recovered in 4 of 10 animals after the first 24 hours by nasal swabs.
No virus could be recovered from the oral cavity. Virus isolated in 4
of 10 animals in the first 24 hours was identified genetically to be the
inoculum virus vector and was detectable in these 4 animals at a level
approximately 10 logs below that of the virus vector inoculum. The
study was halted and no further backpassage was performed. A
shed/spread study recently completed in cattle and swine was also
performed (I. The cattle were administered the
vaccine by IM inoculation, which is the recommended route for

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immunization. All the cattle and pigs remained well during the study
and there was no indication of transfer of virus by FMDV or HAd5
serology in naive animals commingled with vaccinates, nor from nasal
swabs taken from the animals at 7 day intervals throughout the 35-day
post inoculation observation period. The MSV, consistent with its
deliberately designed genotype proved to be completely replication
deficient in cattle.

Although it is clearly unlikely any adenovirus disease will occur, it is
important to point out that only directly administered test animals are
at any risk of adenovirus disease under this proposal as the vaccine has
been proven by direct experimentation to be incapable of shed/spread..

Any directly administered test animals which might experience clinical
adenovirus disease would experience any such disease under full
veterinary supervision and care.

ii The Consequence Rating, (and degree of certainty) for the risk
of adenovirus disease of animals

The consequence of adenovirus disease would most probably be mild
and self-limiting, as discussed above. Therefore the consequence
rating is low, and the degree of certainty is certain.

iii Likelihood Rating, (and degree of certainty) for the risk of
adenovirus disease risk in animals

Adenoviruses are considered to be host-specific in their infectious
range. The MSV was derived from a BBA which originated by
genetic manipulation of a human adenovirus serotype 5 strain, while
the vaccine’s target animal is cattle. In its native state, serotype 5
human adenovirus is normally associated only with relatively mild
upper respiratory disease in man and is not usually considered
infectious for any mammalian species other than man. Wild-type
Serotype 5 human adenoviruses, therefore, are not usually considered
capable of producing infectious disease states in cattle, mice or any
other mammals than man. The hazard to animals, other than man,
posed by wild type serotype 5 adenoviruses seems to be very low.

Beyond the host-range limitation described above, the hazard to
animals associated with exposure to the MSV, and/or product has been
further reduced by GenVec’s genetic manipulation of the human
adenovirus serotype 5 strain prior to use in preparation of the MSV.
The MSV has been rendered replication deficient in normal permissive
mammalian cells because it was constructed, not by insertion of
FMDV genes into the wild-type human adenovirus, but rather by

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insertion of the FMDV genes into GenVec’s proprietary human
adenovirus serotype 5 vector, [NN which served as a replication
deficient BBA. This BBA was previously genetically altered by
GenVec so as to render the BBA replication deficient in normal
permissive mammalian cells by deleting the genetic material coding
for complete |, and P| adenovirus proteins (refer to SIF
for full description of the BBA and GenVec’s construction preparation
and testing of the MSV). Since the MSV is incapable of achieving a
full round of viral replication and infection in normal permissive
mammalian cells, the risk of adenovirus to animals is reduced to the
risk posed by a single aborted round of virus replication after cell
penetration and incomplete cycle of virus specific DNA replication or
protein expression.

GenVec has conducted multiple studies (summarized in the SIF) and
in section III.A.1.b.i which confirm by direct evidence the BBA and
RBA are avirulent in cattle and swine. Additional work in other
species support the same conclusion in respect to other species which
might be incidentally exposed to the RBA.

GenVec, has also, in collaboration with scientists at the BL-3 animal

testing facility
, conducted multiple studies (also summarized in the

SIF) which confirm by direct evidence that the BBA-FMDV construct,
(the recombinant strain which is described as the Regulated Biological
Agent (RBA) in the SIF, and which was used to prepare the MSV) is
replication-deficient and was proven safe in both cattle and in swine.
The [MM studies also demonstrated the RBA strain gives a
reasonable expectation of efficacy in cattle, supporting its
development for use under conditional USDA licensure as an aid in
prevention of FMD. In these J BL-3 vaccination/challenge
studies there was no evidence indicating that the RBA had shed or that
it had spread from any of the animals which had been directly injected
to any of the sentinel naive control animals with whom the principal
animals had been co-housed throughout the study. In addition, in
studies with other highly related BBA-FMDV vaccine constructs for
other FMDV serotypes/subtypes, there was no evidence indicating that
these BBA-FMDV vaccine constructs had shed nor that the vaccine
had spread from any of the 131 animals which had been directly
injected to any of the 43 sentinel naive control animals with whom the
principal animals had been co-housed throughout the studies.

GenVec’s recently completed pre-license studies of calf safety, mouse
safety, back-passage/reversion to virulence, and a shed/spread study
all independently confirm with direct scientific evidence the vaccine’s

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innocuousness and inability to induce adenovirus disease in the
environment.

Based on the above combination of direct and indirect scientific
evidence, the likelihood of adenovirus disease in the test animals is
considered very low, with a high degree of certainty.

c. Risk of recombination of the MSV strain with a wild-type FMDV
during testing under this proposal.

i Discussion of risk

The recombination of the MSV or its derivatives with wild-type
FMDV would require co-infection of a single cell concurrently with
the MSV and with an infectious FMD virus. Furthermore, because the
MSV is a DNA containing virus, and FMDV is an RNA virus with no
DNA component, any homologous recombination is very unlikely. In
the event there were recombinations the event would have the effect of
creating a recombinant FMDV which would have acquired one or both
of the donor FMDV sequences of the MSV. Since neither of these
genes is virulence-associated, the recombinant would be expected to
be of no greater virulence than its wild-type FMDV parent.

Furthermore, the theoretical recombination event would be occurring
within a production facility inside the United States and would
presuppose test animals were undergoing a concurrent undetected
FMD outbreak while under a full program of veterinary care. The
consequence of the recombination would be of no significance in
comparison to the wild-type outbreak of FMD within the United
States. Clearly any such FMD outbreak would be of far greater
concern and of greater significance than the unlikely attendant
recombination event. Any FMD detection within the test facility
would no doubt require complete depopulation and disinfection of the
facility, and potentially of surrounding animal populations, as well.

ii Consequence rating, (and degree of certainty) of risk of
recombination with wild-type FMDV

The consequence of a recombination under such circumstances would
be low, and managed inside the greater scope of an FMDV outbreak

emergency. The degree of certainty associated with the consequence
is certain.

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Page 16 of 26

iii Likelihood rating (and degree of certainty) of risk of
recombination with wild-type FMDV

FMDV is exotic to the US, and the entire veterinary machinery of the
country is employed in monitoring and preventing its introduction.
Direct surveillance testing by USDA is done to detect any introduction
of FMDV into US livestock. Presence of a wild-type FMDV would
be very unlikely within the well-managed and veterinary-supervised
animals used for the proposed testing.

In the event of a successful field safety test outcome, the conditionally
licensed vaccine would only be used if deployed from the
USDA/DHS- managed veterinary vaccine bank. Again, under such
circumstances the likelihood of recombination with FMDV would be
very low, and consequences well managed under USDA emergency
program provisions.

The likelihood rating for the risk of recombination of the MSV with
wild-type FMDV is therefore extremely remote, and categorized as
low, with a degree of certainty categorized as certain.

d. Risk of recombination with wild-type adenovirus

i Discussion of the risk.

For a recombination of the MSV with a wild-type adenovirus to occur,
one of the cells of one of the test animals would need to be
concurrently infected with an adenovirus and the MSV. In addition,
there would have to be concurrent localized DNA melting of both the
wide-type and MSV to allow recombination to occur. Because the
MSV is replication incompetent the likelihood of the two viruses
undergoing recombination from localized DNA melting during
replication is remote. Such an event could theoretically result in the
creation of a recombinant adenovirus which would have picked up one
or other or both of the crippling deletions in the J region of the
MSV. Any such recombinant would be equally as attenuated as the
MSV, and would be of no real detectable consequence.

Alternatively, the MSV strain might theoretically pick up a functional
|| or | | gene from the wild-type adenovirus co-infecting the same
cell. If such an event occurred, the consequence would be creation of
a recombinant with a functional [J or a functional [J gene. Because
functioning || and ff genes are necessary for re-acquisition of
replication competent phenotype, two independent recombination
events in two separate locations within the same genome would be
required in order for the MSV to require replication competence. A

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Page 17 of 26

single recombination in the [J region or in the [Jf region would result
in creation of a single site recombinant which would remain
replication deficient, just as the MSV is replication deficient.

In the extremely improbable event that both an || wild-type
recombination event and an [J wild-type recombination event
occurred within the same MSV virus particle, both events occurring
during the single aborted replication cycle of the MSV, it could be
theorized that the possible result would be creation of a replication
competent human adenovirus serotype 5 expressing the FMDV
proteins. That a human adenovirus having achieved infection of a
bovine cell could then sustain the infection by multiplication and
spread to other cattle is even more unlikely. Human adenoviruses
come in contact with cattle on a frequent basis; yet human adenovirus
disease in cattle is unknown. Thus the creation of the recombinant
inside a single bovine cell from which it could not effectively be
spread would be of essentially no consequence.

Furthermore, if this very unlikely series of very low probability events
occurred rapidly and in sequence, the result would be at most very
mild or inapparent disease of one asymptomatic adenovirus infected
SPF animal. For the recombinant to spread further it would require
that the human adenovirus infect a second bovine. Human
adenoviruses are not known to infect cattle in nature. It is expected,
therefore that even if clinical signs were to develop, the problem
would occur within a single bovine individual..

ii Consequence rating (and degree of certainty) of adenovirus
disease due to recombination

The consequence, if any, would be very low, and would almost
certainly be limited to the test animal in which the co-infection and
subsequent recombination events occurred. The consequence rating,
therefore, for adenovirus recombination is low and the degree of
certainty, based on combination direct and indirect scientific evidence
is certain.

iii Likelihood rating (and degree of certainty) of adenovirus
disease due to recombination

This scenario was discussed specifically in the SIF, and its likelihood
was estimated as less than 1 chance in 10”° virus particle replication
events, and thus is considered low. Since the virus production in any
one cell is approximately ma to [BI virus particles, the probably of the
event is extremely low and the ability to detect such an event almost
impossible by current state of the art methods. The degree of certainty

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September 14, 2009

of the likelihood rating is based on indirect scientific evidence and thus
is considered moderately certain.

2. Public Health Safety

a. Risk to Public Health of Release of the MSV into the human
population

i Discussion of risk

The BBA used for construction of the RBA, which was in turn used
for preparation of the MSV, is a replication deficient human
adenovirus serotype 5 currently under testing for use in medical
products for treatment and prevention of human disease. The BBA
was used for construction of four (4) products currently in human
Phase I-III clinical trials, under FDA regulation. The BBA, and thus
the MSV is considered avirulent for humans.

The consequence of release of the MSV into the human population
(for example by accidental self-injection) would be the possible
asymptomatic seroconversion of the directly exposed personnel to the
BBA proteins and the FMDV | | protein. No shed or spread from
the directly exposed individual would be expected, due to the multiple
gene deletions in the MSV which render it replication deficient in
mammals. Based on direct scientific studies of the BBA in FDA
regulated studies it is expected even accidental self-injection of the
MSV would not result in illness.

Direct backpassage and reversion to virulence studies in cattle, in
addition to shed/spread studies in cattle and swine support the
genotype-based assumption that the MSV is not capable of
establishing itself by shed/spread from an inoculated individual.

ii Consequence Rating (and degree of certainty) of risk of release
of MSV into the human population

The consequence rating is low, and its degree of certainty, based on
the FDA-regulated testing of the BBA is certain.

iii Likelihood Rating (and degree of certainty) of risk of MSV
introduction into the human population

The degree of certainty of the low likelihood rating is considered
certain, based on the direct scientific evidence of the BBA testing and

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the recent pre-license testing of the MSV by GenVec and confirmation
by CVB-L.

3. Environmental safety

The back passage and reversion to virulence studies demonstrate, as predicted by
the literature for similar adenovector backbone agents used in human application,
that the virus vector cannot be reisolated from any preferred sites for adenovirus
replication except from the nasal orifice within the first 24 hours post-inoculation
in 4 of 10 animals (see SIF revised September 9, 2009). Furthermore, the
recovered virus vector presented equivalent genetic identity with the RBA
inoculum, indicating the recovered virus was residual inoculum at the
administration site. The virus titer was reduced by approximately 10 logs from the
applied inoculum to the residual level still detectable after 24 hours. At 48 hours,
no virus was recoverable from oral or nasal samples. Thus, by a preferred site of
exposure to the environment and a preferred site for entry of adenovirus into any
mammalian population, the virus is not capable of replicating.

Moreover, when a study designed to examine shed/spread was performed by
administering the vaccine by the intramuscular route, no virus could be isolated
from direct sampling of the animal’s oronasal cavities. Based on the known
genotypic and phenotypic characteristics of both the BBA and RBA and direct
evidence in these two designed experiments it is very confidently predicted the
altered sequences will not enhance survival in the environment nor increase
resistance to therapeutic agents.

The proposal involves release of a non-shedding, non-spreading, replication
incompetent vaccine containing virus originating from a fully tested MSV into
cattle. Release will occur under a fully supervised field safety protocol. In the
event this test is supportive of the product, a later release of the vaccine into the
US cattle population would only occur if the vaccine’s deployment was
authorized by USDA and DHS as part of an FMD emergency plan. Under these
circumstances the risk to the environment is considered negligible.

B. Release Assessment

1. Location of Test Sites
Three cattle production and testing facilities,

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a. The sites are a combination of beef production and dairy farms that are
under the supervision of cooperating Study Investigators (licensed
veterinarians), with the study at all sites operating under the oversight of a
single Principal Investigator. The staff at each site will be trained by the
Site Investigator and/or the Site Monitor to ensure consistent
understanding of the study conduct, technical observations and study
terminology (e.g., VEDDRA terms). The sites are located in rural areas
isolated from both other animal and human populations. The investigators
are experienced with administration of vaccine and data collection for
field exposure conditions.

b. Cattle production facilities approved by USDA CVB for biologics field
safety testing, per 9CFR 103.3.

3. Personnel at the test sites

a. Each testing site will be supervised by an attending veterinarian (Study
Investigator). The names and contact information for these investigators
will be part of the 9CFR 103.3 application to be filed by PerOs USA Inc.
prior to initiation of the study. GenVec has contracted with

to supply overall management of the multi-site field safety
study. The sites above were chosen because they have the ability to
conduct the study plus the personnel are trained in vaccine and infectious
disease management. In addition, they are experienced with experimental
biologicals and data collection needed within a restricted time frame for
the study.

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4. Experimental design

To briefly summarize, a total of approximately 600 healthy cattle, 3 - 6
months of age will be injected with one or other of two pre-licensing
serials of the product (Code 1FM1.RO0). Injection will be per the label
recommendation. Injection sites will be palpated the following day and 7,
14 and 21 days later. Daily observations for general health will be made.
Any untoward events will be recorded and made part of the final field
safety report to be submitted to USDA APHIS CVB in support of the
pending license application for the vaccine.

5. Potential for escape and dispersal from the identified test sites

a. Detailed genetic analysis of the MSV and its predecessors has confirmed
the adenovirus is non-replicating and avirulent. Direct animal evidence
confirming this expectation for the vector has been gathered in swine,
cattle, and mice. Direct evidence that the MSV is incapable of shedding
from the bovine to which it is directly administered has been gathered by
GenVec, as well. There is no realistic possibility of escape or any realistic

prospect of dispersal of the tested materials from these directly inoculated
cattle.

6. Potential for establishment in the environment of the test site

a. The MSV organism is replication deficient and thus incapable of
establishment in the environment. Direct backpassage and reversion to
virulence studies in cattle, in addition to shed/spread studies in cattle and
swine support the genotype-based assumption that the MSV is not capable
of establishing itself by shed spread from an inoculated individual.

7. Monitoring in and around the test site

a. No further monitoring within or around the testing sites is envisioned at
this time.

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8. Contingency plans in case of adverse event

a. Inthe event an adverse event occurred, the study would be quickly
terminated by humane euthanasia of any affected or suspect test animals
with immediate notification of USDA APHIS CVB PLE and IC. A
complete sanitization and disinfection of the affected facility would be
completed. Full post mortem examinations and complete diagnostic

workups of any suspect animals would be conducted and reported to
USDA APHIS CVB.

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C. Risk Characterization

The detailed risk characterization for this proposal is provided in Table 1. All risks of
identified hazards associated with approval of the proposal to field safety test Foot and
Mouth Disease Virus Vaccine, Serotype |, Live Adenovirus Vector, Product Code
1FMI1.RO is characterized as having a calculated risk rating of low. The overall rating of
the risk of this proposal is accordingly considered to be low.

IV Risk Management

A. Contained release

Not applicable.

B. Environmental release

It is proposed that Foot and Mouth Disease Virus Vaccine, Serotype a. Live
Adenovirus Vector, Product Code 1FM1.RO be released for field safety testing, and if
deployed by DHS/USDA, be used in the field during a federally directed FMD-
emergency management program.

V Risk communication

The applicant understands that this document may be used by USDA APHIS CVB to
further the agency’s obligation to provide transparent risk communication.

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Table 1. a Product Code 1FM1.RO Risk Characterization

Risk Characterization! of Proposal for Field Safety Testing in Support of Application for Conditional Licensure of Foot
and Mouth Disease Virus Vaccine, Serotype |. Live Adenovirus Vector, Product Code 1FM1.R0

Degree of
Certainty
Rating
for
Likelihood

Degree of |-
Consequence | Certainty ‘Risk .

Rating | Ratingfor | Characterization
Consequence | ©

Expected Risk Justification
Risk Ratin for
Rating 8) Rating”

Likelihood
Rating

FMD within
the testing
and/or
production
facility due to
introduction of
complete
FMDV into
animals

Section IT] A

Low Certain Low Certain LL.C.CL.C 1.0000 L Lai

Risk of
adenovirus
disease in
animals

Section IT A

Low Certain Low Certain LL.C.CL.C 1.0000 L Lai

1 Based on USDA guidance document, “Risk Assessment Outline for Use in Preparing Risk Analyses for Biotechnology-Derived Products, The Center for
Veterinary Biologics, USDA/APHIS/VS , October 1, 2005”
* Section III of this document

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Degree of |.
Certainty:
Rating
for
Likelihood.

8 Degree of
Consequence | . Certainty Risk

- Rating Rating for | Characterization
Consequence |

Justification:
for
Rating”

| Expected
Risk
Rating

Risk —

Likelihood
Rating

Risk Rating

Risk of
recombination
of the MSV Low Certain Low Certain LL.C.CL.C 1.0000 | L__ | Section IA
strain with laiti
wild-type
FMDV

Risk of
recombination | 1... | Moderate Low Certain LL.MC.CL.C 7500 r | Section IA
with wild-type laiv

adenovirus

Public Health
Risk of

Release of the . . Section II] A
MSV into the Low Certain Low Certain LL.C.CL.C. 1.0000 L Dai
human

population

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September 14, 2009

Appendix 1: as

</div>

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