Gp41 Peptide Research Articles

Immunological monitoring of patients with melanoma after peptide vaccination using soluble peptide/HLA-A2 dimer complexes.

To facilitate the immunologic monitoring of a peptide vaccine trial, a novel, empty dimeric HLA-A2 molecule (A2 dimer) that could be loaded with peptides was produced. The dimer comprises the extracellular domain of HLA-A2 noncovalently linked to a fusion protein consisting of human beta2-microglobulin joined to the human IgG1 Fc domain. Peptide-loaded dimer complexes were used to assess the function of peptide-specific T cells. HLA-A2 gp100 peptide dimers stimulated interferon (IFN)-gamma production by the gp100-specific TIL-1520 cell line. Gp100/A2 dimer stimulation in combination with intracellular cytokine staining was used to analyze peptide-specific T-cell responses in patients with melanoma after vaccination with the modified gp100: 209-2M peptide in adjuvant. Titration analysis of the amount of peptide-loaded dimer required to stimulate gp100-specific T cells was used to estimate the functional avidity of effector/memory CD8+ T lymphocytes. The number of peptide-specific T cells detected in the peripheral blood of vaccinated patients using this assay was comparable to the number determined by staining with fluoresceinated gp100: 209-2M HLA-A2 tetramers. IFN-gamma production by T cells was comparable after stimulation with peptide-pulsed dimers, T2 cells, or autologous dendritic cells. Peptide-loaded A2 dimers could also be used directly to stimulate T cells in the ELISPOT assay.

Design and synthesis of alpha Gal-conjugated peptide T20 as novel antiviral agent for HIV-immunotargeting.

An efficient chemo-enzymatic synthesis of alpha Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized alpha Gal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide alpha Gal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic alpha Gal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.

Cutting edge: stimulation with the cognate self-antigen induces expression of the Ly49A receptor on self-reactive T cells which modulates their responsiveness.

NK cell self-tolerance is maintained by inhibitory receptors specific for MHC class I molecules. Inhibitory NK receptors are also expressed on memory CD8 T cells but their biological relevance on T cells is unclear. In this study, we describe the expression of the Ly49A receptor on a subset of autoreactive T cells which persist in mice double-transgenic for the lymphocytic choriomeningitis virus-derived peptide gp33 and a TCRalphabeta specific for the gp33. No Ly49A-expressing cells are found in TCRalphabeta single-transgenic mice, indicating that the presence of the autoantigen is required for Ly49A induction. Direct evidence for an Ag-specific initiation of Ly49A expression has been obtained in vitro after stimulation of autoreactive TCRalphabeta T cells with the cognate self-Ag. This expression of Ly49A substantially reduces Ag-specific activation of autoreactive T cells. These findings thus suggest that autoantigen-specific induction of inhibitory NK cell receptors on T cells may contribute to peripheral self-tolerance.

Immunogenicity of HIV-1LAI gp160 and env peptides in squirrel monkey Saimiri sciureus using alumine and experimental adjuvants.

Since the identification of the HIV virus, important advances have been achieved in the definition of potential subunit vaccines. We investigated the immunogenicity of a recombinant gp160 antigen and of two gp41 peptides from HIV-1LAI associated with seven different adjuvant formulations in squirrel monkeys. All animals were immunized twice with gp160 and then with a gp41 peptide using the same formulation. All adjuvants used led to a subsequent antibody response against gp160, and 55% of the animals immunized developed anti-gp160 antibodies that could neutralize the virus in vitro. Specific anti-gp41 antibody response was also observed. Results obtained underlined the key role of the adjuvant formulation in the antibody response against a given part of the immunogen, and indicate that such immunogenicity-related investigation can be carried out conveniently in the squirrel monkey Saimiri sciureus.

Cross-reactive antibodies between HIV-gp120 and platelet gpIIIa (CD61) in HIV-related immune thrombocytopenic purpura.

We have previously demonstrated that immune platelet destruction observed in an AIDS-free HIV-infected patient was associated with the presence of a cross-reactive antibody recognizing both HIV-glycoprotein (gp)120 and platelet gpIIIa (CD61). We have now investigated the presence of such antibodies in other HIV-infected patients, together with the molecular structure of the cross-reactive epitope. Platelet gpIIb/IIIa antibodies were characterized in sera from HIV-infected patients with immune thrombocytopenic purpura by means of an ELISA and a radio-immunoprecipitation procedure (RIP). The platelet antibodies were purified and tested for their ability to recognize HIV-gp. We also tried to characterize the antibody target epitope on HIV-gp120 using recombinant gp and synthetic peptides. IgG with anti-gpIIb/IIIa activity were detected, by means of an ELISA with purified gpIIb/IIIa, in 101/138 (73%) sera from HIV-infected patients with immune thrombocytopenic purpura. The platelet antibodies were purified from 23 sera by absorption/elution on purified immobilized platelet gpIIb/IIIa, and recognition of gpIIIa was confirmed in eight cases with a RIP. Furthermore, the presence of a cross-reactive antibody between HIV-gp120 and platelet gpIIIa was demonstrated in 18/18 patients (including the eight with a confirmed gpIIIa antibody) by the ability of the serum HIV-gp160/120 antibodies to bind to purified gpIIb/IIIa. The cross-reactive epitope was shown to be independent of the carbohydrate moieties of gp120, since deglycosylation of two recombinant (r)-gp120s did not abolish antibody binding. However, the antibody did not recognize synthetic gp120 peptides spanning 355 of the 516 amino acids of gp120, particularly the four regions exhibiting sequences of four or five consecutive amino acids that are identical between r-gp120 and gpIIIa. Our results thus support the hypothesis that the cross-reactive antibody recognizes the conformational structure of gp120. These results strongly suggest that molecular mimicry between HIV-gp120 and platelet gpIIIa may be important in the pathogenesis of immune thrombocytopenia in AIDS-free HIV-infected patients.

Activation of intraperitoneal T cells after stimulation with antigen specific major histocompatibility class (MHC) I tetramer

Introduction: Appropriate trafficking and activation of CD8+ T cells in the peritoneal cavity are essential in the response to a number of insults, including intra-abdominal sepsis and carcinomatosis. The dynamics of the CD8+ T cell response in the peritoneal cavity however, have not been well characterized. Antigen specific MHC I tetramers activate CD8+ T cells directly, without requiring antigen presenting cells, and have been used extensively to analyze CD8+ T cell function in vivo. In this study, we used antigen specific MHC class I tetramer to evaluate intraperitoneal T cell activation and trafficking of CD8+ T cells after adoptive transfer in vivo. Methods: We adoptively transferred (AT) CFSE labeled P14 T cells (TCR specific for LCMV peptide gp33) into the peritoneal cavity of C57BL/6 mice and stimulated them via intraperitoneal route (IP) with PE-labeled MHC I tetramer bound to gp33 or control peptide. At various time points, we harvested peritoneal washings and lymphoid organs and assessed AT lymphocytes by flow cytometry. Results: Five days following adoptive transfer, unstimulated AT cells still constituted over 50% of CD8+ T cells in the peritoneal cavity. Following intraperitoneal injection of antigen specific MHC 1 tetramer, the majority of AT T cells in the peritoneal cavity (compared to AT T cells in other lymphoid organs, such as the spleen) are bound to MHC I tetramer within 2 hours. Even 4 days after intraperitoneal MHC class I tetramer injection, dramatic increases in CD44 expression, representing activation, and vigorous CFSE dilution, representing proliferation, are observed in AT T cells in the peritoneal cavity compared to AT cells in other lymphoid organs. In addition, 50% of the AT T cells activated with MHC class I tetramer, regardless of location, are functionally active, producing interferon gamma, and cytotoxic T lymphocyte activity is detected. Conclusions: This study demonstrates the unique activation, proliferation, and effector phenotype profile of CD8+ T cells following adoptive transfer into the peritoneal cavity and stimulation with antigen specific MHC I tetramer. Further investigation of the intraperitoneal CD8+ T cell response to infection and malignancy in vivo is warranted.

High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens.

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.

Cytotoxic T lymphocyte reactivity to gp100, MelanA/MART-1, and tyrosinase, in HLA-A2-positive vitiligo patients.

Vitiligo is a common depigmentation disorder thought to result from autoimmune destruction of melanocytes. Recent studies suggest a role for cell-mediated immune responses to melanocyte differentiation antigens, including gp100, MelanA/MART-1, and tyrosinase, in vitiligo pathogenesis. This study investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in HLA-A2-positive patients with vitiligo. Melanocyte-specific T cell responses were measured ex vivo via enzyme-linked immunospot assay following stimulation with MelanA/MART-1, tyrosinase, and modified gp100 epitopes. Antigen-specific T lymphocyte reactivity to gp100 peptides was seen in 15 of 17 (88%) patients, with many demonstrating very high reactivity at levels comparable with those observed with common recall antigens. Reactivity to gp100 was noted to be associated with disease activity. Antigen-specific T lymphocyte reactivity to MelanA/MART-1 and tyrosinase peptides was not observed ex vivo in our patients, and only one patient demonstrated responses to MelanA/MART-1 and tyrosinase peptides following in vitro re-stimulation. Our findings implicate T cell reactivity to gp100 in patients with active disease and support the concept of an immunopathologic mechanism in vitiligo, in which cell-mediated responses to normal melanocyte antigens play a crucial part.

Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain . Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601–613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400–429 of human T cell lymphotrophic virus-I (HTLV-I), β-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.

The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20.

A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. One of them, T20 (aa 638-673), was found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (HR1) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from HR1, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action consistent with a previous fluorescence study. It was also found that peptides capable of solubilizing T20 dispersion have a high content of helix, as well as beta-strand, conformation in aqueous solution. Specificity of T20/HR1-derived peptide binding was ascertained by using a scrambled sequence of a T20-active peptide and a plateau in scattering reduction of T20 suspension with variation in the concentration of a T20-active HR1 peptide. Implications on the mechanism of T20 inhibition and the sequence of folding of the gp41 core structure are discussed.

Degradation of the immunogenic peptide gp100 280–288 by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100 280–288 (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100 280–288 is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100 280–288 severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.

Functional characterization of CTL against gp100 altered peptide ligands.

In this study, four modified gp100 peptides were designed by combining amino acids from the melanoma peptide antigen gp100((209-217)) with preferred primary and auxiliary HLA-A *0201 anchor residues previously identified from combinatorial peptide library screening with recombinant HLA-A*0201. These modified peptides demonstrated stronger binding affinity for the HLA-A*0201 molecule compared to wild-type gp100 peptide. Nine CTL lines generated from patients immunized with the g209-2 M peptide and one CTL line from a non-immunized patient were tested for the ability to respond to these modified gp100 peptides. Stimulation of CTL by two of four modified peptides induced higher levels of IFN-gamma secretion than the wild-type gp100 peptide, demonstrating that higher peptide binding affinity for HLA molecules does not necessarily equate to functional activity of CTL. Two major and one minor CTL recognition pattern were observed, irrespective of previous peptide immunization, suggesting that multiple, rationally designed modified tumor peptides for the same epitope stimulate a broad CTL response by activating multiple CTL capable of cross-reacting with the natural antigenic peptide.

Comparing modified and plain peptide linked enzyme immunosorbent assay (ELISA) for detection of human immunodeficiency virus type-1 (HIV-1) and type-2 (HIV-2) antibodies

Serological diagnosis of human immunodeficiency virus (HIV) based on detection of HIV antibodies is one of the easiest, cheapest and simplest assay. Synthetic peptides corresponding to immunodominant regions of envelope glycoprotein (gp41, V3 loop for HIV-1 and gp36 for HIV-2) were used in the present study, to detect the anti-HIV antibodies in sera of Sexually Transmitted Diseases (STD), Tuberculosis (TB), Anti-Natal Care (ANC) patients. About 550 serum samples were tested using Enzyme Linked Immunosorbent Assay (ELISA) technique. The human sera positive for antibody to HIV-1 and HIV-2, reacted to different degrees with these peptides when used as a plain peptide with or without CGG motif/biotin motif at the amino terminus. The selected sequences are of Indian strain with ‘C’ serotype. The results showed a 100% sensitivity and specificity for V3 loop peptide and 98% sensitivity and specificity for gp41 peptide containing CGG moiety while the plain peptides showed similar sensitivities but low specificity's, i.e. 98% for V3 loop peptide and 42% for gp41 peptide when reacted with HIV-1 positive sera. The presence of biotin at the amino terminus did not provide any beneficial effect in increasing the sensitivity although the specificity was enhanced for both the peptide sequences, i.e. gp41 and V3 loop peptide. Furthermore, the gp36 peptide containing CGG moiety detected the HIV-2 sera with 100% sensitivity and 98% specificity while the sensitivity and specificity of gp36 plain peptide was reduced to 98 and 90%. Thus the study overall highlighted the importance of synthetic peptides containing CGG moiety as a capture antigen in detecting both HIV-1 & 2 sera using an indigenously built ELISA system which is simple, cheap, sensitive and cost effective for rural areas.

Generating neutralizing antibodies, Th1 response and MHC non restricted immunogenicity of HIV-I env and gag peptides in liposomes and ISCOMs with in-built adjuvanticity

For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2d, H-2b, H-2k and H-2q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-γ and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development.

Cell-Surface-Expressed HIV-1 Envelope Induces the Death of CD4 T Cells during GP41-Mediated Hemifusion-like Events

Cells expressing the HIV-1 envelope glycoprotein complex (gp120/gp41, Env) induce the death of target cells either after cell-to-cell fusion or after cell-to-cell contact in a fusion-independent fashion. Here, we demonstrate that Env-induced death of single cells (including primary CD4 T cells) required gp120 and gp41 function. The gp41 peptide C34, which blocked syncytium formation, completely inhibited the death of single target cells by specifically acting on gp41 function. Moreover, Env-induced single cell death was exclusively observed in CD4 cells and was associated with specific gp41-mediated transfer of lipids from the membrane of Env-expressing cells to the target cell but not with detectable cytoplasm mixing (complete fusion). We conclude that after gp120 function, gp41 mediates close cell-to-cell contacts, thereby triggering cell death in single uninfected cells in the absence of detectable cell-to-cell fusion.

Synthesis of maleimide-activated carbohydrates as chemoselective tags for site-specific glycosylation of peptides and proteins.

An array of maleimide-activated mono- and oligosaccharides were synthesized to permit site-specific glycosylation of cysteine-containing peptides and proteins. Maleimide-activated monosaccharides, in which the native alpha- or beta-O-glycosidic linkages found for nonreducing terminal sugars of native glycoproteins are preserved, were prepared using 2'-aminoethyl glycosides as the key intermediates. In addition, a native high-mannose type oligosaccharide, Man(9)GlcNAc(2)Asn, was converted into its maleimide-activated form by taking advantage of the existing amino group in the Asn portion. The application of these maleimide-activated carbohydrates was exemplified by the site-specific glycosylation of a 36-mer HIV-1 gp41 peptide, T20, which is a potent inhibitor against HIV infection. The chemoselective ligation was found to be rapid, highly efficient, and essentially quantitative. Tagging the biologically active peptide with a mannose and/or oligomannose moiety will be useful for targeting the drug to macrophage and dendritic cells, which are primary targets for HIV-1 infection and are expressing mannose- and oligomanose-specific receptors on their surface. In combination with site-specific mutagenesis, the maleimide-activated carbohydrates can serve as generally applicable tags for site-specific glycosylation of proteins via the highly efficient maleimide-thiol ligation reaction.

Conformational Partitioning of the Fusion Peptide of HIV-1 gp41 and Its Structural Analogs in Bilayer Membranes

Experiments have shown that the ability of the HIV-1 virus to infect cells can be greatly diminished by deactivation of the N-terminal (fusion) peptide of its glycoprotein gp41. Deactivation can be achieved by the deletion of several amino acid residues, or replacement of a hydrophobic residue with a polar residue, to form mutant variants of the wild-type peptide. We report Monte Carlo simulation studies of a simplified peptide/membrane model, representing the interaction of an HIV-1 fusion peptide (FP) and four closely related mutagens with a lipid bilayer. In agreement with experimental results, we show that FP inserts deeply into the bilayer at ∼ 40° to the bilayer normal. We also show a previously unreported behavior of membrane peptides, namely their equilibrium partitioning between several distinct conformations within the bilayer. We quantify this partitioning behavior and characterize each conformation in terms of its geometry, energy, and entropy. The diminished ability of FP mutagens to hemolyse and aggregate red blood cells due to their partitioning into unfavorable conformations, is also discussed. Our analysis supports a negative curvature mechanism for red blood cell hemolysis by FP. We also suggest that the small repulsive forces between surface-adsorbed peptides in opposing membrane surfaces may block aggregation.

HIV-1 gp120 proteins and gp160 peptides are toxic to brain endothelial cells and neurons: possible pathway for HIV entry into the brain and HIV-associated dementia.

Breakdown of the blood-brain barrier is commonly seen in patients with human immunodeficiency virus (HIV)-associated dementia, despite the lack of productive HIV-infection of the brain endothelium. Through this damaged blood-brain barrier, HIV and HIV-infected monocytes/macrophages infiltrate the brain and further infect microglia and brain macrophages. Neuronal cell death and dysfunction are the underlying cause of HIV-associated dementia, but no productive HIV-infection of neurons has been documented. It is likely that secreted viral products play a major role in blood-brain barrier damage and neuronal cell death. The aim of the present study was to examine the effect of HIV-1 gp160 peptides and gp120 proteins on brain microvascular endothelial cells and neurons from both human and rats. Four of the 7 gp160 peptides tested evoked significant neurotoxicity. Two different full-length recombinant HIV gp120 proteins (HIV-1CM235 gp120 and HIV-1MN gp120) also induced neuronal and brain endothelial cell death, and concentrations as little as 1 ng/ml evoked pronounced morphological changes in these cells and marked cytotoxicity. This study suggests that HIV proteins and peptides that are shed in vivo may be directly involved in blood-brain barrier damage and neuronal cell death in HIV-associated dementia.

Carbohydrate-centered maleimide cluster as a new type of templates for multivalent peptide assembling: synthesis of multivalent HIV-1 gp41 peptides

This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.

Serum IgA of HIV-exposed uninfected individuals inhibit HIV through recognition of a region within the alpha-helix of gp41.

HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro. To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals. Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication. IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the alpha-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5. These results have important implications for the development of vaccines and therapeutical strategies against HIV infection.