Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.     

Lack of antibodies to HTLV-II in patients with dementia in Italy

A study was conducted of the reactivity of 85 plasma samples from HIV-1 seropositive persons living in Brazil. Five HIV-1 plasma samples had synthetic peptides from the V3 region isolates (IIIB, MN, SC, WMJ-2, and RF). There was a low reactivity (59%) with HIV-1 isolate MN derived PND peptides, while most studies report a 90% or higher reactivity. Brazilian HIV-1 seropositive sera had lower reactivity with MN isolate PND peptides than sera from other countries. Researchers used 5 HIV-1 MN isolate PND peptides from other sources to analyze the same plasma to confirm the study's results. The HIV-1 MN isolate PND peptides had different amino acid sequences, but all had the GPGR top of the V3 loop. 52% of the Brazilian plasma recognized V3 peptides, 55% for 48 peptides, 56% for C52 peptides, and 49% for C53 pep tides. Only 27% of the Brazilian plasma recognized the C54 peptide. There was considerable overlapping reactivity, however. For example, 65% of the V3 MN reactive plasma also recognized the C52 MN peptide and the C53 MN peptide. The overlap between C52 MN and C53 MN was 69%. These findings suggest that, in the Brazilian study population, at least 2 different anti-MN-PND antibody specificities directed against amino acid sequences including the GPGR top of the V3 loop exist (an epitope including PNY[NKRKRIHIGPGR] and the epitope including [KRKRIHIGPGR]AFY). 86% of the plasma reacted with at least 20% of the MN PND peptides, which equals reactivities in other studies. 96% of the Brazilian plasma were positive for neutralizing antibodies based on overlap of peptide recognition and HIV-1 MN isolate neutralization (titers = 1:100-1:52,600). Recognition of the V3 loop of the HIV-1 MN isolate in sera from HIV-1 positive persons in Brazil has two different antibody specificities. Most of the Brazilian plasma recognize the N terminal arm, which consists of the GPGR top of the V3 loop.     

Immunogenicity of synthetic HIV-1 gp120 V3-loop peptide-conjugate immunogens

A successful prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine must elicit an immune response that will prevent establishment of the persistent viral infection. The only response shown to be effective in this regard is virus-neutralizing antibody directed against the viral gp120 hypervariable V3-loop region. Conjugate immunogens, containing cyclic peptides representing the V3 determinant covalently bound to a carrier protein, were capable of eliciting virus-neutralizing antibodies. The consistency of the response was related to peptide size. The smaller cyclic peptides, expressing relatively conserved sequences from the V3-loop apex, were poor inducers of neutralizing activity. In contrast, the largest cyclic peptides mediated neutralizing responses that were similar to those observed and previously reported for intact gp120 immunogens. A cyclic synthetic peptide expressing most of the prototypic HIV-1 MN variant V3 determinant warrants further study as a potentially effective vaccine immunogen.     

Interaction of human immunodeficiency virus type 1 envelope glycoprotein V3 loop with CCR5 and CD4 at the membrane of human primary macrophages

We show that infection of primary monocyte-derived macrophages (MDMs) and blood lymphocytes (PBLs) by human immunodeficiency virus type 1 (HIV-1) R5 strains, but not that of PBLs by X4 strain HIV-1LAI, is inhibited by beta-chemokines RANTES and MIP-1alpha. A biotinylated disulfide-bridged peptide mimicking the complete loop of clade B consensus V3 domain of gp120 (V3Cs), but not a biotinylated V3LAI peptide or a control beta-endorphin peptide of approximately the same molecular weight (MW), was found to bind specifically to MDM membrane proteins, in particular two proteins of 42 and 62 kDa migrating as sharp bands after electroblotting onto Immobilon, and this was specifically inhibited by anti-V3 antibodies. When biotinylated V3Cs was incubated with intact MDMs, which were then washed and lysed, and the resulting material was incubated with streptavidin-agarose beads and electroblotted onto Immobilon, fresh V3Cs also bound to proteins of the same molecular weight recovered in the V3Cs-interacting material. This binding was inhibited by anti-V3 antibodies, and no binding occurred with the control peptides. V3Cs also bound to soluble recombinant CD4, and CD4 monoclonal antibody Q4120 specifically recognized the V3Cs-interacting 62-kDa protein, which should thus correspond to CD4. Recombinant radiolabeled RANTES, MIP-1alpha, and MIP-1beta, but not IL-8, also bound to a 42-kDa protein on the membrane of MDMs as well as to the V3Cs-interacting 42-kDa protein, and excess unlabeled V3Cs inhibited such binding. This protein was also recognized by antibodies to CCR5, the RANTES/MIP-1alpha/MIP-1beta receptor. These data show that V3Cs binds to MDM membrane proteins that comprise CD4 and CCR5, and that multimolecular complexes involving at least gp120 V3, CD4, and CCR5 are formed on the surface of MDMs as part of V3-mediated postbinding events occurring during HIV-1 infection.     

Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.     

Crystal structure of the principal neutralization site of HIV-1

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.     

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.     

Inhibition of anti-V3 domain antibody binding to human immunodeficiency virus type-1-infected cells by sulfated polysaccharides

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is an immunodominant region. Anti-V3 domain antibodies neutralize both HIV-1 infection and syncytium formation. The V3 domain has a high density of positive charge which is a potential binding site for anti-HIV-1 sulfated polysaccharides. To investigate the inhibitory effect of sulfated polysaccharides on the binding of anti-V3 domain antibody, fluorescence-activating cell sorting analysis was performed using two kinds of antibodies, NEA9284 (purified, 0.25 micrograms/ml) and 0.5 beta (ascite, 2.0 mg/ml), and HIV-1-infected CEM cells. When the binding assay with a 1:100 dilution of each antibody was performed in the presence of dextran sulfate, heparin, and inositol hexasulfate at concentrations which are antiviral, the compounds did not inhibit the binding of either antibody. As the antibody concentration was decreased with higher dilution, dextran sulfate was able to reduce antibody binding by 50-60%. Thus, antagonism of anti-V3 domain antibody binding by sulfated polysaccharides is not as extensive as reported previously by several groups.     

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.     

Sonolucent peripancreatic masses: differential diagnosis and related imaging

The third variable region (V3) of the HIV-1 gp120 envelope molecule appears to represent a target for naturally occurring neutralizing antibodies in HIV-1-infected individuals. In this report, we examined the extent of antibody cross-reactivity to a panel of V3-based synthetic peptides in six inbred strains of mice following repeated immunization with a baculovirus-derived recombinant gp160 (rgp160) preparation formulated with alum. The amino acid sequence of the rgp160 used in these immunizations was based upon the HIV-1 IIIB (LAI) isolate. Following five injections with rgp160, all six strains developed antibodies to the homologous IIIB-based V3 peptides, designated 304-321 and RP135. However, antibody cross-reactivity to the other nonhomologous V3 peptides was either undetectable or limited among the strains of mice examined. No in vitro neutralizing activity against HIV-1 was observed in sera from any of the six inbred strains of mice that were examined. These results suggest that repeated immunization of mouse strains with a rgp160/alum formulation leads to nonneutralizing antibodies directed against the V3 region which remain predominantly type specific.     

Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA

The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.     

Kinetic constants of alpha-methyl-D-glucoside transport in the chick small intestine during perinatal development

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.     

gp 120s derived from four syncytium-inducing HIV-1 strains induce different patterns of CD4 association with lymphocyte surface molecules

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.     

Activation of interleukin 4- and interleukin 6-secreting cells by HIV-specific synthetic peptides

Peptides were synthesized in which the type-specific determinant of the V3 loop region of gp120 (SP10) was expressed C terminal to a conserved T helper epitope (T1) on the same molecule. These T1-SP10 peptides can stimulate both cell-mediated and humoral immune responses. The current work used a novel approach to study the nature and specificity of the response elicited by these peptides. Cytokine-specific ELIspot assays were used to examine the number, kinetics and fine specificity of cells induced to secrete IL-4 and IL-6 in mice immunized with T1-SP10 peptides. Results indicate that the peptides activated cytokine-secreting cells in a dose-dependent manner in vivo. In vitro restimulation experiments demonstrated that both the SP10 and T1 regions contributed to this activation. Consistent with previous studies, mice sequentially immunized with peptides expressing different V3 loop regions generated B cell responses that were larger and more cross-reactive than those induced by a single peptide. Sequential immunizations had less effect on the number or specificity of the cytokine-producing cells.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Study of the V3 loop as a target epitope for antibodies involved in the neutralization of primary isolates versus T-cell-line-adapted strains of human immunodeficiency virus type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIVMN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIVMN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Potent neutralization of primary HIV-1 isolates by antibodies directed against epitopes present in the V1/V2 domain of HIV-1 gp120

Although it is known that some human immune sera possess potent neutralizing activities for primary viruses, the identity of the target epitopes mediating this neutralization is unknown, and currently available immunogens have not been able to induce such activities. Using recombinant fusion glycoproteins expressing native V1/V2 domains of gp120 we have found that sera from a subset of HIV-1-infected humans contain antibodies that recognize broadly conserved V1/V2 epitopes. Such antibodies were isolated from one human serum by affinity chromatography on a column containing a V1/V2 fusion protein, and shown to efficiently neutralize several macrophage-tropic HIV-1 isolates. Rodents immunized with the purified V1/V2 fusion protein produced antibodies reactive with unrelated V1/V2 fusion proteins and with heterologous gp120s. V1/V2-specific immunoglobulins isolated from sera of these animals by affinity chromatography also possessed potent neutralization activity for several primary HIV-1 isolates. These results indicate that the V1/V2 domain of HIV-1 gp120 contains conserved epitopes that mediate potent neutralization of primary viruses, and suggest that subunit vaccines that efficiently induce such antibodies may provide protective humoral immunity against clinically relevant HIV-1 isolates.     

Immunization with human immunodeficiency virus type 1 rgp120W61D in QS21/MPL adjuvant primes T cell proliferation and C-C chemokine production to multiple epitopes within variable and conserved domains of gp120W61D

Human immunodeficiency virus type 1 (HIV-1) gp120W61D-specific T cell lines (TCL) were generated from an HIV-1-seronegative volunteer who received rgp120W61D in QS21/MPL adjuvant with emulsion. TCL were challenged with pools of consecutive, overlapping peptides spanning the gp120W61D sequence and then with the individual peptides of the immunostimulatory pool. T cell epitopes were found within both variable and conserved domains, and there was no evidence of a single immunodominant epitope. The two most frequently recognized peptides were located in the C1 domain and in the C-terminal region of the V3 loop. Several TCL were shown to recognize multiple peptides from nonoverlapping regions. Peptides from both conserved and variable domains were capable of inducing MIP-1alpha, MIP-1beta, and RANTES production. When tested against the equivalent peptide from the HIV-1IIIB sequence, however, TCL were able to tolerate only minor conserved changes in the amino acid sequence.     

Neutralization of human immunodeficiency virus type 1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity

We investigated the relative importance of binding site occupancy and epitope specificity in antibody neutralization of human immunodeficiency virus (HIV) type 1 (HIV-1). The neutralization of a T-cell-line-adapted HIV-1 isolate (MN) was analyzed with a number of monovalent recombinant Fab fragments (Fabs) and monoclonal antibodies with a range of specificities covering all confirmed gp120-specific neutralization epitopes. Binding of Fabs to recombinant monomeric gp120 was determined by surface plasmon resonance, and binding of Fabs and whole antibodies to functional oligomeric gp120 was determined by indirect immunofluorescence and flow cytometry on HIV-infected cells. An excellent correlation between neutralization and oligomeric gp120 binding was observed, and a lack of correlation with monomeric gp120 binding was confirmed. A similar degree of correlation was observed between oligomeric gp120 binding and neutralization with a T-cell-line-adapted HIV-1 molecular clone (Hx10). The ratios of oligomer binding/neutralization titer fell, in general, within a relatively narrow range for antibodies to different neutralization epitopes. These results suggest that the occupancy of binding sites on HIV-1 virions is the major factor in determining neutralization, irrespective of epitope specificity. Models to account for these observations are proposed.     

Recognition of a small number of diverse epitopes dominates the cytotoxic T lymphocytes response to HIV type 1 in an infected individual

Mitogen-activated T cell lines may be reproducibly used to identify relatively conserved HIV-1 epitopes that dominate CTL recognition of HIV-infected cells. Using a combination of nested truncations of HIV-vaccinia recombinants encoding HIV-1LAI Env and overlapping peptides that span the coding regions of the HIV-1 SF2 subclone of env, gag, nef, rev, and tat, we have mapped the immunodominant, relatively conserved CTL epitopes recognized by 25 HIV-seropositive individuals with CD4 counts between 100 and 500/mm3 and no history of AIDS opportunistic infection. We could characterize at least 1 peptide CTL epitope recognized by the T cell lines of 18 of 25 of the subjects; the T cell lines from 2 additional subjects recognized HIV-vaccinia presenting targets, but no dominant peptide epitope was identified. CTL epitopes were most frequently encoded by gag (recognized by 16 of 25 patient T cell lines), followed by nef and env (11 of 25 each), and the RT region of pol (9 of 25). Tat and Rev were rarely the sites of CTL epitopes. The identified epitopes occurred predominantly in relatively conserved regions of HIV-1. The mean number of HIV peptides identified at a single time for each cell line was 2.7 +/- 1.7. Although no single peptide dominated CTL recognition in more than four individuals, clusters of epitopes were found in the N-terminal region of gp160 and in two central regions of Nef. The dominant HIV-1 CTL epitopes in infected patients were not predictable on the basis of MHC expression and varied widely in an MHC-diverse population.