Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

520 Abstracts 31125-31129

12th World AIDS Conference

tiating effects of the triple drug regimen. We have previously shown that triple
combination therapy fails to enhance anti-HIV-1 CD4~ T cell responses. More
prolonged triple drug combination therapy or additional strategies may be needed
for full reconstitution of HIV-1 specific T cell function in late stage patients.
31125 Rare occurrence of CTL escape mutations in HIV-1
infection
Alicja Trocha, K. Hartmann, S. Kalams, B. Walker, R. Johnson. Massachusetts
General Hospital, AIDS Research Center, 149 East 13th Str, Charlestown, MA,
USA
The extensive sequence variation exhibited by HIV-1 has been proposed to be a
mechanism of immune escape, but conflicting data exist regarding this point. We
have examined the relationship between sequence variation in HIV-1 and recognition by CTL specific for multiple epitopes in the gag, polymerase and envelope
proteins of HIV-1 in two HIV-1 seropositive individuals. Using both CTL clones and
bulk CTL assays, we identified multiple CTL epitopes in each individual studied,
ranging from 8 to 12 epitopes per subject. Recognition of these CTL epitopes
over time in bulk CTL assays was relatively stable, although in some cases loss
of recognition of CTL epitopes was observed. Analysis of sequence variation
within these epitopes at multiple different time points revealed several different
patterns. In the majority of epitopes studied, no significant sequence variation
was observed (11/16 epitopes) or sequence variants that arose did not affect
CTL recognition (2/16 epitopes). In two instances, an increasing prevalence of
an unrecognized sequence was observed. For one of these epitopes, incubation
of naturally-infected CD4 lymphocytes with a CTL clone resulted in a preferential
outgrowth of viruses not recognized by this CTL clone. In another instance, an
immunodominant CTL response recognized a minor sequence variant, while the
dominant sequence in vivo was poorly recognized. Taken together, these results
suggest that in subjects with a polyclonal CTL response, that evolution of unrecognized CTL variants is uncommon. Furthermore, these data suggest that escape
mutations are not required for disease progression and that other mechanisms of
immune evasion appear to play a role.
HIV sequence variation may be a mechanism for HIV to escape surveillance.
Two subjects recognizing 8 and 12 epitopes respectively were studied over time
and epitope specific CTL clones were tested for in vitro inhibition of viral replication. The low frequencies of poorly recognized epitope variants suggest that
escape mutations are uncommon and not required for disease progression.
31126 Analysis of LPS-mediated inhibition of HIV-1 infection of
macrophages: p38 MAPK is the critical regulator
Michael Bukrinsky, N. Reiling, G. Zybarth, G. Franchin, B. Sherry. The Picower
Institute, 350 Community Dr. Manhasset, NY 11030, USA
Cells of the monocyte/macrophage lineage may be among the first targets of
HIV-1 in patients, and also serve as reservoirs for the virus during the course
of infection. Lipopolysaccharide (LPS) has been shown to induce expression of
HIV-1 in monocytoid tumor cell lines, but to protect primary macrophages from
viral infection in vitro. We investigated the effects of LPS on pre-integration events
of HIV-1 infection of monocyte-derived macrophages (MDMs) using PCR analysis
of viral DNA. Addition of LPS at the time of infection stimulated entry of HIV-1
into MDMs, but inhibited synthesis of late reverse transcription products, strongly
repressed nuclear import of the viral DNA, and inhibited production of HIV-1 in
a long-term culture. Pre-exposure of MDMs to LPS, on the other hand, caused
a rapid decrease of virus entry, associated with disappearance of HIV-1 coreceptors from the cell surface. Anti-CD14 monoclonal antibody and Lipid A partial
structure (compound 406), when added prior to incubation with LPS, rescued
HIV-1 infection of MDMs, indicating that the inhibitory effect of LPS is mediated
through the CD14 receptor. The effect of LPS was also reversed by disruption of
the p38 mitogen-activated protein kinase (MAPK) pathway. On the other hand,
the inhibitory effect of LPS on HIV-1 infection of MDMs was mimicked by muramyl
dipeptide (MDP), which was found to induce activation of p38 MAPK. The anti-HIV
effect of p38 MAPK activation in MDMs is in contrast to the situation in T cells,
where activation of p38 MAPK stimulated viral replication.
Conclusions: Our results demonstrate importance of macrophage activation in
anti-HIV responses of the immune system. While activation of T cells is required
for productive HIV-1 replication, activation of macrophages makes them resistant
to infection. These consideration should be taken into account when designing
novel anti-HIV therapeutics and vaccines.
1603*/31127 New HLA-A11 HIV-1 subtype E cytotoxic T
lymphocyte (CTL) epitopes identified in studies of
thai HIV-infected female sex workers
Kyle Bond1, B. Sriwanthana1, T.W. Hodge1, C.P. Pau1, A.S. Degroot2,
B.M. Jesdale2, T.D. Mastro3, N.L. Young3, K. Limpakarnsanarat3,
J.M. McNicholl1. 1 CDC 1600 Clifton Road MSA25, Atlanta GA. 30333; 2Brown
University School of Medicine, Providence RI, USA; 3HIV/AIDS Collaboration

Nonthaburi, Thailand
Background: While many CTL epitopes have been identified for HIV-1 subtype B,
no data are available regarding the CTL epitopes recognized by HIV-1 subtype E
infected individuals. In regions where subtype E predominates, such as Thailand
and much of Southeast Asia, HLA-A11 is found in 30-50% of persons. Using
a peptide-based approach, we determined HIV-1 subtype E CTL epitopes in 11

HLA-A 11-positive female sex workers from northern Thailand who were subtype
E HIV infected.
Methods: Possible 9-10 amino acid HLA-All-binding CTL epitopes were
predicted in sequences of CM243 and 93TH253 subtype E strains using the
computer-based prediction program EpiMatrix. HLA-A11 binding of these peptides was determined in a flow cytometric T2-HLA-A11 stabilization assay. Of
those tested, 22 bound and were considered candidate All CTL epitopes. PBMC
from HIV-infected donors were expanded with these peptides in the presence of
IL-2 and IL-7 and were tested for reactivity in CTL assays using peptide-pulsed
51Cr-labelled autologous B cell lines at effector-to-target ratios of 60:1, 30:1, and
10:1. A positive response was defined as lysis of peptide-pulsed targets at a level
of >10% over that of the non-pulsed targets.
Results: HLA-All-restricted HIV-specific CTL reactivity was detected in 8 of
11 HIV-infected persons to pol (7 of 8), env (5 of 9), gag (3 of 8), and nef (5
of 8). Six pol, 6 env, 1 gag, and 2 net All epitopes were recognized, some of
which were conserved between HIV-1 subtypes B and E, while others had one
or more amino-acid differences between subtypes. The regions containing all 5
previously known HLA-A11 subtype B CTL epitopes were recognized, although
substitutions were observed in some cases.
Conclusions: Some HIV-1 subtype E-infected persons in Thailand recognize
regions of HIV not previously known as HLA-A11 CTL epitopes; some recognize
known CTL epitopes that are conserved between subtypes B and E. These data
suggest that some existing HIV-I vaccines may be immunogenic across genetic
envelope subtypes while the identification of new immunogenic regions in HIV-1
may permit the improvement of existing or the development of new HIV vaccines
for Southeast Asia.
602*/31128 Cross-clade cytotoxic T cell response to HIV-1
proteins among HLA disparate North Americans
and Thais
Julia Lynch1, M. Desouza2, M. Robb3, S. Nitayaphan4, L. Markowitz2, D. Birx3
J. Cox2. 113 Taft Court Suite 200, Rockville MD 20850; 3 Walter Reed Army
Institute of Research, Washington DC; 5Henry M Jackson Foundation, Rockville
MD, USA; 2Henry M Jackson Foundation, Bangkok; 4Armed Forces Res Institute
for Medicine, Bangkok, Thailand
Objective: To compare the frequency and magnitude of same clade and crossclade CTL responses to four HIV-1 proteins (Gag, Pol-RT, Env and Nef) among
HLA defined clade B infected North Americans and clade E infected Thais.
Methods: The CTL population (effectors) was expanded from PBMC of 10 B
infected NA and 12 E infected Thais using a 14 day in vitro stimulation with cells
expressing each protein. Autologous targets were created with EBV transformed
B cell lines infected with vaccinia virus constructs containing HIV gene insertions.
Lysis of targets was measured by Chromium release at different effector to target ratios. CTL response was measured to both homologous and cross-clade
proteins. All subjects were HLA typed at the A and B loci by molecular typing
techniques.
Results: HLA distributions for study subject North Americans (NA) and Thais
were distinctly different and representative of their origin. CTL responses were
detected against all proteins but were most frequent to Gag in both groups (5/10
NA, 8/9 Thais). Cross-clade Gag responses were seen in 9 of 10 responders
tested. Pol-RT was recognized less frequently by Thais (2/9) than North Americans (5/10). Nef and Env were infrequently recognized by both groups. Cross
clade protein recognition was relatively common for Gag, Pol-RT and Nef but rare
(1/6) for the highly diverse Env.
Conclusion: Gag is likely to be an important antigen in a broadly immunogenic
vaccine. The limited cross recognition of Env suggests that inclusion of clade E
Env may be prudent for a vaccine to be tested in Thailand. Variability in CTL
recognition between HLA disparate populations suggests that population specific
CTL data is needed to adequately prepare for vaccine trials outside of North
America and Europe.
S31129 Oral inoculation with HIV-2 elicits a Gag-specific cytotoxic
T-cell response in juvenile but not neonatal macaques
Arnd Martin Herz1, John Lynch2, M.N. Robertson2, A. Schmidt3, B. Kennedy3,
L. Kuller3, W.R. Morton3, P.D. Greenberg2. 1Children's Hospital and Medical
Center, Seattle, WA; 2University of Washington, Seattle, WA; 3Regional Primate
Research Center, Seattle, WA, USA
Background: Thirty percent of human infants progress to disease rapidly following infection by HIV. Although HIV-specific cytotoxic T-lymphocytes (CTL) are
thought to be important in moderating the course of this infection, infants may be
unable to mount an appropriate cellular immune response. Since the majority of
children are thought to be infected peripartum by mucosal routes, we have developed an animal model of maternal-neonatal transmission using oral inoculation
of newborn macaques. Here we test the ability of primate neonates to respond to
a pathogenic HIV-2 isolate inoculated via the mucosa.
Methods: Neonatal and juvenile macaques were inoculated orally with HIV-2.

Lymphocytes were stimulated twice with autologous cells infected with a vaccinia
construct expressing the Gag protein and tested in a standard chromium release
assay.
Results are presented in the table.
Conclusions: Neonatal macaques are unable to generate an immune response sufficient to attenuate infection by HIV-2. Both neonatal and juvenile
animals developed antibody responses, but differed in their ability to produce