Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Monday, July 8, 1996

Mo.A.100 - Mo.A.104

Mo.A. 100
CHEMICALLY INACTIVATED WHOLE HIV VACCINE INDUCES CELLULAR
RESPONSES IN MICE
Adldawe MD, Fabrizzi F.*, Dourmashkin R., Wilson 5, John S Oxford. London Hospital
Medical College, London UK and 151 Castelvecchio Italy
Objectives:To investigate the immune response in mice to an experimental whole virus
chemically inactivated HIV- I vaccine.
Methods and Results: Groups of female mice (Balb/c) were immunised with two doses
(I 0pg and I pg) of a whole HIV MN virus grown in C8166 cells.The vaccine production
procedure involves using a multiple step inactivation of HIV- I virus containing supernatant
with two independent treatments of 0.2% Beta-propiolactone (BPL), concentrated and
purified by ultra-filtration and ultra-centrifugation with 20%-60% sucrose density gradient
respectivelyVirus purification was confirmed by Western Blot and Electron microscopy
Binaryethylenimmne (BEI; 10mM) was then used in additional inactivation of the virus followed by stabilisation with 0.05% Formaldehyde. Animals were boosted with vaccine at
three week intervals and after three boosts sera and splenocytes were removed.
Splenocytes were separated into CD4+ and CD8+ T-cell subpopulations by panning with
specific antibodies and re stimulated in vitro for 6 days with either gp 20,orV3-loop peptides derived from MN virus. Major histocompatibility (MHC) class I and II restricted HIV- I
env-specific CTL activity was determined using 51ICrlabelled murine P8 15 (H-2d) and A.20
(H-21A) cell as targets pulsed with V3-loop peptide or gp 120. Sera were assayed for reactivity to V3-loop peptide and rgp I 20 using ELISA and for virus neutralisation using syncytium
reduction in C8 I166 cells. Splenocytes taken from mice immunised with this vaccine demonstrated HIV- I env specific cytotoxic activity but no CTL activity was detected in control
mice. High antibody titres to whole virus and to recombinant gp I 20 were also detected in
the sera of animals immunised with the lowest dose of vaccine (I/pg).These antibodies also
cross-neutralised MN, IIIB, and RF viruses.
Conclusions:The chemically inactivated HIV vaccine was found to be highly immunogenic
in mice, inducing both Neutralising antibody and HIV- I env-specific cytotoxic T-lymphocytes. Previous studies (Race E, et al.Vaccine 1995 vol. 13 number 16. ppp. 1567-1575)
have established the inactivation kinetics using the four step methodology and in excess
of 20 log 10 TCID50 of virus is destroyed.The vaccine therefore fulfills the initial basic
requirements of safety and ability to induce a B and T cell immune response, at least in a
laboratory model.
Mohamed D.ADDAWE Department of Academic Virology and Retroscreen Ltd. London
Hospital Medical College 64 Turmee Street, London EI 2ad
Mo.A.10 I1
EFFICACY EVALUATION OF CONVENTIONAL DUAL-SUBTYPE HIV VACCINE.
Yamamoto Janet K, Mison, M, Elyar, J,Tellier, MC, Pu R. University of Florida, Gainesville, FL, USA
Objective: To evaluate the immunogenicity and prophylactic efficacy of dual-subtype infected
cell vaccine against homologous and heterologous FIV subtype challenges in domestic cats.
Methods: Dual-subtype FIV vaccine was developed from two singly-infected (subtype A or
D) cell lines that were inactivated and mixed with threonyl-MDP adjuvant. Specific pathogen
free (SPF) cats were immunized SC with either dual-subtype or single-subtype vaccines at a
monthly interval (4X). All cats were challenged IP 4 wks after the final immunization with
either 500 ID50 of heterologous subtype B strain or 50 ID50 of homologous subtype A or
D strains. All cats were monitored for antiviral immunity and virus infection.Virus infection
was determined by virus isolation, proviral PCR, and by monitoring changes in FIV-specific
antibody profile.
Results: Significant levels of antiviral antibodies (including virus neutralizing antibodies) and
FIV-specific cytotoxic T lymphocyte (CTL) activity were detected after immunization with
conventional vaccines. Cross-subtype CTL activities were detected in a number of these
cats. As of 24 wks post-challenge (pc), 3 of 5 (60%) cats receiving dual-subtype vaccine
were protected from high-dose challenge with heterologous subtype B. All cats (n-6) vaccinated with either single-subtype A or D vaccines were not protected from similar subtype
B challenge. Further, all cats immunized with single-subtype A vaccine (n-2) were protected
against homologous FIV strain, but those immunized with single-subtype D vaccine (n-2)
were not protected against homologous FIV strain. Results from the immunogenicity study
indicate that the level of Subtype D virus antigens in the single- and dual-subtype vaccines
was at least 6-fold lower than the level of Subtype A virus antigens. Thus, vaccine efficacy
can be broadened by adding low doses of other FIV subtype antigens to the prototype
subtype A vaccine.
Conclusions: The single-subtype vaccines do not protect cats against heterologous subtype
challenges. More importantly dual-subtype FIV vaccine provided broad-spectrum protection
against a heterologous subtype.
J.K.Yamamoto, Univ. of Florida, Dept. of Pathobiology College of Vet. Med., PO Box 110880,
Gainesville, FL, USA 3261 I;Telephone: [phone] ext.3943 Fax: [phone]
Mo.A. 102
ADENOVIRUS HOST RANGE MUTANT-SIV RECOMBINANT VACCINE TRIAL IN
RHESUS MACAQUES
Bo e Suzan LI, Lubeck M2, Kalyan N3, Cheng S2, Richardson EI, Markham P4, Miller C5,
Udem S, Robert-Guroff M I. NCI Bethesda MD2Wyeth-Ayerst Radnor PA3WyethLederle Pearl River NY4ABL Kensington MD5CRPRC Davis CA
Objective: To evaluate a prime-boost vccine regimen using an adenovirus host range
mutant (AdShr)-SIVsm envelope recombinant and native SIV251 gpI20 for protective efrcacy against vaginal transmission of SIV.
Methods: Six adult female macaques were immunized orally and intranasally at 0 weeks and
intratracheally at 12 weeks with AdShr-IV recombinant.They were boosted at 24 weeks
with syntex adjuvanted gp I 20. Four controls received the AdShr vector and syntex adjuvant; 2 controls were unimmunized. Immune responses were monitored every 2-4 weeks.Tcell proliferative responses to SIV envelope protein and peptides were assessed by thymidine incorporation and IL-2 production. Cytotoxic Tlymphocyte (CTL) activity was evaluated by Cr5 1-release using autologous B cell targets infected with vaccinia-SIV env recombinants or control vaccinia. Serum antibodies were monitored by ELISA against whole disrupted SIV antigen. Antibodies in nasal and vaginal swabs were assessed by ELISA against
native SIV gp l 20.

Results: T-cell proliferative responses to SIV envelope peptides were observed sporadically
in 1/6 macaques following the Ist immunization and in 3/6 following the second. Significant
SIV-specific CTL activity was not observed.Transient serum antibody levels to SIV envelope
were first detected in all 6 immunized macaques after the 2nd immunization. Antibody levels were significantly boosted and became persistent following the gp I 20 inoculation. SIVspecific IgG antibodies were detected in nasal swabs of 2/6 macaques following the 2nd
immunization. Following the protein boost, all 6 macaques exhibited IgG antibodies in nasal
swabs and 5/6 in vaginal swabs. IgA antibodies were not observed; IgM antibodies in nasal
swabs appeared in 2/6 macaques following the protein boost.
Conclusions: Ad5hrSIV recombinant immunization and subunit boosting effectively elicited
humoral and mucosal immune responses.The presence of antibodies in nasal and vaginal
secretions is particularly encouraging. Following a 2nd protein boost at 36 weeks, these
macaques will be challenged vaginally with SIV251 at 40 weeks (March, I 996). Results of
that challenge will be reported.
Suzan L. Buge, NIH, 9000 Rockville Pike, Bldg. 37/Rm 6A I I, Bethesda, Maryland, 20892
Tel: [phone] Fax: [phone]
Mo.A. 103
IMMUNIZATION WITH SIVmne ENVELOPE (gp160) VACCINES PROTECTED
MACAQUES AGAINST INTRARECTAL CHALLENGE BY UNCLONED VIRUS.
P Polacino I,V. StallardI,J. Klaniecki2, C. Brown3, R. Watanabe I,W.R. Morton I, R. E.
Benveniste4, S. L. Hu 1.2 I Washington RPRC, Seattle, WA; 2Bristol-Myers Squibb, Seattle, WA;
3Henry Jackson Foundation, Rockville, MD; 4National Cancer Institute, Frederick, MD; USA.
Objective: Envelope (gp I 60)-based vaccines, when used in a live virus priming and subunit
protein boosting regimen, protected macaques from intravenous and intrarectal challenge
by a cloned homologous virus SIVmne E I IS. In the present study we investigated the
breadth of the protective immunity elicited by the envelope antigen against intrarectal challenge with uncloned SlVmne.
Methods: Six Mcocid osciculoris immunized with envelope (gp I 60), were previously challenged and protected from cloned E IIS infection.They were boosted again with gpl60 and
rechallenged intrarectally with 2-20 animal infectious doses of uncloned SIVmne. Six naive
animals were included as controls. Infection was monitored by nested-set PCR, PBMC coculture, viral load by semiquantitative PCR, plasma viremia, and in situ hibridizotion (ISH).
Lymphocyte subset, and SIV-specific antibodies titers were also determined.
Results: PCR analysis of PBMC DNA showed that all 6 control animals became infected
after challenge with the uncloned virus while 5/6 of the immunized animals were protected.
ISH and DNA PCR analyses showed virus-positive lymph nodes in all six controls starting at
week 2. Plasma viremia was transiently detected in some of the control animals. All controls
seroconverted after the challenge.The 5 immunized and protected animals remained PCR
negative and did not show any sign of infection in their lymph nodes. SIV-specific IgG was
detected on day of challenge (DOC) in mucosal secretions of immunized animals examined.The only immunized animal that became infected had the lowest serum antibody titer
at DOC as measured by ELISA. An anamnestic response was observed in this animal, indicating the presence of viral replication.
Conclusions: Envelope (gp I 60)-based vaccines protected macaques against intrarectal challenge by cloned and uncloned SIVmne, indicating that protection against mucosal infection is
achievable by systemic immunizations. Our result also indicates that protection may be
more easily achieved against mucosal, rather than blood-borne infections, since the same
regimen protected against infection by the cloned virus, but not the uncloned virus, by
intravenous inoculations.
Patricia Polacino, PhD. 1-321, HSB, Box 357330 University of Washington, Seattle,WA
98 195,USA.Telephone: [phone]
Mo.A. 104
IMMUNE RESPONSES IN CYNOMOLGUS MACAQUES INFECTED WITH
PATHOGENIC OR ATTENUATED SIV.
Rud, Erling W.*, Sheering, A*, Bogdanovic, D*, Ko, D*,Vogel,T*, Cook, N*, Hall, G**,
Cranage, MP*, Parenteau, M***, Beausoleil, N***, Fournier, J* *. Health Canada, *LCDC,
**ARD, Ottawa, Canada. **CAMR, Salisbury, UK.
Objective:To determine the immune responses induced by infection of macaques with
either an attenuated or pathogenic variant of SIVmac32H and ultimately to correlate these
to the protection induced against subsequent heterologous challenge with virus derived
from SIVsmm.
Methods: Three groups of 8 cynomolgus macaques were infected with virus derived from
SlVmac32H(pj5), SIVmac32H(pC8) or mock infected.The C8 molecular clone is a naturally
attenuated variant containing a 12 bp deletion in the nef/3'LTR region.To determine the
breadth of this protection we will challenge these macaques with virus derived from
SIVsmm(pBJI14).The virus load in these infected macaques was determined by limiting dilution of PBMCs and cocultivation with C81 66 cells. CD4/CD8 analysis was performed by
flow cytometry Proviral DNA and viral RNA loads in circulating PMBCs and plasma were
determined by limiting dilution of PBMC DNA and nested set PCR and bDNA analysis of
plasma associated RNA. Isolated PBMCs were used in the following immunological studies:
CTLp frequencies to various SIV proteins; the TCR repertoire; cytokine pattern of expression; lymphocyte proliferation to PWM, cynomolgus EBV and SIVmac gp120 and Mixed
lymphocyte Reactions (MLRs).The macaques were also followed by hematology blood biochemistry and weight gain.
Results: Two weeks post inoculation the macaques infected with C8 had a lower virus load
then those infected with J5.The virus load decreased much quicker in C8 infected
macaques.The macaques responded similarly to PWM, cynomolgus EBV and in MLRs and
the SIV infected macaques responded similarly to SIVmac gpI20.The V repertoire of the
TCR, in general, showed no selection for a specific subtype of V expression.The CD4/CD8
ratios did not vary over normal biological variation.The CTLp frequencies to SIVmac gp I 60
and SIVmac RT the antibody titres directed against SIVmac gpI20, and the cytokine profiles
are all being examined at present.
Conclusions: The immune responses in macaques infected with either the attenuated or
non-attenuated v:riants of SIVmac32H do not seem to differ: It seems that the macaques

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