Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

ThA 1573-ThA 1576

TRACK A: BASIC SCIENCE

ThA 1573 ADOPTIVE TRANSFER OF A NEF-SPECIFIC CTL CLONE INTO hu-PBLSCID MICE AND AN HIV-INFECTED PATIENT Koenig Scott'; van Kuyk Rt;
Jones G'; Torbett Bt; Mosier Dt; Brewah YA'; Leath S*; Davey VJ'; Yannelli J~;
Rosenberg S~; Fauci AS'; Lane HC'; *MedImmune Inc, Gaithersburg MD; tMBI
LaJolla CA; 'NIAID/NCI/NIH Bethesda MD
Objective: We have previously demonstrated that nef-specific CTL can inhibit HIV replication in vitro.
Preliminary studies indicated that adoptive transfer of CTL to SCID mice reconstituted with human peripheral
blood leukocytes (hu-PBL-SCID)just prior to HIV challenge can prevent infection in 20% of the animals. In
the current studies, we examined the protective effects conferred by multiple transfers of the CTL clone prior
to and following HIV challenge of hu-PBL-SCID mice. In a Phase I clinical study we examined whether the
adoptive transfer of this CTL clone to the seropositive donor from whom it was derived would be safe and
determined its effects on viral replication and immunological function.
Materials & Methods: A CTL clone, HLA-A3 restricted and specific for peptide QVPLRPMTYK, was
expanded in vitro with irradiated PBLs, CD3, and IL-2. In hu-PBL-SCID mice, CTL were injected i.p. for
three days prior to and following challenge with HIVmsB. Infection was monitored for one month by the
polymerase chain reaction and by p24 production in cocultures of peritoneal cavity cells or spleen cells with
phytohemagglutanin stimulated PBL. In our clinical study, 26 x 10 CTL were infused in escalating divided
doses followed by four days of infusions of IL-2. Laboratory parameters monitored included HIV-specific CTL
responses in bulk and limiting dilution assays, cultures of plasma for HIV production, serum p24 levels,
lymphocyte subsets by FACS, and proliferative responses to mitogens and antigens.
Results: In hu-PBL-SCID mice, 9 of 12 controls became infected with HIV. One of 12 animals receiving
CTL showed evidence of infection. Nef-specific CTL activity was not detected 3 days after CTL transfer to
hu-PBL-SCID mice. In our clinical study, infusion of a CTL clone was well tolerated. No increase in HIVspecific CTL activity was observed after any of the cell infusions. Transient elevations in CD4' and CD8'
counts were seen during IL-2 infusion but returned to baseline in 2 weeks. No decreases in p24 antigen were
noted; modest decreases in HIV plasma titers were found during the two weeks following infusion of cells.
Conclusions: Adoptive transfer of HIV-specific CTL may be beneficial in preventing acute infections and may
transiently decrease virus load in HIV' individuals. Additional studies will need to determine if frequent of
administrations of CTL can achieve a sustained anti-viral effect.
Scott Koenig, M.D., Ph.D. 301-417-0770
MedImmune, Inc. 301417-6289 (fax)
35 West Watkins Mill Road
Gaithersburg, MD 20878
ThA 1575 LIMITED T CELL RECEPTOR GENE USAGE IN HIV-1 ENVELOPE SPECIFIC
CYTOTOXIC T LYMPHOCYTES FROM AN INFECTED INDIVIDUAL.
Kalams. Spyros*; Johnson R.P.*; Trocha A.*; Dynan M.J.*, Kurnick J.T.**;
Walker B.D.* *Infectious Disease Unit, Mass. General Hosp., Boston,
MA,USA. **Dept. of Pathology, Mass. General Hosp., Boston, MA, USA..
Obiective: To determine the T cell receptor (TCR) gene usage ofcytotoxic T lymphocytes (CTL) with
known epitope specificity and class I HLA restriction from an HIV-l infected individual.
Methods: Peripheral blood mononuclear cells obtained from a seropositive individual were cloned at
limiting dilution in the presence of IL-2, feeder cells, and a CD3-specific monoclonal antibody as a
stimulus to T cell proliferation and were never exposed to exogenous viral antigen in vitro. Clones
were tested for HIV- -specific cytotoxicity against EBV transformed autologous lymphoblasts
infected with recombinant vaccinia viruses expressing HIV-1 genes as well as a control vaccinia
virus. CTL epitopes were further defined using autologous EBV lymphoblasts incubated with
synthetic HIV-1 peptides. cDNA was prepared from approximately 5x106 cloned T cells. PCR was
then performed with 5' specific oligonucleotides from the variable regions of 22 different Va genes
and 20 different Vp genes in conjunction with a 3' primer from the constant region of the respective
TCR a or P gene. PCR products were reamplified and sequenced directly using the dideoxy chain
termination technique.
Results: Four distinct envelope-specific CTL clones from two different time points (20 months apart)
from the same individual were isolated. A nine amino acid peptide was identified as the minimal
epitope for these HLA B 14 restricted CTL clones. All four of these clones utilize Val4 and Vp4 TCR
genes. Sequencing of the Vp4 gene of two of these clones has revealed identical diversity and joining
regions, the regions thought to encode for an area of the TCR critical for binding to antigen/HLA
complex.
Conclusion: This study demonstrates that HIV-1 envelope-specific CTL from this individual of a
given epitope specificity and HLA restriction exhibit restricted T cell receptor usage, which is
maintained over time. Deciphering the role TCR gene rearrangement plays in the CTL recognition of
HIV-1 epitopes will aid in the understanding of the HIV-l-specific cell-mediated immune response.
Spyros Kalams, M.D. Infectious Disease Unit, Room 5234
Massachusetts General Hospital, 149 13th St., Charlestown, MA
02129, USA 617-726-5772, FAX [phone]
NOTES

ThA 1574 IDENTIFICATION OF CYTOTOXIC T LYMPHOCYTE EPITOPES AND
ANALYSIS OF SEQUENCE VARIATION IN A LABORATORY WORKER
INFECTED WITH HIV-1 IIIB.
Johnson. R. Paul*; Trocha, A*; Dynan, M*; D'Aquila, R*; Blattner, W**; Walker,
BD*. * Mass. General Hosp., Boston, MA, USA; **NIH, Bethesda, MD, USA.
Objectives: To identify cytotoxic T lymphocyte (CTL) epitopes using target cells expressing homologous
viral proteins and to analyze the extent of sequence variation in HIV-1 DNA coding for these CTL
epitopes.
Methods: Peripheral blood mononuclear cells (PBMC) were obtained from a laboratory worker infected
with HIV-1 IIIB. CTL clones were derived by a previously described technique and used as effector cells
in a standard chromium-release assay. Target cells consisted of EBV-transformed lymphoblasts infected
with recombinant vaccinia viruses expressing homologous HIV-1 proteins. Specific epitopes recognized
by these CTL clones were identified using target cells expressing serial truncations of HIV-1 proteins and
further defined using synthetic HIV-1 peptides to sensitize target cells. HIV-1 DNA coding for CTL
epitopes was amplified by nested PCR of limiting dilutions of lysates of unstimulated PBMC from this
subject Multiple clonal DNA fragments were sequenced directly at each time point.
Results: HLA class I-restricted CTL clones were isolated which recognized HIV-1 gag, nef, reverse
transcriptase and envelope proteins. Epitope mapping using recombinant vaccinia viruses and synthetic
peptides demonstrated CTL epitopes in a relatively conserved region of gp41 and in two different regions
of pl7. Two of these epitopes were further fine mapped to 9 amino acid minimum epitopes. Typespecific anti-HIV-1 envelope clones were also obtained which recognized an epitope contained in aminoacids 204 to 287 of gpl20. Sequencing of multiple clones of HIV-1 gag DNA demonstrated amino acid
variation in areas immediately adjacent to, and outside, known CTL epitopes.
Conclusions: Because the recombinant vaccinia viruses used in these experiments express HIV-1 proteins
which were derived from the IIB strain, analysis of CTL obtained from this laboratory worker represents
a unique opportunity to identify type-and group-specific CTL using targets expressing homologous viral
proteins, particularly for the envelope glycoprotein. Multiple CTL epitopes were identified, including
epitopes in variable as well as relatively conserved regions of HIV-1. Analysis of sequence diversity
revealed amino acid variation in regions not known to contain CTL epitopes and in a region immediately
outside a CTL epitope.
R. Paul Johnson, M.D. Infectious Disease Unit, Room 5234
Massachusetts General Hospital, 149 13th Street., Charlestown, MA
02129, USA. 617-726-5772, FAX [phone]
ThA 1576 CHARACTERISATION OF SEVERAL PROMISCUOUS CTL EPITOPES
IN THE C-TERMINAL REGION OF NEF. F.Hadida*, A. Samri*
A.Hosmalin* R.Spohn+ G.Jung+ P.Debr6* B.Autran*.
*Lab.Cell. Immunol, H6p.Piti6-Salpetriere, Paris,
France. + Inst.Org.Chem;Eber.Karls Univ. Tlbingen,FRG
Obiective: NEF is an early regulatory protein known for its high
immunogenicity. We have previously described an immunodominant region
for cytotoxic T lymphocytes (CTL) in its C-terminus (AA 182-206). The
aim of this study was to determine whether: 1/ this region contained
several CTL epitopes as predicted by theoretical models, 2/ these
epitopes were presented by several HLA molecules and therefore were
of interest at a population level.
Methods: HIV-specific CTL lines of sero+ patients were restimulated
in-vitro with a panel of 8 to 16 AA-long synthetic peptides and
lipopeptides (from ANRS and TUb. Univ.) corresponding to the HIV1-BRU
NEF: AA 182-206 region. Effector CTL from splenic lymphocytes of
sero+ patients were tested against autologous and HLA-matched EBV
cell lines coated with synthetic peptides.
Results: Several distinct peptides were specifically recognized by
CTL: AA 182-189, AA 186-193, AA 188-195, AA 192-199. Several HLAclass I molecules efficiently presented either identical or distinct
peptides: HLA Al, A10, A24, B5, B8, B35.
Conclusion: The C-terminal region of NEF (AA 182-206) contains
several T cell epitopes recognized by CTL, in agreement with 3
distinct predictive models. Several class-I MHC molecules are able to
present epitopes in this region suggesting: 1/ The existence of
promiscuous CTL epitopes in this region, which contains also B and T
helper cells epitopes; 2/ The interest of the COOH-terminus part of
NEF for induction of CTL responses within a broad spectrum of MHC
presenting molecules, in vaccine strategies.
F. Hadida, Laboratoire d'Immunologie Cellulaire, URA
CNRS 625, CERVI, H6pital Piti6-Salp4triere, 83, bld
de 1'H6pital 75013, Paris, France, Tel: [phone]; FAX: [phone].
NOTES

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