Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

20 Abstracts WePeA5771-WePeA5775

XIV International AIDS Conference

WePeA5771I Susceptibility to HIV transmission and AIDS
progression: role of toll-like receptor 4
S.R. Kleeberger', A.E. Jedlicka2, E.C. Seaberg2, D.A. Schwartz3. 1National
Institute of Environmental Health Sciences, 111 T W Alexander Dr, Building 101,
D240, National Institute of Environmental Health Sciences, Research Triangle
Park, NC 27709, United States; 2Johns Hopkins University, Baltimore, United
States; 3Duke University, Durham, United States
Background: Toll-like receptors (TLR) have recently been shown to be important
modulators of innate immunity, a critical component of resistance to HIV infection and AIDS progression. TLR4 has been implicated as a determinant of HIV
infection and viral replication in vitro. TLR4 also mediates responses to Gramnegative bacteria. Two hypotheses were tested: (1) a functional polymorphism
in TLR4 negatively associates with HIV-1 infection; (2) the TLR4 polymorphism
confers resistance to disease progression in HIV-infected individuals.
Methods: Specimens from 501 men were tested: 402 HIV seroconverters (cases)
and 99 high-risk HIV negative men (controls). Among seroconverters, 379 had
a HIV seroconversion window less <1 yr and AIDS onset window <1 yr (welldefined cases). Individuals were genotyped by single-stranded conformation polymorphism (SSCP) analysis for a 896 A to G TLR4 polymorphism that confers
resistance to endotoxin. Polymorphisms were confirmed by sequence analysis.
Prevalence of the TLR4 mutation was compared between cases and controls
(hypoth. 1). Time from seroconversion to AIDS and kinetics of CD4, CD8, and
HIV RNA following seroconversion were assessed for the 379 well-defined cases
(hypoth. 2).
Results: The TLR4 polymorphism was not associated with HIV transmission.
However, men heterozygous (+/-, n=36) or homozygous (-/-, n=3) for the TLR4
polymorphism had significant protection against AIDS onset during the first 8 yr
following seroconversion (RR=0.44, 95% C1=0.21-0.89) compared with men homozygous for the wild-type allele (+/+). All TLR4 -/- men did not develop AIDS
(min. FU=10.7 yr). Median HIV RNA following seroconversion was lower in -/men compared to +/- and +/+ men, but small numbers precluded formal analysis.
Conclusions: Results indicate TLR4 is an important determinant of AIDS progression in HIV-infected individuals, and may provide a potential therapeutic target for treatment of AIDS.
Presenting author: Steven Kleeberger, 111 T W Alexander Dr, Building 101,
D240, National Institute of Environmental Health Sciences, Research Triangle
Park, NC 27709, United States, Tel.: [phone], Fax: [phone], Email: kleeberl @niehs.nih.gov
WePeA5772I Long-term survivors in Nairobi: complete HIV-1
RNA sequences and immunogenetic profiles
G. Fang', C. Kuiken2, B. Weiser', S. Rowland-Jones3, F. Plummer4,
C.H. Chen', R. Kaul3, A.O. Anzala5, J. Bwayo5, J. Oyugi5, B. Gaschen2,
D. Lang2, S. Philpott', S. Beddows6, Q. Sattenau6, C. Ramirez Kitchen7,
E. Paxinos8, H. Burger'. IWadsworth Center, Wadsworth Center, New York
State Dept. of Health, 120 New Scotland Avenue, Albany, NY, United States;
2Los Alamos National Lab, Los Alamos, United States; 3Oxford University
Oxford, United Kingdom; IUniversity of Manitoba, Winnipeg, Canada;
5University of Nairobi, Nairobi, Kenya; 61mperial College, London, United
Kingdom; 7University of California, Los Angeles, United States; 8 Virologic, San
Francisco, United States
Background: The pathogenesis and pace of HIV-1 disease progression reflect
the interactions among viral genomic sequences, host genetics, and immune responses. Slowly progressive HIV-1 infection has been studied in the West, where
clade B HIV-1 is predominant. In Africa, clade B is rare, and multiple viral clades
and recombination are common. In Nairobi most female sex workers become
infected and progress to AIDS rapidly, but we have identified a group of HIV-1 -infected women who were untreated long-term survivors (LTS, > 11 years).
Methods: To investigate the determinants of pathogenesis, we obtained complete plasma HIV-1 genomic sequences and immunogenetic profiles from 7 LTS
including 2 long-term non-progressors (LTNP).
Results: Most HIV-1 was subtype A, but subtypes C and D were also found.
Three of the 7 women including a LTNP had recombinant HIV-1 genomes composing 2 subtypes. Computational analyses did not reveal any clear sequence
changes likely to cause attenuation and the immune response was variable. Functional studies demonstrated that all HIV-1 strains utilized CCR5. Neutralizing antibodies to viral strain MN but not to an African strain were detected in 6 LTS. Determination of the complete HIV-1 sequence and the HLA types (A and B) made
it possible to predict the CTL epitopes. Functional CTL assays were performed in
4/7 women, with strong responses seen in 1 LTNP and positive responses seen
in 2 other LTS. The LTS were genetically characterized for CCR5, CCR2, and
SDF-1-3 UTR. Significantly, the 2 women who were homozygous for the CCR2
641 mutation were the LTNPs. Major drug resistance mutations were not detected.
Conclusions: These data suggest that interactions between HIV-1 and immune
responses are complex, making determination of complete HIV-1 sequences in

conjunction with genetic and immunologic studies a priority for investigation of
pathogenesis and the correlates of protection.
Presenting author: Fang Guowei, Wadsworth Center, New York State Dept. of
Health, 120 New Scotland Avenue, Albany, NY, United States, Tel.: [phone], Fax: [phone], E-mail: [email protected]

WePeA5773 HIV-1 long term non-progression is associated
with low viral replication
J.F. Morlesel, N.A. Qazi', G. Hardy2, R. Abbas', B.G. Gazzard', N. Imami2.
' St. Stephen's Centre, Chelsea & Westminster Hospital, St Stephen's Centre,
Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9TH,
United Kingdom; 2Dept of Immunology, Chelsea & Westminster Hospital,
London, United Kingdom
Background: The progression of HIV-1 infection is slower in a group of patients
deemed the long term non progressors (LTNPs). In these LTNPs the HIV-1 specific helper-T lymphocyte (HTL) responses are more vigorous than in normal progressor patients. It is believed that these responses are capable of suppressing
ongoing viral replication and hence controlling the progression of disease. We
have recently validated the use of 2-LTR HIV DNA circles as a measure of ongoing replication. The aim of this study was to determine whether vigorous T-cell
responses are associated with reduced viral replication.
Methods: Ten male LTNP patients naive to antiretroviral therapy (ART) defined
with evidence of HIV-1 infection for >10 years and a rate of fall of CD4 count
<80 cells/yr and RNA load <50 copies/mL were studied. PBMC were isolated
by Ficoll centrifugation and HIV-1 nef, tat, p24 and gpl20 lymphoproliferative responses were assessed. The 2-LTR circles were assayed using real time quantitative PCR.
Results: The 2-LTR circle copy number in the LTNPs was <10 copies/106 PBMCs
and HIV-1 specific HTL responses were present.
Conclusions: The LTNPs had vigorous HIV-1 specific lymphoproliferative responses which were associated with low levels of ongoing viral replication as
assessed by the 2-LTR HIV-1 DNA copy number. This suggests that LTNPs are
able to maintain low levels of viral replication through preserved specific antiHIV immune responses. Furthermore, the 2-LTR HIV-1 DNA copy number maybe
a useful surrogate marker for successful antiretroviral treatment in normal/rapid
progressors.
Presenting author: Nad Qazi, St. Stephen's Centre, Chelsea and Westminster
Hospital, 369 Fulham Road, London SW10 9TH, United Kingdom, Tel.: +44 (0)20
8846 6182, Fax: [phone], E-mail: [email protected]
WePeA5774 Characterization of a defect in the vif gene of an
unusually slow growing strain of HIV-1
M.A. Farrow, J.L. Sullivan, T.C. Greenough, M. Somasundaran. University of
Massachusetts Medical Center Department of Pediatrics Program in Molecular
Medicine Center for AIDS Research, 373 plantation st, biotech //II suite 318,
worcester, ma 01605, United States
The study of viral isolates from long term non-progressors (LTNP) often provides
insight into naturally occurring attenuating mutations. LTNP viruses have been
shown to harbor extensive mutations in the vif gene. Virus produced from restrictive cells in the absence of functional Vif is defective at a step post-penentration,
pre-integration in the viral life cycle. The Vif of a slow-growing LTNP patient isolate, that showed a vif-dependent phenotype in T cell lines, was genotypically
and phenotypically characterized. Clinical data that was collected on this LTNP
for twenty years revealed a consistent pattern of low viral burden and stable CD4
counts. Viral vif sequences were obtained following amplification and sequencing
of samples from cocultured virus, patient cell provirus, and plasma virus over a
range of ten years by standard PCR techniques. Persistent mutations found in the
patient vif as well as WT vif were used to construct in-frame dsRed fusion proteins
that were transfected into MAGI cells to determine expression and localization of
the Vif proteins by fluorescence microscopy. To assess the contribution of the mutations to the replicative fitness of the virus, the mutations were introduced into the
WT molecular clone NL4-3 by PCR mutagenesis. Replication was monitored by
measuring p24 levels following primary infection. Genotypic analysis of the LTNP
vif gene revealed a unique substitution of KKRK, a potential NLS, for RKKR at
positions 90-93. This mutation was persistent in samples over at least ten years.
Fluorescence microscopy revealed a punctate distribution of WT Vif in the cytoplasm. In contrast, the KKRK Vif was concentrated in the nucleus. The infectivity
of the WT NL4-3 was normal, but the KKRK-NL4-3 produced no detectable virus
for up to one month following infection. The presence of a functional NLS in the
Vif protein results in mislocalization of Vif from the cytoplasm to the nucleus and
abrogates viral replication.
Presenting author: melissa farrow, 373 plantation st, biotech II suite 318,
worcester, ma 01605, United States, Tel.: [phone], E-mail: mellisa.farrow
@umassmed.edu
WePeA5775 The "Milano" cohort of long-term non progressors
(LTNP): a 7-year follow-up study
S. Ghezzi', G. Morsica', E. Vicenzi', F Pacciarini', E. Santagostino2,
A. Gringeri2, G. Carminati3, M. Cusini3, A. Lazzarin', G. Poli'. 'San Raffaele

Scientific Institute, San Raffaele Scientific Institute, Italy; 2University of Milano,
Milano, Italy; 31RCCS "Maggiore Hospital" Milano, Milano, Italy
Background: LTNP are fundamental for understanding the nature of a protective
immune response. A cohort of 47 individuals belonging to different risk categories
was established in 1994 (Milano cohort).
Methods: All individuals were periodically for:CD4+ T cell counts, plasma
viremia, PBMC-associated HIV DNA load, HIV isolation and phenotype,