AD-AtOP 926 DEVELOPMENT AND EVALUATION OF FIENO-NTLV-III HYBRID 1VIRUS FW NO-CVTOPRT.. (U) BIOTECH RESEARCH LABS INCROCKVILLE N N T LUBET ET AL. 29 OCT 87

7-96-C-62-94 F/6 611 Nll6/15 Itll

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a) IDEVELOPMENT AND EVALUATION OF

0) ADENO-HTLV-III HYBRID VIRUS AND NON-.

00 CYTOPATHIC HTLV-III MUTANT FOR VACCINE USE ,p

Annual Report

by p

Martha T. Lubet and Sandra K. Dusing

October 28, 1987

Supported by

US ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-86-C-6284 DTICELECT .

FEB 2 6 1988 ,Biotech Research Laboratories, Inc. S

Rockville, Maryland 20850

Approved for public release; distribution unlimited.b

The findings in this report are not to be construed asan official Department of the Army position unless sodesignated by other authorized documents.

I

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SECURITY CLASSIFICATION OF THIS PAGE F A

Form Approved -. *REPORT DOCUMENTATION PAGE OMB No. 0704-0788

la. REPORT SECURITY CLASSIFICATION lb. RESTRICTIVE MARKINGS ,. , .

Unclassified Unclassified %

Za. SECURITY CLASSIFiCATiON AUTHORITY 3. DISTRIBUTION /AVAILABILITY OF REPORT

2b. DECLASSIFICATION/DOWNGRADING SCHEDULE Approved for Public Release andDistribution Unlimited

4. PERFORMING ORGANIZATION REPORT NUMBER(S) . MONITORING ORGANIZATION REPORT NUMBER(S).

6a. NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION

Biotech Research Labs., Inc. (If applicable)

6c. ADDRESS (City. State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

1600 East Gude Drive

Rockville, MD 20850 , ,

8a. NAME OF FUNDING/SPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S. Army Medical (If applicable)

Research & Development Commal d Contract No. DAMD7-86-C-6284

8c. ADDRESS (City, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERSPROGRAM PROJECT TASK WORK UNIT

Fort Detrick, Frederick, MD 21701-5012 ELEMENT NO. NO. 3M2- NO. ACCESSION NO.

623105 623105H29 AD 014

11. TITLE (Include Security Classification)Development and Evaluation of Adeno-HTLV-III Hybrid Virus and

Non-Cytopathic HTLV-III Mutant for Vaccine Use12. PERSONAL AUTHOR(S)

Lubet, Martha Turner and Dusing, Sandra Kay

13a. TYPE OF REPORT 13b. TIME COVERED 114. DATE OF REPORT (Year, Month, Day) 15. PAGE COUNT

Annual FROM 9/29/86TO 9/30/87 87/10/28 46

16. SUPPLEMENTARY NOTATION

17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP

06 03 AIDS, Vaccine, DNA Recombinant, Antibody, T-Cell .--

06 13 "'19. ABSTRACT (Continue on reverse if necessary and identify by block number) _rcnz as

- Acquired immunodeficiency disease syndrome (AIDS) was initially recognized as a

separate disease in 1981. Results from research groups in France and the United States

determined that a previously unknown virus called HIV is the primary aetiological agent

of AIDS.

Two HIV vaccines, a recombinant Adeno-HIV hybrid virus and a recombinant vaccinia ""

HIV will be tested. The recombinant Adeno-HIV virus is being developed as part of this

proposal. The vaccines will be tested in two species of monkeys, chimpanzees and African

green monkeys. Vaccinated animals will be challenged with a defined dose of HIV virus.

Assessment of vaccine efficacy against the virus challenge will include T4/T8 ratios,

Interleukin-2 production, HTLV-111 serology and ability to detect infectious HTLV-III

virus in peripheral blood cells. T-cell mediated immunity will be assessed by monitoring

cvtotoxic T-cell activity and antigen-induced PBL proliferation.

In Phase II of this proposal, we will focus on whether generation and cloning of

HIV specific killer T cells could be used to protect humans from AIDS.

20. DISTRIBUTION/AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATIONUNCLASSIFIED/UNLIMITED 0 SAME AS RPT 0 DTIC USERS Unclassified

22a. NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (Include Area Code) I22c OFFICE SYMBOLVirgini,, Miller (301)663-7325 SRD-RMI-S

DDFormI47L, JUN 86 Previous editions are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

DD... 47.............................................................. .;

TABLE OF CONTENTS

DD Form 1473, Report Documentation Page

I. Summary .. . .. ... ... .. ............... i

II. Foreword ........ ............... .. ii

III. List of Figures and Tables

A. Figures ........................ 1

Bo Tables ............................ 1 16

IV. Body of the Report

A. Statement of the Proposal ........ 3

B. Background ......................... 4

C. Rational........ ..................... 6

D. Experimental Methods................. 9

E. Results and Discussions ............ 12

F. Conclusions............................ 19

G. Recommendations .................... 19

H. Figures .......................... 21-33

I. Tables ................................ 34-39

V. Literature Cited -............... 40 o.on For

VI. Distribution List.. ....................... . .

t#, L v_1A or

t

SUMMARY

The immune response of African Green monkeys, baboons

and chimpanzees to HIV has been monitored in infected andvaccinated animals. Anti-HIV antibody was characterizedfor its ability to neutralize HIV virus and its reactivityin the HIV Western blot assay. Antigen-induced cellularproliferation was also studied. Preliminary experimentsto establish protocols for measuring cytotoxic T cellresponses in primates were conducted. Standardizationof the conditions leading to reproducible infection ofAfrican Green monkeys with HIV established that this spe-

cies could be infected most reproducibly by intravenousadministration of HIV 2. 50 primates of various species

*, were tested for "natural" infection with HIV. Only onemangaby showed a reproducible anti-gpl60 reaction withHIV 1 but not HIV 2.

Construction of plasmids containing gpl20 sequencesnecessary for development of the rAd2 vaccine have beencompleted. Both gpl20-specific RNA and proteins havebeen detected in transient expression experiments usingHeLa cells transfected with the gpl20-plasmids indicating

that the HIV env sequence contained in these plasmids istranscribed and translated in mammalian cells. Basedsolely upon plaque size on CV-I cells, it appears thatisolates of rHIV 1 gpl20/Ad2 have been obtained fromrecombination between the plasmid pAD.ENV.NDI and wt Ad2.

3%

I.w.

FOREWORD

In conducting the research described in this report, theinvestigator(s) adhered to the "Guide for the Care andUse of Laboratory Animals", prepared by the Committeeon Care and Use of Laboratory Animals of the Instituteof Laboratory Animal Resources, National Research Council(DHEW Publication No. (NIH) 78-23, Revised 1978).

The investigator(s) have abided by the National Institutesof Health Guidelines for Research Involving RecombinantDNA Molecules (April 1982) and the Administrative Prac-

tices Supplements.

Citations of commercial organizations and trade namesin this report do not constitute an official Department P

of the Army endorsement or approval of the products orservices of these organizations.

.'

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LIST OF FIGURES AND TABLES

FIGURES PAGE

1. Local Reaction of Monkeys Immunized with

Recombinant Vaccinia-HIV virus ...... 21

2. Restriction Digestion Pattern of pAD.MLPgpl20 ..................................... 22

3. Strategy for Construction of Vaccine

Plasmid pAD.MLP.NDl ...................... 23

4. Analysis of Plasmid pENV.NDI .............. 24

5. Analysis of Plasmid pAD.ENV.NDI.............. 25

6. Analysis of Plasmd pAD.ENV..................26

7. Analysis of Plasmid pAD.MLPgp60 ........... 27

8. Analysis of Plasmid pENV(160)NDI .......... 28

9. Accumulation of HIV gpl20 in TransfectedHeLa Cells ................................. 29

10. Dot Blots of RNA from Cells Transfectedwith pAD.ENV.NDI and pAD.ENV .............. 30

11. Construction of Recombinant HIV gpl 2 0/Adenovirus 2 .............................. 31

12. Morphology of Plaques Formed by PutativeRecombinant HIV gpl20/Adenovirus 2 on d

CV-I cells ............. . ............. 32

13. In Situ Hybridization of CV-1 Cells Infec-ted with Putative Recombinant HIV gpl20/Adenovirus 2 ................................... 33

TABLES

1. Follow-up Studies of African Green MonkeyInfected with HIV ............................. 34

• 2. Immune Response of Monkeys to HIV .......... 35

3. Assay for Standardization of HIV Infection

of CercopIthecus .......................... 36

1°.

LIST OF FIGURES AND TABLES (Cont'd)

TABLES PAGE

4. Immune Response to Recombinant Vaccllnia-

U ~~~H IV Vaccine......... .............................. 37

5. Cell-Mediated Gytotoxicity Against CellsCarrying HIV env Antigens................... 38

6. Selection of Recombinant HIV env/Adeno-virus 2................ ........................ 39 A

I.-

2

2J'

BODY OF PROPOSAL

A. Statement of Problem:

Acquired immunodeficiency disease syndrome (AIDS)was initially recognized as a separate disease in 1981.Results from research groups in France and the UnitedStates determined that a previously unknown virus

called LAV in France and HTLV-III in the United States

is the primary etiological agent of AIDS. Recentlythis virus has been renamed HIV.

AIDS is a highly lethal disease and presents aserious medical, social and economic problem of globaldimensions. The World Health Organization estimatesthe total number of AIDS cases worldwide at 100,000 %with 5 to 10 million asymptomatic HIV-infected indivi-duals (1). In the United States alone, more than36,000 cases had been reported by mid-1987 with a

mortality rate of greater than 50% (2). James Curronof the U.S. Center for Disease Control, Atlanta, Geor-gia, estimates that, within 5 years, AIDS will be theleading cause of death among young and middle-aged men(2). In the past, the major mode of AIDS transmissionwas considered to be through homophilic sexual practices

and via contaminated needles associated with drugabuse. However, evidence is accumulating that hetero-sexual transmission with attendanct maternal-fetal infec-tion is, likewise, an important mode of HIV transmission,thus, putting the entire population "at risk". The spread

of AIDS into the general population underscores theurgency of developing an effective vaccine against HIV asimmunoprophylaxis appears to be the most rational andeffective means to control the spread of this disease.

Up to 100% of patients with AIDS and pre-AIDS syn-

drome have anti-HIV 1 antibody in their sera (3), sug-gesting that anti-HIV 1 antibody is not sufficient toprotect patients from development of AIDS. The aim ofthis proposal is to study the role which T cell media-ted immunity and anti-HIV antibody play in preventingor ameloriating HIV infection and the development of aneffective HIV vaccine.

Monkey models studied include chimpanzees andAfrican Green monkeys. Chimpanzees are the only non-human primate that can be reproducibly infected with HIV(2,4,5). Preliminary experiments by one of us (D.Zagury)indicate that the African Green monkey can be used asan animal model to study HIV infection. Studies inAfrican Green monkeys will establish the strain and

dose of HIV virus and the route of infection. Chimpan-

zees and African Green monkeys will be vaccinated with

3N N

&'2A Ni! %

a recombinant adeno-HIV virus and vaccinia-HIV hybridvirus. Assessment of vaccine efficacy against vaccinevirus challenge includes T4/T8 ratios, HIV serology,and ability to detect infectious HIV virus in peripheralblood cells. T cell mediated immune responses will bemonitored by cytotoxic T cell activity and antigen-induced cellular proliferation.

B. Background:

AIDS is a recently identified disease character-ized by increasing deficiencies in the body's cell

mediated immune response in previously normal patients(3). A reduction in the number of helper T lymphocytes

(OKT4+) is usually involved and is accompanied bymultiple opportunistic infectious and/or malagancies(7). A syndrome designated AIDS related complex (ARC)has been identified in groups at risk. The dominantclinical expression in ARC is unexplained chroniclymphadenopathy or leukopenia involving a reduction inOKT4+ cells. Minor cutaneous infections, diarrhea,weight loss and fever may be associated with ARC (8).Results from research scientists in France and theUnited States strongly suggest that a previously unknownvirus now called HIV 1 is the primary aetiologicalagent of AIDS. Western Blot analysis reveals that AIDSand ARC patients serum contains antibodies to all orsome of HIV proteins (9).

Chimpanzee Is the only non-human primate known tobe susceptible to infection with HIV (4,5). Infectedchimpanzees develop a transient severe lymphoadeno-pathy. Virus can be recovered from peripheral bloodlymphocytes and bone marrow but not the plasma, saliva

or cerebralspinal fluid (10). Although all HIV inocu-lated chimps remain persistently infected, a persistentdecrease in T4+ cells does not develop and the chimpan-

zees remain clinically well (11).

Preliminary experiments by Dr. Daniel Zaguryindicated that Africian Green monkeys (C. aethiops)infected with HIV virus by intraperitoneal (IP) injec-tion develop viral infection. Some animals showedevidence of infection as detected by reverse transcrip-tase activity, ability to transmit HIV infection toperipheral blood lymphocytes and the presence of HIVgenome in their peripheral blood lymphocytes. Somemonkeys had a serum antibody response to HIV. Becausenot all of infected animals could be shown to beinfected, this model requires further characterization.

When a foreign body invades the body, a variety ofimmune reponses, including antibody and cytotoxic T

cells, can be induced. In order to be successful, a

4

%UK.

vpccine must elicit a immune response that is neutra-lizing or protective. Although anti-HIV antibodies mayneutralize at the intial stages of viremia, they do notappear to be protective since patients with high titersof anti-HIV antibodies develop AIDS (12). Cytotoxic T

cells can play an important role in host defense againstviral infectious (13) and vaccination leading to cellmediated immunity to HIV may be desirable becausecytotoxic T cells could protect from further cell tocell viral spread. HIV specific cytotoxic T lymphocytesin seropositive individuals have been demonstrated (14,15). However, the role they play in AIDS is not known.T cell receptors present a heterodimeric reactivitywhich is directed against both self-major histocompati-bility antigens and processed antigenic determinants.In contrast to antibodies, which may be serotype specific,cytotoxic T cells exhibit broader antigenic specification.This accounts for cross reactions observed by cytotoxicT cells of autologous targets infected by differentstrains of virus with serologically distinct influenzavirus (16,17). Thus a vaccine inducing cytotoxic Tcells against one subtype of HIV may destroy cellsinfected with other HIV subtypes.

The objective in the development of a HIV vaccineis to produce, with minimal side effects, an immuneresponse of long duration in the host that will provideeffective protection against viral infection and thesubsequent onset of clinical AIDS. The worldwide pre-valance of AIDS also makes it important that such avaccine be easily administered and relatively inexpen-sive to produce.

The majority of viral vaccines now in use are

preparations of either live, attenuated or killedvirus. Concerns for reversion to virulence and the

risk of reactivation of intact virions or proviral DNAmake these approaches to a HIV vaccine untenable. Atpresent, subunit vaccines utilizing immunogenic viruspeptides or proteins derived from purified virions orby recombinant DNA technology appear to provide thebest approach to development of a safe and effectivevaccine. These subunit components may be administeredin association with adjuvants or carriers, incorporatedinto artifical "membranes" or delivered by means ofrecombinant viruses. Several recombinant vaccinia-"IVviruses have been developed and tested in chimpanzees(18-22) and humans (23). Chimpanzees vaccinated withvaccinia-HIV demonstrate very low titers of neutrali-zing antibodies (18). Therefore the development of analternate HIV recombinant vaccine may prove valuable.For a variety of reasons outlined below, we have proposedto utilize human recombinant (r) adenovirus (Ad) as avector for expression of the HIV glycoprotein, gpl20.

5

2., e.

Bivalent immunization with live, attenuated Ad

type 4 and type 7 has proven to be both safe and effec-

tive in military recruits over a period of 20 years

(24,25,26). This vaccine, which is administered orally

in enteric-coated capsules, liberates virus into the

intestine where a subclinical infection is established

that confers a high degree of immunity. Successful

immunization with Ad types 1, 2 and 5 by this route

have also been demonstrated (27). The fact that admi-

nistration of live vaccines by the gastrointestinalroute may facilitate spread of the Ad (28) may be advan-

tageous in immunizing against HIV if a suitable, rela-

tively non-pathogenic strain of adenovirus is used as

vector. The majority of Ad infections result in self-

limiting and short-lived clinical manifestations although

prolonged asymptomatic or latent infections may occur

(29). It is possible that reactivation of latent adeno-

virus, as has been proposed to occasionally occur in

rubella infections (30), may provide a mechanism for a

natural HIV "booster" immunization in the case of the

rAd vaccine.

Ad structural proteins are synthesized in large

quantities during infection and at least 80% of viralhexon, penton and fiber are not incorporated into

progeny virus but remain in the infected cell in theform of readily soluble multimers (31). Theoretically,

exogenous DNA sequences stably integrated into the Ad

genome under the control of the major late promoter

might also be expressed at a high level to provide a

source of immunogen.

Ad is very stable and can tolerate temperatures of

4-36*C and pH 5-9 with minimal loss of infectivity,

thus, alleviating many problems associated with vaccine

transport and storage especially in under-developed

countries.

The technology necessary to propagate large quanti-

ties of Ad to produce an enteric-coated vaccine is pre-

sently available so that production of a rAd vaccineshould not require the development of new manufacturing

processes.

This project is designed to investigate the feasi-

bility of using infectious Ad to produce gpl 2 0 or otherHIV immunogen and to determine whether these HIV anti-

gens would be presented in such a way as to elicit

protective immunity in the host.

C. Rationale

Since chimpanzees are the only non-human primate

whose ability to be infected with HIV is well documen-

6

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ted, our vaccination protocols have been changed toinclude chimpanzees. However we feel that further

characterizing the African Green monkey as an alterna-tive model is important since the chimpanzee model hasseveral limitations. Chimpanzees are difficult toobtain in large enough numbers to conduct a vaccination

protocol. They are expensive to maintain. Althoughchimpanzees can be chronically infected with HIV, theyremain clinically well (10). African green monkeys mayprove to be a useful model since enough animals toconduct a vaccination trial can be obtained and main-tained at a relatively low cost. Preliminary experimentswith Africian green monkeys established that thisspecies can be infected with HIV. Future experimentationrevealed that using the original protocol, not allmonkeys responded with full evidence of infection andan anti-HIV immune response. Full evidence of infectionwas defined as reverse transcriptase (RT) activity insupernatants of peripheral blood lymphocytes (PBL),immunofluorescent staining of PHA activated PBL, HIVtransmission to T cells and an intergrated RIV genomicpattern southern blot DNA hybridization. Experimentsto determine the protocol necessary to infect 100% ofAfrican green monkeys were begun. Parameters studiedincluded:

a) Varying the strain of African green monkey.Three strains, Cercoithecus aethiops, C.ascarius and C. cerebus were tested;

b) Varying the HIV strain, HIV I and HIV 2 '.were tested;

c) Varying the route of infection. Intrave-nous (IV) and intraperitoneal (IP) routeswere investigated;

%

d) Varying the number and dose of virus.

We have not conducted any studies with HTLV-III-X1O-1 mutant virus since vaccination with live mutantHIV virus is not practical due to possibility of rever-sion to virulence. Therefore, the immune response torecombinant vaccinia-HIV virus containing gene sequen-ces to HIV gpl60 has been studied. Because T cells canplay an important role in host defense against viral

infectious (13), it is important to investigate T cellmediated immunity in vaccinated or infected monkeys.Both T cell mediated lysis of infected targets andantigen induced cellular proliferation have been inves-tigated. Serological studies have also been conducted.

Investigations into cytopathogenic mechanisms ofHIV infection have been conducted by comparing results

7

1% N1

in chimpanzee and human models. The work conductedwith humans is being funded through other resources.Chimpanzee HIV infected T cells have been isolated by a

specific rosetting technique to determine if they l

differ from human infected T cells.

The rAd for vaccine purposes is obtained through

recombination between homologous regions of viral DNAand a plasmid construct containing HIV sequences. Adtype 2 (Ad2) was selected as the vector because this

serotype has been extensively characterized on the DNA,RNA and protein level and is easily propagated in thelaboratory. The viral early region 3 (E3), which isnot essential for Ad2 replication in vitro, is replacedwith the desired HIV sequences. Nucleotide sequences

encoding gpl20, the major external glycoprotein exposedon the surface of HIV, were used in the initial constructof the rAd plasmid vector. Cpl20 was selected becausesurface glycoprotein are the major retroviral antigen(32) and are essential for infectivity. Precedence has

shown that immunization with retroviral glycoprotein(s)elicit both neutralizing and cytotoxic antibodies. TheHIV gpl20 DNA fragment used to construct the rAd vectorextends from a SspI site 65 base pairs (bp) upstream ofthe initial ATG of the envelope coding sequence to asecond SspI site at nucleotide 7567 of HIV strain HXB2.This fragment includes 87% of the gpl20 coding sequence

plus the 30 amino acid (aa) leader. Because the HIVtransmembrane protein, gp 4l, may also be a potentialimmunogen, a second vector which includes the entireHIV env gpl60 sequence has also been constructed. Inall constructs, the gpl20 or gpl60 coding sequences are "flanked 5' by the Ad major late promoter including thetripartite leader and 3' by an Ad polyadenylationsignal and VA RNA coding region.

Chimpanzees have been the sole animal model in

which to conduct valid tests of potential vaccinesbecause these animals are susceptible to HIV infection,

react immunologically, and simulate the lymphoadeno-pathy syndrome. Powever, the proposed rAd vaccinecannot be evaluated in chimpanzees because human Ad donot replicate well, if at all, in monkey cells, and,virus replication is essential for expression of theHIV sequences. This block to Ad replication in simian

cells appears to occur at the RNA splicing level (33)and is overcome by the carboxy terminus of the simian

virus (SV) 40 T antigen (Ag)(34). Therefore, theinitial rAd vaccine construct contains approximately

500 bp of 3' SV40 T Ag coding sequence plus 200 bp ofearly SV40 DNA driven by the SV40 promoter. Thisfragment with Sall sites on either end can easily beremoved and the plasmid religated to provide a construct rminus SV40 sequence for the human vaccine. The presence

8

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of SV40 sequences also provides a means to selectrecombinant Ad2 progeny virus based upon plaque size.Theoretically, rAd containing the SV40 fragment should preplicate more efficiently and rapidly on the CV-l VAfrican green monkey kidney cell line and producelarger plaques.

To facilitate homologous recombination into the AdE3 region, the vaccine plasmid construct contains flan-king Ad sequences with 1051 bp of E3 and protein pVIIlcoding region upstream and 1513 bp of E3 and fibercoding region downstream of the gpl20-SV40 sequences.

Ad2 and vaccine plasmid DNA are cotransfected intothe 293 Ad EIA-transformed human embryonic kidney cell pline and the progeny virus plaqued on CV-I cells. Thelargest plaque isolates are selected and plaque-purn- ..

fied prior to preparation of virus stocks in HeLacells. Virus-infected cells are analyzed for thepresence of HIV-specific DNA and RNA and for expressionof gpl20. The rAd is administered in enteric-coatedcapsules to African green monkeys which are monitoredfor evidence of viral infection, pathogenicity andvirus shedding as well as for a rise in complementfixing (CF) and neutralizing antibody (Ab) titer directedagainst both Ad2 and HIV gpl 2 0.

DSimilar methods will be used to construct a human

form of the rAd lacking SV40 T Ag. The deletion of .this viral sequence will necessitate the selection ofthe desired rAd progeny on the basis of HIV gpl20sequences or protein detected in infected cells.Because Ad2 is endemic with neutralizing antibody found 0in 60% of the U.S. population (35), it is essential to N"change the Ad serotype in order to develop an effective p

human vaccine. Two approaches can be taken: 1) use amore rare and relatively nonpathogenic Ad serotype toconstruct the rAd vaccine virus or 2) exchange the Ad2sequences encoding the fiber, which is primarily respon-sible for type-specific neutralizing antibody (36),

with that of a rare strain. Therefore, the final liverAd vaccine virus will consist of HIV gpl20 sequencesexpressed from the Ad major late promoter inserted in %the E3 region of a modified Ad2 or non-Ad2 virus vector.

D. Experimental Methods:

Monkey units: Two unit of animals are maintained,the first at the primatology center of CNRS in Ville-juir Paris (directed by Dr. Pierre Dubouch) and thesecond at INRB in Kinshasw (directed by Mrs. DelphiMessinger, an American zoologist). Newly acquiredprimates are anesthetized with ketamine, T-B tested inthe left eyefold and given an overall physical exam.

9 D

Periodical checks are made for parasites and animalsare wormed as needed. Animals are provided access towater and ire fed a cake of wheat, corn and soy flourwith peanuts, milk, millet and bananas, other fruit,bread, palm nuts and various greens.

Magnesium ion dependent reverse transcriptase (RT)actively was measured according to the methods ofPopovic et al. (37). Anti-HIV antibody was measuredusing commercially available ELISA assays (Abbott,Chicago, IL). Positive sera were confirmed by a WesternBlot Assay purchased from Du Pont (Wilmington, DE) andretested in the ELISA assay to determine the titer.Neutralizing antibody assays were performed accordingto the methods of Robert-Guroff et. al. (38).

T4 and T8 cell counts were determined by specificrosetting (39). IL-2 production in PBL was measured bymethods of Zagury et al. (40). Antigen dependent cellmediated proliferation was measured by the methods ofZarling et. al. (41). Cell mediated cytotoxicityassays were conducted according to the methods ofCerrotini and Brunner (42). Assays using vaccinia-RIVrecombinant virus infected targets were done as describedby Plata et. al. (43 ).

Vaccination and infection protocols varied and are

described in the results section.

HIV infected chimpanzee and human PBL for electronmicroscopy studied were obtained 4-6 days after T 4 + cellsare infected with a high MOI of HIV. Only 15 - 30% of thecells expressed viral antigen as detected by immuno-fluorescence (IF) assay. Cells expressing HIV antigenwere obtained by incubating the cells with human anti-HIVsera which has a high titer of anti-envelope antibodiesand then rosetting the cells with bovine red bloodcells coated with anti-human IgG (44).

DNA Manipulations

Bacterial plasmid DNA preparation, enzyme diges-tions and agarose gel electrophoresis were carried outby standard techniques (45). Plasmid DNA was used totransform Escherichia coli (E. coli) DH5 cells (pAD.MLPgpl20) or HB1OI cells (a ll other plasmids). Ampicillinresistant transformants were screened by colony hybridi-zation with a 3 2 p-gp120 probe (gpl20-containing plasmids)or on the basis of size and restriction endonucleasedigestion pattern (gpl60-containing plasmids). Restric-tion endonucleases were purchased from BRL (Caithersburg,MD) and used according to the manufacturer's recommen-

dations.

10

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'' .. . . . . . . . . . . . . . . .-.. . . . . . . . .,. . .. t

The plasmid, -actin 2000, containing 2108 bp of-actin gene sequence cloned into the Hindlll site of

pBR322 was kindly provided by Steve Hughes (NCT-FCRF,Frederick, MD). Ad2 DNA was purchased from BRL (Gai- Il

thersburg, MD)..e

Cell Culture and DNA Transfection

HeLa and COS-I cells were obtained from G. Crouse(Emory University, Atlanta, CA); 293 cells were provided ,by T. Shenk (Princeton University, Princeton, NJ); and -

CV-I cells were obtained from G. Pavlakis (NCI-FCRF, L

Frederick, MD). All cell lines were maintained as .monolayers in Dulbecco's modified minimal essentialmedium (DMEM) with 1 gm/L glucose (GIBCO Laboratories,Grand Island, NY) supplemented with 10% fetal calfserum (GIBCO) and were passaged by disassociation with ,,

trypsin-EDTA (GIBCO).

Cell monolayers at approximately two-thirds conflu-

ency were used for transfection. Culture medium waschi,nged 3 hours before the addition of DNA. Calciumphosphate-DNA precipitates were prepared according tothe methods of Graham and Van der Eb (46) and Wiglet etal. (47) and used at a concentration of 2 ug DNA/35 mm :

dish unless otherwise noted. The precipitated DNA was

allowed to remain in contact with the cells for 6 hours,after which time the DNA-containing medium was removed

and replaced with fresh culture medium. For transient 2expression experiments, expression time was 48-72 hours ,unless otherwise stated.

Viruses and Virus Infection

Ad2 (VR 846) and Ad 31 (VR 1109) were obtained fromthe American Type Culture Collection (Rockville, MD) and

propagated in HeLa cell monolayers. Infected cells andculture medium was frozen and thawed three times and p

the cell-free supernatant from a 3000 rpm centrLfugation

was used to prepare virus stocks. Wild type (wt) Ad2 '

and tAd2 were plaqued on 293, HeLa and CV-I cell monola- ,-yers. Virus dilutions were prepared in phosphate buf

feled saline (PBS) with 2% heat-inactivated fetal calf.

serumea and bsorbed to PBS-washed cell monolayers for90-120 minutes at 37C. Infected HeLa cells were over-layed wrth modified Eagle medium (GIBCO) containing 4

heat-inactivated fetal calf serum and 0.7% Noble agar e

(Difco Laboratories, Detroit, MI). Plaque assay medium

for CV-I and 293 cells were supplemented with 10%t ctryptose phosphate (GIBCO) and 25 mM MgCp 2g Additionalplaque assay medium was added at 3 day intervals post-infection (PI) with 0.02% neutral red (GIBCO) incorpo-rated into the final overlay for plaque visualization.Plaques were scored at 7 days PI on 293 cells and at

toaEd

I.

13-15 days PI on CV-I cells.

RNA Isolation and Analysis

Total cellular RNA was isolated from transfected "S

cells by lysis in buffer containing 4M guanidium thio-cyanate, 50 mM Tris-HC1, pH 7.6, 10mM ethylene diaminetetraacetic acid (EDTA), 2% sarkosyl and 2% -mercapto-ethanol (personal communication, S. Hughes, NCI-FCRF,Frederick, MD). Phenol-chloroform-extracted and ethanol-precipitated RNA samples were suspended in 50 mM Tris-HCl,pH 7.5/1 mM EDTA and spotted on nitrocellulose. Hybri-dization with 3 2 P-labeled gpl20 and -actin probes wascarried out at 42°C overnight. Nick Lranslated probeswere prepared by the method of Maniatis, et al. (45).

In Situ Hybridization -

Ad-infected cells propagated in tissue culture

chamber/slides (Miles Scientific, Naperville, IL) werefixed in 70% ethanol-20% acetic acid for 10 minutes at-20°C and hybridized at 105°C for 5 minutes to a 2,4

dinitrobenzyl-diaminohexane (DNB)-labeled (48), HaeIII-digested pAD.MLP.gpl20 probe. After reacting withrabbit anti-DNB serum and alkaline phosphatase- conjugatedgoat anti-rabbit IgG, hybridized sequences were visual-ized with fast red (Vector Labs., Inc., Burlingame, CA).

E. Results and Discussions:

1. Standardization of conditions leading to repro-

ducible infection of cercos.

Initial experiments with Cercos infected withHIV I showed that some but not all monkey were

infected with HIV I (Table 1). Some animals (701and 706) injected with viral particles were infectedas evidenced by RT activity but no HIV antibody

response was detected. Animals injected withantologous HIV infected PBL became infectedwith HIV and anti-HIV antibodies could be detected.However, further experiments failed to show thatAfrican green monkeys could be reproducibly infected(Table 2). Similar results were obtained in baboons.(Table I and 2). Because African green monkeys aremuch easier to purchase and less experience tomaintain than chimpanzees, a systematic effort todevelop an HIV African green monkey model wasundertaken. Several infection protocols are beinginvestigated to determine optimal infection proto-cols. Parameters investigated are: 1) species ofCercus; 2) HIV 1 or HIV 2 infection; 3) Varying

the route of infection (IV or IP) and 4) varyingthe number of challenges. Pesults are summarized

12

°o . , ., , , o °o o • , o ° . ° - ,.5 .- ° ° o - ,° .•° . o -. .. ° . °. :. . o ° - °

in Table 3. Both HIV 1 and HIV 2 can infect cercos.However infection with HIV 2 yields a higher reversetranscriptase. IV infection (see monkeys C25, C34and C39) is much more efficient than IP infection.IP infection requires high doses of HIV and repeatedinfection (3-4 times). This model appears to be analternate model to the chimpanzee model. Additionalexperiments to extend and reproduce these resultsare currently being conducted.

2. Screening of primate colony for natural retro-

virus infection.

Several species of primates are being screenedfor antibodies to HIV 1 or HIV 2 by Western blotanalysis. Animals being investigated at INRB are:

Cercopithecus: Cerobus: Colobus: Pan:

Aethiops Atterimus Angolensis TroglodytesAscanius Galeritus PaniscusCephusNamlyniNeglectus1 'HoestiPogoniasWolfi

A total of 50 animals have been screened and onlyone animal, a subspecies of crested mangaby (goldenbellied mangaby) was positive. This animal #1105was bled to determine if virus could be isolatedfrom his PBL. This experiment is currently inprogress. Dr. Zagury is attempting to buy 20-30golden bellied mangaby to start trials to determinethe parameters necessary to infect this species.

3. Recombinant Vaccinia-HIV Virus

Five chimpanzees have been vaccinated withrecombinant vaccine HIV virus in five divideddoses on shaved backs (Fig. 1) Four animals werevaccinated on the 11th of June and one on the 29thof June. They will be boosted with autologous HIVinfected PBL's (1/3 of dose by IV slow drip, 2/3by scarification on the back). Vaccinated monkeyshad a fever for 2-3 days following vaccination and hadswollen axillary glands. Vaccinated chimpanzeesand control (vacctnia vaccinated) will be challangelwith virus.

13

17 - 1-: 1

The immune response of Cercos, baboons andchimpanzees to recombinant vaccinia-HIV viruseswas investigated (Table 4). All the vaccinated

monkeys developed antibodies to HIV envelopeantigen. However, no neutralizing antibody wasdetected. Antigen induced cell proliferation wasinduced in the animals tested.

Since in autologous HIV infected cell popula-

tion, only 10-20% express viral antigen on theirmembrane, it is difficult to prepare a homogeneouscell population of target cells expressing HIV C.signals. Three target cell population are currently

under investigation to determine their usefulnessin cytotoxic T cells assays of chimpanzee PBL.HIV infected H9 cells may be used as target if thethe chimpanzee shares the same phenotype as H9 cells(Al or A3 phenotype). Murine P815 cells transfected

with genes for HLA-A2 histocompatible phenotype

and HIV envelope genes have been produced. Thiscell line has been successfully used as targets inthe human model (Table 5). Epstein Barr virustransformed autologous B cells infected with recon-binant vaccinia-HIV virus may be used as targets.30 chimpanzees from Kinshasa Zoo are being typed

for histocompatibility phenotype.

5. Electron microscopic studies of HIV infected

chimpanzee T4 cells

Human and chimpanzee cultured T4 cells wereinfected with high MOI of HIV. After 4-6 days,15-30% of the cells express viral antigens as

detected by immunofluorescence. Cells expressingviral antigen were obtained by treating the cellswith AIDS serum and rosetting the cells with bovinered cells coated with anti-human IgG. Electronmicroscopic analysis of the rosetted cells showthat most human cells produce viral particleswhereas most chimpanzee cells do not.

6. Construction of HIV 1 gpl20 plasmids

The plasmid pAD.MLPgpl20, which contains HIVgpl20 sequences and the Ad2 major late promoter,and plasmid F10-7, which contains the SV40 earlypromoter (49), were kindly provided by George Mark(NIH, Bethesda, MD). To construct pAD.MLPgpl20,Dr. Mark excised a 1408 bp SspI fragment consisting

of the gpl20 translational start, signal sequence,and coding sequences for 456 of 518 aa from plasmid

14

%""

I%pHXB2 which contains the cloned HIV 1 strain HXB2 e%

genome (50). After addition of BamHI linkers,this fragment was ligated into BamHl-digestedplasmid pAD.MLP (51) adjacent to the Ad major latepromoter and flanked 3' by a polyadenylationsignal and Ad VA RNA coding sequences. The plasmidstructure was verified by restriction endonuclease

mapping (Fig. 2).

The derivation of additional plasmids, pENV.ND1

and pAD.ENV.NDI, necessary for development of theHIV-I gpl20/Ad2 vaccine is depicted schematicallyin Fig. 3. The plasmid pOB.NDl was constructed bycleaving SV40 DNA with SspI and EcoRV to obtain a1433 bp sequence extending from SV40 map position0.1-0.28. This fragment, ND1, includes the sequenceencoding the helper function for the growth of Adin monkey cells. After addition of BglIl linkers,this fragment was inserted into BamHl-digestedplasmid F10-7 adjacent to the SV40 promoter sequence.The resultant plasmid, pFlO-SVT, was cut at anunique BamHl site and Sail linkers were added.Subsequent digestion with Sail yielded an 1180 bpfragment, OB.NDI, containing the SV40 helper func-tion (0.14-0.28 map units) and the SV40 promoter.Because the Sall site was found to be missing

in the pAD.MLPgpl20 construct, an adjacent Sphlsite was utilized with the addition of Sail linkersfor insertion of the OB.NDI fragment to yield

plasmid, pENV.NDI (Fig. 4).

Commerical Ad2 DNA was digested with Spel andScal; and the 5.2 kilobase band containing the AdE3 region plus a portion of the adjacent pVII andfiber-coding regions were ligated into the Xbaland Hincll sites of pUC-19 to yield pUC-AD2. Thisplasmid was digested with BgllI to remove 2635 bp P%of Ad sequence leaving 1051 bp at the 5' end and1523 bp at the 3' end to facilitate recombination.pENV.NDI was digested with Pvul, Nrul and Sachl to

yield a 3704 bp fragment, ENV.ND1 containing SV40

sequences and the Ad late promoter driving the HIVgpl20 gene. After treatment with Mung bean nuc-lease, the 3704 bp fragment and BgllIdigestedpUC-AD2 vector were ligated to yield pAD.ENV.NDI(Fig. 5). This is the plasmid that was used toconstruct the IIIV 1 gpl20/Ad2 hybrid by in vivo

recombination.

To obtain a plasmid construct free of SV40sequences suitable for human vaccine use, pAD.ENV.NDI was digested with Sall to remove the OB.NDlfragment. After ligation to regenerate an uniqueSall site, the resultant plasmid, pAfl.FNV (Fig. 6)

15

1ml.

contains the HIV gpl 2 0 gene driven by the Ad majorlate promoter flanked at the 5' and 3' ends by theAd sequences necessary to facilitate recoirhination

into wt Ad2.

7. Construction of 1IV-I gpl60 Plasmids

Several potentially important antigenic epi-topes are deleted or partially deleted from thetruncated gpl20 fragment used in the originalvaccine plasmid construct including the highly

conserved fusion peptide sequence in the N-terminal %

hydrophobic region of gp 4 l (52). Thus, our next

objective was to construct vectors containing the

entire HIV gpl60 sequence. To do this, pBHIO,which contains the HIV BHI0 genome in a pSP64 evector (53), was digested with Sall and Xhol to %give a 3108 bp fragment extending from a pointjust 5' of the start of the tat III coding sequence

and extending approximately 100 nucleotides beyondthe end of the envelope coding sequence. Thisfragment was gel isolated and treated with Mungbean nuclease. Plasmids pAD.MLPgpl20 and pENV.NDIwere digested with BamHI to remove the gpl20sequences and the gel-isolated vector was treatedwith Mung bean nuclease and calf intestinal phos-phatase. The blunt end gpl60 fragment and pAD.MLPgpl20 or pENV.NDI vector were ligated; and pAD.MLPgpl60 and pENV(160).NDI transformants containing

an intact gpl60 fragment in the proper orientationwere selected by mapping with diagnostic restric-tion enzymes (Fig. 7 and 8).

8. Transfection and Transient Expression of HIV gpl20Plasmids

To test for gpl20 expression, pAD.MLPgpl20(10 ug/IO0 mm dish) was transfected into HeLa, KB,CV-I, COS--l, BHK 21, DXB-11 CHO and A549 humanlung carcinoma cell lines. Total cellular RNA wasextracted from the transfected cells and subjectedto slot-blot hybridization analysis using a 3'P- %labeled gpl 2 0 fragment as probe. Gpl20-specificRNA was detected in all cells with HeLa, KB, COS-I

and A549 cells exhibiting the strongest hybridi-zation signals (data not shown). RNA extracts from

non-transfected control cells or cells transfectedwith pAD.MLP showed only background hybridization

signals. Positive results were also obtained withNorthern blots of RNA extracted from feLa cellsafter DEAE dextran-facilitated transfection (54)with pAD.mLPgpl20, pENV.NDI, or pAD.FNV.NDI DNA.

HeLa cells transfected with the three plasmids

N N

containing gpl20 were also analyzed for the presence P

of gpl20 protein by indirect immunofluorescencestaining and the alkaline phosphatase/anti-alkalinephosphatase staining procedure developed at BiotechResearch Laboratories. Approximately 5% of cells

transfected with pAD.MLPgpl20 exhibited a positive

fluorescent signal whereas mock-transfected HeLacells were negative (Fig. 9). The presence ofgpl20 was also demonstrated in HeLa cells trans-

fected with pENV.NDI and pAD.ENV.NDl (Fig. 9)although the percentage of positive cells (1-5%)

was less than that observed with the pAD.MLPgpl20construct. These cytochemical results combinedwith the RNA hybridization data suggest that gpl20sequences are expressed in HeLa cells transfected

with these plasmids.

The OB.NDI fragment included in the vaccineplasmid construct contains SV40 sequences which

should overcome the block to processing and trans-lation of Ad-specific RNA in monkey cells (33).In addition, enhancer sequences which constitute a 1!

portion of the SV40 early promoter (55) includedin this fragment may also affect the level oftranscription of gpl20 sequences. To investigatewhat effect these SV40 sequences might have onexpression levels of gpl20, blotting studies withtotal RNA from human and monkey-derived cell linestransfected with pAD.ENV.NDI or the OB.NDl-minusderivative, pAD.ENV, were carried out. Righerlevels of gpl20-specific RNA were detected in cellstransfected w~th the OB.NDI fragment-containing

construct (Fig. 10).

Interestingly, the level of gpl20-specific

RNA was higher in monkey cells than in human cellswhen either construct was used. No gpl20 RNA

sequences were detected in control cells exposedonly to CaPO 4 without plasmid DNA. The RNA fromall cells, whether transfected with plasmid DNA orcontrol, reacted with approximately equal intensityto a 3 2 P-labeled -actin probe. No conclusions canbe drawn from these preliminary studies concerningthe role of SV40 sequences on gpl20 expressionother than gpl 2 0 is transcribed in monkey celllines irregardless of the presence of the OB.NDIfragment.

9. Construction and Selection of Recombinant HIV-Ienv/Adenovirus 2

pAd.ENV.NDI and Ad2 DNA were cotransfectedinto 293 cells (Fig. 11) which were monitored forthe production of progeny virus based upon the

17 I

%'!1

development of typical adenovirus cytopathiceffect (CPE). Alternatively, 293 cells were trans-fected with pAD.ENV.NDl and 18 hours later wereinfected with wt Ad2. At 48 hours after transfec-tion, CPE was almost complete in 293 cells that

*. had been infected with wt Ad2 with or withoutpAD.ENV.NDI. In contrast, only limited CPE wasobserved in 293 cells transfected with Ad2 DNA +pAD.ENV.NDI. CaPO 4 transfection control cells andcells which had been transfected with only pAD.ENV.NDI showed no evidence of cytopathology.

A portion of each transfected cell lysate wasa-passaged three times through CV-I cells to, theore-

tically, enrich for recombinant Ad2 most efficientfor replication in monkey cells. Cell lysates aderived from the original 293 cells used fortransfection as well as lysates from 3 passages inCV-l cells were plaqued on CV-I cells (Table 6). a

No plaques were observed with control cell lysateswith the exception of a lysate of pAD.ENV.NDl-trans-

fected cells which had been passaged through CV-lcells. An isolate of this unknown virus has been

.P

saved for further characterization. At 10 daysPI, the largest plaques were picked from allsamples, solubilized and used to infect CV-I cells(Table 6). The infected CV-I cells were harvested

upon the development of complete CPE and 9 of 49CPE-positive cell cultures were selected forplaque purification. Although the largest plaqueswere consistently selected, each subsequent plaquingstep results in a mixture of both large and smallplaque isolates (Fig. 12). However, size homoge-

neity within each isolate does appear to be increa-sing with each additional plaquing. Note that

progeny virus derived from infection of 293 cellswith wt Ad2 produced plaques on CV-l cells in the

initial selection and continued to replicateeffectively in this monkey cell line. The reasonfor this is not known but possible explanationsInclude experimental error or an illegitimate

recombination event between input wt Ad2 andendogeneous SV40 sequences in the CV-l cells which

were originally derived from African green monkeykidney.

10. Detection of gpl20 Sequences in rAd-InfectedCells

CV-l cells were infected with the putativeHIV-I gpl20/Ad2 recombinant virus isolates selected

for plaque purification and fixed for in situ hybri-zation when the cells showed characteristic Ad CPE.Cells in all infected cultures reacted positively

18

%

P k

with the DNB-labeled pAD.MLPgpl20 probe (Fig. 13).No hybridization was detected in non-infected

control cells. These results provide some evidencefor the presence of gpl20 sequences in the virus-infected cells. However, the specific hybridizationobserved may also be attributable to Ad major latepromoter sequences present in the plasmid probe.

F. Conclusions

The immune response of African green monkeys, ba-boons and chimpanzees to HIV has been monitored in

infected and vaccinated animals. Anti-HIV antibody wascharacterized for its ability to neutralize HIV virusand its reactivity in HIV Western blot assay. Antigeninduced cellular proliferation was also studied.Preliminary experiments to establish protocols formeasuring cytotoxic T cell responses in primates wereconducted. Standardization of the conditions leading toreproducible infection of African green monkeys withHIV established that this species could be infected mostreproducibly by intravenous administration of HIV 2.50 primates of various species were tested for "natural"infection with HIV. Only one mangaby showed a reprodu-cible anti gpl60 reaction with HIV 1 but not HIV 2.

Construction of plasmids containing gpl20 sequencesnecessary for development of the rAd2 vaccine have beencompleted. Both gpl20-specific RNA and proteins havebeen detected in transient expression experiments usingHeLa cells transfected with the gpl20-plasmids indicatingthat the HIV env sequence contained in these plasmids istranscribed and translated in mammalian cells. Basedsolely upon plaque size on CV-I cells, it appears thatisolates of rHIV I gpl20/Ad2 have been obtained fromrecombination between the plasmid pAD.ENV.NDI and wt Ad2.The preliminary nature of other data presented hereinmakes it impossible to draw any additional conclusions.

E. Recommendations

1. Alternative screening method for HIV-l gpl20/Ad2

The possibility that rAd2 may be constructed whichdo not contain the SV40 OB.NDI fragment makes it neces-sary to develop an alternative to the present selec-tion method based upon plaque size. We recommend thatan in situ hybridization technique be evaluated basedupon the procedure of Villarreal and Berg (56) forhybridization in situ of SV40 plaques.

2. Modified Ad2 serotype for human vaccine purposes

Ad2 hexon induces primarily group-specific CF

19

antibody which usually disappears rapidly after acuteinfection (57). In contrast, Ad2 fibers are largelyresponsible for type-specific neutralizing antibody (36)which is long lasting (57) and may be of greater concernin the development of a live Ad2 vaccine. We recommendthat the feasibility of exchanging Ad2 fiber codingsequences with that of a less common Ad serotype beconsidered. If the decision is made to continue withthe proposed exchange of hexon coding sequence, werecommend the use of a serotype other than "oncogenic"Ad3l because of possible regulatory complications.

3. Simian immunodeficiency virus (SIV) and HIV-2model system for AIDS

Proposed studies in which HIV 1 gpl20/Ad2 is admi-nistered to African green monkeys are designed to demons-trate that subclinical infection with the rAd is estab-lished with attendant development of humoral and cell-mediated immunity. However, these animals cannot bereproducibly infected with HIV I to determine if thisimmune response might be protective. We recommend thatsimilar rAd constructs using SIV env gene sequences beconsidered for use in macaques, which develop AIDS-likedisease after infection with virulent SIV strains. Thiswould provide a valid and much less expensive animalmodel system in which to test the efficacy of live Advaccines. Similarly, when the HIV 2 African greenmonkey model is established, rAd containing HIV 2 envgenes can be administered and the monkeys challengedwith HIV 2.

20

I

.7~ ~ ~ -J !~ -~ ~ jr '~y Y 'J(~.X J~Y K'~YWJCW~.p ~ ~ ~ - .E -.m -~ -~ -- ~ - .~

p.

L/,

o

U

p.

uJ ~

W ~) -~ tw..

p -.

0

0

p-4

.S

pp

pp

ppp.

.4

Sd.

p.

~'J.

s.d.

.p.

4-

I

21-- C-....4-s. ~ - -. V' 4

4'

4pp*~4 -4.44.*4* 44

4- ~ P' ~ ~ '4 -' -- p4- - .%%~

1 2 3456789109111213

GiAM HI

Barn HI

Fig. 2 A. Restriction digestion pattern of A.L

gp12O. Enzymes: 1. BamHi; 2. SaIl; 3. NruI;4. EcoRi + Bamil; 5. BamilI + Nrul; 6. BglI1;7. EcoRl + Xhol; 8. XhoI; 9. HindlIl + Bam~l;

*10. ZpnI + BamHl; 11. Hincil + BamHI; I2. KpnI;13. No enzyme. B. Restriction map of pADMLPgp 12 0.

1 22

SY40 P

BI I

linkers

%

?Fj 3 B~nm I

Sal [ linkerSeal Sph I

fflhIIf1 ~Sph IOB.NDI Sad I hnkers P

Nru k " "?

p-...-V.

p' p ADZ

Sac II AdZ

Belli SC I

S I II 3/.9eson

-inc II t

PAD",

Fig. 3 Strategy for construction of vaccineplasmidpAD. ENV. NDI. SV40 promoter (P), jJ-j ]J , andSV40 T Ag fragment ND, were combined

into fragment Of.NDI,fiW!I 1. OB.NDI was p

ligated to HIV gpl20 coding sequences underthe control of the Ad major late promoter,

, derived from pAD.MLPgpI20. To facilitate

recombination, an Ad fragment encompassing E3,I; , was isolated and digested with appro-

priate restriction endonucleases to permitinsertion of AD.MLPgpI20 and OB. NDI sequences.See text for specific details.

23

I

".. " ,'. )% , . -. .. .' -. ,. -. , - .. . . N . -•*

A B-

1 2 3 4 5 6 7 8 910111213kb

94

Sp Sp4.4 P ,Bg

PENV.- K

NDI

Bg

" i!.

S1.4 H

0.9

pENV._

Fig. 4 Analysis of plasrnid pENV. NDI. A. Plasmid DNAwas digested with restriction endonucleasesand fractionated on a 0.8% agarose gel.Lane 1 kSX-HaelI standard; 2, \-HindIllstandard; 3, BanHI; 4, Sall; 5, HindIIl; 6,SspI; 7, Bglll; 8, PstI; 9, Kpnl; 10, NruI;11, NruI + KpnI; 12, PstI + Kpnl; 13, uncutplasmid DNA. B. Structure of pENV.NDI withrelevant restriction endonuclease cleavagesites: B, BanHi; S, Sall H, Hindlil; Sp,

Symbols: _____ , Igp12O fragment;Ad major late promote, and tripartite leader;

,SV40 promoter and T Ag sequence;,Ad VA sequences; ____,p-BR322

vector.

24

Ilk

TW.

A 23 4 5 6 78 91011

kb

9.4 sS+ '/Sp

4.4 i . B

pAD.ENV. Bg

HB

2.0X Bg

1.4 B

0.9

Fig. 5 Analysis of plasmid pAD. ENV. NDI. A. PlasmidDNA was digested with restriction endonuc-leases and fractionated on a 0.8% agarosegel. Lane l,-HindlIl standard; 2, X-HaeIllI

-" standard; 3, Ba HI; 4, Kpnl; 5, SmaI; 6, Sail;7, BgIII; 8, Hind 111; 9, Sspl; 10, XmnI;11, uncut plasmid DNA. B. Structure ofpAD.ENV.NDI with relevant restrictionendonuclease cleavage sites: B, BamHl; K, KpnI;Sm, Smal; S, Sall; , Bgll ; H Hindlll;Sp, Sspl; X, Xmnl. Symbols: T ,Splgpl 2O fragment; Ad major late promoterand tripartitb leader; , Ad VA sequences;

SV40 promoter and T Ag sequence;i A Ad E3 flanking sequences; ,

pUCl8 vector,.

25

1 2 3 4 5 6 7 8 91011 p

kb

9.4 I

4.4

N-

2.0

1.4

0.9

EIB .4

BBV

BB m B

Sp. B"""

Bg

KD ..ENV

; K ".-.'

pAD.ENV

H Bg

H ~IAHSS

Fig. 6 Analysis of plasmid pAD.ENV. A. Plasmid DNAwas digested with restriction endonucleasesand fractionated on a 0.8% agarose gel. -.

Lanes: I. A-HindIII standard; 2. X-Haelllstandard; 3: BamHl; 4. Kpnl; 5. Smal; 6.Sal ; 7. BgllI; 8. HMndlII; 9. SspI; 10.Xmnl; 11. uncut plasmid DNA. B. Structureof pAD.ENV. with relevant restriction endonu-clease cleavage sites: B, BamHI; K, Kpnl;Sm, SmaI; S, Sall; Bg, BglII; H, HndlII;"Sp,. -Sp; x, XmnI. bols: ISapHIV gpl20 fragment; - Ad ma o latepromoter and tripartite leader; , AdVA sequences ; , Ad E3 flankingsequences; _, pUCI8 vector.

26

W

Akb

9.4

4.4

1.4.

0.9

BB

Ng B

Bg

Fig. 7 Analysis of plasmid pAD.MLPgpl6O. A. PlasmidDNA was digested with restriction endonuc-leases and fractionated on a 0.8% agarose gel.Lane 1. A -HindIII standard; 2. X-HaeIIIstandard; 3. BanHl; 4. H in d I I1 5. Sspi;V6. P&jIII; 7. KpnI.; 8. NruL; 9. Sall; 10. -

KpnI + PstI; 11. PstI; 12. NruI + KpnI.B. Structure of pAD.MLPgpl6O with relevantrestriction endonuclease cleavage sites: B, -

BamHl; H, HindlII; Sp, Sspi; Bg, EglIl; K,nP~l'i N, NruI; S, Sall; P, PstI. S b ~Is:T 1I,SalI-Xhoi gp16O fragment; ,Ad

Ma o late promoter and tripartite leader;* , ~Ad VA sequences; ____,pBR322

vector.

* 27

rK~h7WJTf~"F .X -IJ7 1 W'rr UW IYJVJW-.&wv WV w PIEmUX I Or~u PUIN~~v ~ 'WJJY U rT .- W 'i rT vTJ j . , _ s rWiwy ~r-VW,o.

1 2 3 4 5 6 -7 .8 9 1011

Akb

9.4 a

4.4

2.0 :

1.4- -*.

0.9

B "-"

St

Bga

pENV.

(16O)NDI - 139B .. .""S p <.

N B Sp

Bg

Fig. 8 Analysis of plasmid pENV(I60)NDI. A. Plasmid

DNA was digested with restriction endonuc-i,

leases and fractionated on a 0.8Z agarose N

gel. Lane I, -Hindlll standard; 2, OX-Haelll

standard; 3, Ba.MHl; 4, Hi__dIll; 5, SspI ;--6, t4Bg A I I ; 7 . Kp _n- - 8 . N r _ l ; 9 o S s _ I I ; 1 0 . S s _ I I '+ BamHl; II. Nrul + Kpnl. B. Structure of

I.

pENV160)NDI with relevant restriction endonu-

clease cleavage sites: St, Sst8; for other

abbreviations, see legend to Fig. 7. S___ymbols: '-.gl. L all-Xhoi gp160 frsgnt; 2MA, 4X-Ha

Ad ma or late promoter and tripartite leader;

Ad VA sequence; S SV4O0promoter and T Ag sequence; , pBR322vector.

28

.,.I

icl

U-.

Fig.9 Acumultio of IV g120 n tansfctedHe~

cels Becnrlcls(A rclstas

Fig.Cll 9er Accumulatione ofwVg Oih Eransce Hlea

fectecewith pAD.MLcgpd2wi(B)por ENV.NDI ()were ftied iniacetoe to64hours an tinedlAb usbing reence seru frseod AIDS patint a

(A271) tas primar Ab. nBisndnay gosatani-huma Abt citonjluatethfoluorescein.omo

4cmlexof a-ndlkaln phosphate- t-lkln

29 U

AD.ENV. AD. ENV. Control"-NDI-

.'.

,gp12O

He La

293

Cos •

CV-I 1 0.. i pactin

He La !

€p

-, 29 3"

Fig. 10 Dot blots of RNA from cells transfected with,.pAD. ENV. NDI and pAD. ENV. Cellular RNA (10 ug)

was applied to a nitrocellulose filter and 2hybridized to a nick translated gp120 fragment

or -actin fragment. The blots were autora-

diographed overnight with two intensifying ,sc reens. '

30.'

AI.NV ......C nro

'~~7v~~~Wv~~wvIrvw W!W%. w,% V%; V% '%WJW v-~wj q~w ' . w Wi - V -Wj -W -'i -WV,. u1W -j- rw.

[-.

[A

CONSTRUCTION OF RECOMBINANT HIVenv/ADENOVIRUS 2 ,.-

(DP V i14 Z Can,

Capo,

293 celts 293 ceus

18 hrs

ufect with wt Ad2

wt Ad2rAd2

CV-lIx3

Piqcue on CV- I ceilsrAd2 = large plaque

plaq'ue puriicatonr - . characterizat:cn,

Fig. 11 Construction of Recombinant HIV gpl20/Adeno-virus 2. Human 293 cells were cotransfectedwith pAI).ENV.NDI and Ad2 DNA or transfectedwith the plasmid and infected with wt Ad2 as

described in the text. A portion of theprogeny virus was passaged through CV-1 cellsto enrich for the desired recombinant virus.Putative recombinant viruses were selected on

the basis of large plaque size in CV-I cellsand plaque-purified three times prior to

characteri zat ion.

31

Aa C

a

C !'r "

Foosy

'," i-

b d tr

Fig. 2oog of plaques A.Forsed byqu puiecareom.B cnan H plOaduenpurifica2ion. Viruscellat.sMonoeayerive ofro CV9 cells erane-

etedwihvs o eraid with agr ediuman

* pad incubate fo-nece 12 -15 dasetll s.

zsoatn of paqudes sae rthlauh prc-

islats enrihrte deed frre93cllsinan-

virus prior to selection.

32e

b " [, ,..-

.w." -"I

O %

j"t.

A"[: t . ,- .

Fig. 1 3 i , i d z - c i

v i u 2 ,. 1-iu h y r d z t i n w s p e f r e

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Table 2 IMMUNE RESPONSE OF MONKEYS TO HIV 1

Neutralizing (Index ofAnimal Treatment I HIV Western Blot Antibody 2 Proliferation) 3

160 120 41 31 24 18

AEl cells + + + + + + 10,52 ND

AE2 virus + cells + 0 0 0 + 0 4 ND

AE3 virus + cells ---- N.D.---- ND 5 ..

NE5 0 0 0 0 0 0 0 ND ND

ASI cells + + + + + I Ing 6

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AS5 0 0 0 0 0 0 0 ND 1

BI virus + cells + + + 0 0 0 ND NO

B2 virus + + + 0 0 0 ND 0

B3 virus 0 0 0 0 0 0 0 NDI

B4 0 0 0 0 0 0 0 0 1

1 Monkeys were infected by administration of viral supernatants or in vitro

infected autologous PBL.

2 Ability of antibody to neutralize HIV 1 strain B was measured according to

methods described in text. Results are expressed as reciprocal of thetiter.

3 Antigen is HIV I strain B.

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4.4

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a .... ~'p

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- ~--%.. .-- - - - - - ~ -p ~