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

Mo.A.153 - Mo.A.161

Monday, July 8, 1996

Mo.A. 153
HIGH DOSE HIV-I MN RECOMBINANT GPI60 (RGPI160) VACCINE INDUCES
ANTI-V3 MN,AND IGG 1-4 AND IGA ANTI-RGP160 ANTIBODIES
Gorse, Geoffrey * 1, McEhrath MJj, Belshe RB*, Corey L -, Matthews Tt, Eil Mb, Kennedy
D*J, Frey SC, Hsieh Rt, Walker MC.j i. *Saint Louis University and VAMC, St. Louis, MO;
INIAID AIDS Vaccine Evaluation Group, USA; ~IMMUNO-AG,Vienna, Austria; DAIDS,
NIAID, NIH, Bethesda, MD), U.S.A.
Objective: To evaluate the safety and immunogenicity of a mammalian cell-produced HIV- I
MN rgpI160 vaccine (IMMUNO-AG) at a high dose in low-risk, HIV- I uninfected volunteers.
Methods: In an ongoirng, couble-blind, randonmized study subjects received 800 pg MN
rgpl60 at 0,1, and 6 months (N=8) or at 0,2, and 8 months (N=8); 4 subjects received
placebo. Subjects (N- 19) are receiving a fourth dose 6 months after the third. Clinical and
laboratory safely, serum a pti-r-gp160 and V3 MN binding (ELISA) and HIV-I MN neutralizing
antibodies, and lymphocyte proliferation are being measured.
Results: Local injection site pain of moderate severity on average occurred in all 20 subjects. Self limrited, mild to moderate systenmic symptoms occurred in 5 vaccinees and 3 controls. Binding antibody resonses 14 days post third vaccine dose were:
Schedule Anti MN rgp 160 Reciprocal GMT Anti-V3 MN Antibody Mean O.D
(No. Positive/No.Tested) (No. Positive/No.Tested)
(Months) I G I IgG 2.g 16G3 IgG4 IgA
0,1,6 263 566 100 200 200 59 0.28
(8/8) (8,'s') 8/8) (8/8) (8/8) (8/8) (1/8))
0,2,8 3200 800 i 9 200 400 71 0.62
(4 1/ 4/g) (3/4 (4/4) (4/4) (4/4) (6/7),Additional resIcts ir 3 v,,c,nes perdin, iI>-j 01. Neutralizin antibody assays of sera after
the third and fourth dosesar pending High levels of lymphocyte proliferation to MN
rgp 160 were induced in 8 vaconees tested,( far (Mean Stimulation Index post thi-d dose
S93.4+50.1), indicating T cell memory to vaccine antigen.
Conclusions:The vaccine was safe, and inducedT cell memory and anti-rgp 160 IpG (subclasses 1-4) and IgA binding astibodies. Compared to 200pg, the 800 ag MN rgp 160 doses given
at 0,2, and 8 months better stimulated anti-V3 MN antibody. Anti-V3 antibody has correlated
with neutralizing antibody against HIV- I lab strains. In earlier studies, 200 pjg doses of MN
rgp 160 induced neutralizing antibody against HIV I MN even in the absence of anti-V3 MN
antibody Anti-V3 antibody induced in thris study may increase functional activity of these sera.
Geoffrey J. Gorse, M.D., Division of Infectious Diseases, Saint Louis Univ. Health Sciences
Center 3635 Vista Ave. (FDTF-8N), St. Louis, MO 631 I10, USA: Phone: [phone];
FAX: 314-771 --3816; gorsemd(sluvca.slu.edu
Mo.A. 154
CHIMERIC HUMAN IMMUNODEFICIENCY VIRUS GAG PARTICLES AS AN ANTIGEN
PRESENTING VEHICLE
Hoshikawa Nariyoshi*,*,Yasuda A","**, KurataT", Kojima A". *Kosei Hospital,Tokyo,
Japan; **Dept. of Pathology NIH,Tokyo, Japan; *** Nippon Zeon Co. Ltd., Kawasaki, Japan
Objective: Viral antigens usually show the highest immunogenicity when they form virus-like
particle structures. In this study we examined whether human immunodeficiency virus (HIV)
Gag particles work as immunogenic carriers for a hepatitis C virus (HCV) core epitope.
Methods: The HIV- I gag gene (BH I10) was deleted of the p2 region by introduction of KpnI sites at its 5'- rand 3'. ends and then Kpn-I digestion. Synthetic oligonucleotides encoding
the N-terminal 28 amino acids of HCV core (C28) were inserted at the Kpn-I site to create hybrid gag-C28 genes.The hybrid genes were expressed by recombinant vaccinia virus
(RW). Expression and re ease of hybrid proteins were analyzed by immunoblotting. Particle
formatiorn was exarmined by transmission and scanning electron microscopy of RVV infected
cells. Antibody responses in RVV infected mice were determined by ELISA.
Results: RVVinflected cels expressed Gag proteins with the increased molecular mass
when analyzed with anti p7 and anti p2t monoclonal antibodies.The larger Cag proteins
also reacted specifically to sera from ICV-infected patients and fhom rabbits immunized
with recombiniant HCV core. Gag-like particle formations prepared from culture niediurr
by ultracentrifugation contained HCV core-specific and Gag-specific proteins. Extracellular
Gag-like particles were observed by electron microscopy of RW-infected cells. Mice inoculated with RVV induced anti C28 as well as anti pl7 antibody responses.
Conclusions: We showed that a Gag-C28 mosaic protein assembles into recombinant Calike particles and that Gag-C28 particles process marked immunogenicity in mice.These
results suggest that HIV (_ag particles can be useful for carrying immunogenic epitopes and
vaccine vehicles.
N. Hoshikawa, Dopt. of Pathology NIH, 1-23 i,Toyama, Shinjyuku-ku,Tokyo, Japan
Telephone: 3 51'785 i i I Fix: 3-5285-1 189 email: nhoshika(anih.go.jp
Mo.A. 155
IMMUNOGENICITY OF A LIVE RECOMBINANT CANARYPOX VIRUS EXPRESSING
gp 20tm-MN / gag / PROTEASE-LAI (vCP205) BOOSTED WITH A p24E /V3-MN
PEPTIDE (CLTB-36} IN HIV NEGATIVE VOLUNTEERS (ANRS VAC 03)
Salmon Door ni -, Excle JL2, Finkelztejn LI, Chapurs L., Heshmati F, Glckrnan JC3,
Autr~an B3, Merynrer 82, Klein M12, Sicard UI. AGIS and ANRS. IH~p. Coccino, Paris; 2pasteur
Mdrieux-Connaught, Marnes La Coquette: 3H6p. Pitid-Salpetriere, Paris - France
30 healthy volunteers at low risk for HIV infection were immunized with vCP205 at 10
TCIDSO and or CLTB-36 240 pjg, and raridomly assigned.
Results at month 7 of lymphoproliferative response (LPR) and cytotoxic T lymphocyte
activity (CTL) were as follows:

Neutralizing ant hcdie, to llV-I MN were detected in 5/15 volunteers of groups C
and D one month after the 4th injection, but none of the sera could neutralize an HIV-I
primary isolate.
In conclusions: I 'se n i n ary results suggest that: CLTB-36 is poorly immunogenic; p24E
is not an immunotraiim- rI helper epitope in this population; 4 x vCP205 are able to
induce both hur n, l,: ' NI-lular responses; CTL activity although low, is broadened against
env, gag and pol,, 1'/cO4+ mediated and HLA-I restricted in some subjects. Booster
injections did not ennrnce responses. Priming with higher coses of vCP205 and boosting
with subunits such as p! 20,p24 or pseudovirions might be worth testing in humans.
Salmon Dominique, Hlpital Cochin, Medecine Interne, 27 rue du faubourg, 750 I Paris,
France.Telephone: [phone], Fax: [phone], email: [email protected]
Mo.A. 156
HIV- I PR55gag VIRUS-LIKE PARTICLES ALLOW THE ACCESS OF POLYPROTEINS AND
DEFINED EPITOPES TO THE MHC-I PRESENTATION-PATHWAY
1 Ludwig Deml, 2Reinhold Schirmbeck, 2Jdrg Reimann,1 Ha-s Wolf, 1RalfWagner.
I Universitit Regensburg, Regensburg, Germany; 2Universitlit Ulm, Ulm, Germany
Objective: In many viral infections, cell mediated immunity, in particular the cytotoxic T-cell
response, plays a key role in the control of a viral infection.There is striking evidence to suggest that this may be also in the case of an HIV infection.Tnerefore we tested the capacity
of different types of chimeric Pr55gng VLPs of traficking defined CTL epitopes or complex
polyproteins to the MHC-I presentation pathway
Method: We developed an autologous, safe and non replicating carrier system, basing on
recombinant, non infectious HIV I Pr55 lipoprotein particles (VLPs) To expand the
immrnunogenicity of these VLPs, defined domains within the Pr55 polyprotein were replaced
by selected CTL eptopes such as the third variable loop V of the HIV- I envelope protein
gp I20 (type I VLPs). Alternatively chirmreic HIV- I envelope proteins were stably anchored on
the surface of these VLPs by a heterologous type I transmembrane moiety of the EBV envelope protein gp350/220 (type 11VLPs).To test the capacity of VLPs to induce a V3 loop specific MHC-I restricted CTL response, different VLP pr-eparat ons were produced in insect cells
by using recombinant baculoviruses and puried by sedimentation through a sucrose cushion.
Results: Immunisation of BALB/c mice with different types of chimeric Pr55yng/V3 VLP
resulted in the induction of a strong CD8+ CTL response in vwo, irrelevant of the position
of the V3 loop within the comlex carrier component. Different routes of immunsation
(s.c., i.p., i.v.) as well as antigen doses in a range of 1-20 ja have been proven as equally
effective. In contrast only a week CTL-response was elicited by VLP adsorbed to Alum or
emulsified in IFA.
Conclusions: Pr55 based lipoprotein particles resemble a novel and potent immunogen,
which efficiently mediates the transport of small peptides and complex polyproteins to the
MHC-class I antigen presentation pathwayThe importance of these data for the development of a second generation of HIV candidate vaccines will be discussed.
Ludwig Deml, Institut fir Mledizinische Microbiologie und Hygiene, Universitit Regensburg, Franz
Josef Straub Allee I I, D-93053 RegensburgTel. +49 94 I 944 6480, Fax. -49 94 I 944 6402
Mo.A. 160
LIMITED DETECTION OF HIV-I SUBTYPES BY THE ROCHE AMPLICOR
DIAGNOSTIC POLYMERASE CHAIN REACTION ASSAY
Gleeson, Tim, Montpetit, M. Health Canada, Ottawa, ON, Canada
Objective: To determine the effects of primer-template mismatches on Roche Amplicor
[Iignoseic polymerse chain reaction (PCR) results for HIV- I strains not belonging to
genetic subtype B.
Methods: Computer analysis (OLIGSAN program) of homology between the Roche
Amplicor PCR primer pair SKI3 I/5K462 and 100 HIV I strains from the 8 Group M and
Group O subtypes of HIV I revealed considerable mismatching. Site- directed mutagenesis
of cloned SK43 1 and SK162 amplification primers was pedformed to generate the mismatches found in the computer analysis of primer homology. Amplif cation and detection
of the original and r.nutagenized targets was performed initially according to the rnanuficturer's instructions.Targets incorrectly diagnosed were subjected to further amphfication
attempts using different reaction conditions to identify suitable PCR reaction parameters
for proper diagnosis
Results: Concer ns about the limited detection of HIV- I strains and subtypes by the Roche
Amplicor HIV I PCR systemn h ave been confirmed. While hlimited divergence at the 5' ends
of the primers had little etIect on diagnostic accuracy the widespread mismatching found in
a number of subtypes severely limits the diversity of HIV- I detected by the Roche Amplicor
system.The use of the same primers in the Roche Amplicor Monitor System designed for
viral load measurement may cause falsely low readings for patients with viral strains not
members of subtype B.
F. Gleeson. HIV Genetics, 1000D I Virus Building, Ottawa, Ontario. K IA OL2 Canada.
Tel: [phone] Fax 6 I 3 957-7258 email: mmontpetitt.hphbhwc.ca
Mo.A. 16 I
THE EFFECTS OF MUTATIONS ON HIV- I QUANTITATION BY RT/PCR
Christopherson, Cindy U.D., Kwok, S. Roche MolIecular Systerms, Alameda, CA, U.S.
Primer design and a pcawtionk conditions s-re p-evioustr/shown ti effect 3 terminal mismatch tolerance (Kellog et. al., NAR 1990). In this study we investigated the effect of nternal primer'template nisratches on reverse transcription 'RT)-PCR and of probe-template
mismatches on probe capture eficiency
Templates were constructed by PCR that contained mutatans that represent various HIV-I
subtypes in either the upstream primer binding region (5K462), the downstream primer
binding region (SK43 I), or the probe bindng region (SKI02). RNA transcripts were generated by in vitro transcription of the amplifed DNA products.The effects of mismatches
were determined by a quantitative RT/PCR assay A maximum of 6 mutations in the
upstream primer 5 in the downstream primer and 5 in the probe region were examined.
DNA ampliication efficiency was not affected by as many as 4 mutations, but a decrease in
eficiency was seen when 5-6 mutations were present. Mismatch tolerance was signifcantly
improved by lowering the annealing temperature and/or by incorporating unconventional
bases that stabilize DNA duplexes. In the worst case, the presence of 5 mutations in the RT
primer reduced product yield by i 0-fold, but a "step-up RT whereby the temperature

Cellular immune responses at month 7
L PR (net cpm > 1000) CTL

(E /T:

Vaccine regimen gp I 60 p24 p24E V3 CLI B-36 env gag
A CLTB-36 x 3 n =5 - - 1I/5 3/5 4/5 2
B vCP205 x 2 and 7/10 3/10 0/10 2/10 3/10 - I
CLTB 36 x 2 n= 10
C vCP205 x 3 n-=10 7/10 3,/10 0/10 1/10 I/h0 2 2
[) vCP20S x 2 3 /d
vCP205 + CLTB 36 x 2 n 5 3/5 3/5 1/5 3/5 4/5 2 2

3

120/ I -
pol No
- 2/5S
2 1/10