Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 "tier 2" viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.     

Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gp160 humoral immune response in HIV type 1-infected chimpanzees

The fine specificities of antibodies produced against human immunodeficiency virus type 1 (HIV-1) gp160 were examined in sera from 23 HIV-1-infected chimpanzees. These animals had been infected with one of six isolates of HIV-1. Sera were screened by enzyme-linked immunosorbent assay for reactivity against seven synthetic peptides corresponding to regions of gp160. Chimpanzees appear to remain healthy after infection with HIV-1, suggesting that these animals may prevent extensive spread of the virus in vivo through immunologic mechanisms. Antibody specificity to gp160 epitopes may play a key role in the defense against HIV-1-related disease. Approximately one-half of all chimpanzee sera contained antibodies reactive with peptide 846-860, which corresponds to the carboxyl terminus of gp41. Less than 10% of sera from HIV-1-infected humans that were examined contained antibodies reactive with peptide 846-860, suggesting that this region is not highly immunogenic in humans. Of the human sera containing antibodies reactive with this peptide, all were from individuals classified as Walter Reed stages 1 to 3. No sera from humans with advanced stages of the disease contained antibodies reactive with peptide 846-860. Peptide 600-611, which reportedly reacts with nearly all sera from HIV-infected humans, was reactive with less than one-half of sera from HIV-1-infected chimpanzees. The observed differences in antibody reactivity to gp160 peptides in sera from HIV-1-infected chimpanzees and humans suggest that each may generate antibodies against differing sets of HIV-1 epitopes. These differences may contribute to the lack of disease progression in chimpanzees after infection with HIV-1.     

Antigenic activity of three chimeric synthetic peptides of the transmembrane (gp41) and the envelope (gp120) glycoproteins of HIV-1 virus

The antigenicity of three chimeric synthetic peptides (Qm, Qm-16, and Qm-17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587-617) and the different epitopes of the gp120 envelope protein (495-516), (301-335), (502-516) of human immunodeficiency virus (HIV-1), separated by two glycine residues, was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HIV-1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti-HIV-2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C-terminal region of HIV-1 gp120 (495-516) combined with gp41 (587-617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV-1 virus.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.     

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.     

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.     

Characterization of monoclonal antibodies against the human immunodeficiency virus matrix protein, p17gag: identification of epitopes exposed at the surfaces of infected cells.

Eight monoclonal antibodies reactive with the matrix protein of human immunodeficiency virus type 1 (HIV-1), p17gag, were isolated from rats which had been immunized with solubilized HIV-1 lysate. The epitope specificities of these antibodies were determined with a series of synthetic peptides representing overlapping regions of p17. Six of the antibodies were mapped to three distinct regions of p17, while two antibodies (G11g1 and G11h3) reacted only with intact recombinant p17, suggesting that they were directed against conformational or discontinuous epitopes. All the antibodies bound to HIV-infected cells which had been permeabilized with acetone, but only G11g1 and G11h3 reacted with live HIV-infected cells. Specificity studies with diverse virus strains demonstrated that these two antibodies recognized distinct epitopes, one which was group specific for HIV-1, and one which was shared with HIV type 2 and simian immunodeficiency virus. Binding competition studies indicated that these epitopes were proximal in native p17. Despite their reactivity with intact cells, these two antibodies did not possess appreciable virus-neutralizing activity. These results indicate that a form of p17 is expressed on the surfaces of live HIV-infected cells which is accessible to some, but not all, antibodies against p17. These cell surface molecules may play a role in the generation of antibodies against p17gag that are characteristic of early stages of HIV infection, and they may act as natural targets for the immune system and as potential targets for immunotherapy of HIV-infected cells.     

Evaluation of the potency of the anti-idiotypic antibody Ab2/3H6 mimicking gp41 as an HIV-1 vaccine in a rabbit prime/boost study

The HIV-1 envelope protein harbors several conserved epitopes that are recognized by broadly neutralizing antibodies. One of these neutralizing sites, the MPER region of gp41, is targeted by one of the most potent and broadly neutralizing monoclonal antibody, 2F5. Different vaccination strategies and a lot of efforts have been undertaken to induce MPER neutralizing antibodies but little success has been achieved so far. We tried to consider the alternative anti-idiotypic vaccination approach for induction of 2F5-like antibodies. The previously developed and characterized anti-idiotypic antibody Ab2/3H6 was expressed as antibody fragment fusion protein with C-terminally attached immune-modulators and used for immunization of rabbits to induce antibodies specific for HIV-1. Only those rabbits immunized with immunogens fused with the immune-modulators developed HIV-1 specific antibodies. Anti-anti-idiotypic antibodies were affinity purified using a two-step affinity purification protocol which revealed that only little amount of the total rabbit IgG fraction contained HIV-1 specific antibodies. The characterization of the induced anti-anti-idiotypic antibodies showed specificity for the linear epitope of 2F5 GGGELDKWASL and the HIV-1 envelope protein gp140. Despite specificity for the linear epitope and the truncated HIV-1 envelope protein these antibodies were not able to exhibit virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine.     

Expression,purification, and characterization of an anti-RBCFab-p24 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood

A fusion protein A41Fabp246 consisting of Fab of an anti-RBC MAb A41 and amino acids 8-153 of HIV-1 p24 (p246) fused at the C-terminus of A41Fd was purified following assembly of A41Fdp246 with A41 LC in vitro, using a denaturation- renaturation protocol and a 4-step column chromatography procedure. The highly purified, monomeric A41Fabp246 was then evaluated for hemagglutination-based detection of anti-p24 antibodies using sera from HIV-infected individuals. This derivative of p24 is devoid of maximum homology region and the C-terminal domain of p24, which is responsible for oligomerization of p24, but retains full complement of immunodominant epitopes. This new fusion protein in combination with fusion proteins consisting of monovalent fragment of another anti-human RBC antibody fused to immunodominant regions of envelope glycoproteins of HIV-1 and HIV-2 should be useful in preparing a cocktail of reagents for highly sensitive detection of anti-HIV antibodies in whole blood.     

Epitopes on proliferating cell nuclear antigen recognized by human lupus autoantibody and murine monoclonal antibody

The immune epitopes of proliferating cell nuclear antigen (PCNA), also called cyclin, were analyzed by determining the reactivity between PCNA peptide fragments and anti-PCNA antibodies from lupus patients, murine monoclonal antibody (19A2), and rabbit anti-NH2-terminal peptide antibody. Limited digestion of PCNA/cyclin with Staphylococcus aureus V8 protease resulted in several peptide fragments. Five fragments of 30, 20, 15, 14, and 13 kDa were reactive with rabbit anti-NH2-terminal peptide antibody denoting that they contained the NH2-terminal peptide. The 30- and 20-kDa fragments reacted with 19A2 but the others did not. Lupus sera reacted with 17- and 15-kDa peptide fragments allowing their classification into three groups. Two of eight sera (type A) reacted only with the 17-kDa fragment. Two others (type B) reacted with both the 17- and 15-kDa fragments and the remaining four sera (type C) reacted only with the 15-kDa fragment. The sera reacting with the 15-kDa fragment also reacted with the 20-kDa fragment, but the sera reactive only with the 17-kDa fragment did not, indicating that the 17-kDa fragment was not a degradation product of 20-kDa fragments. The 19A2 epitope resided in the region between 15 and 20 kDa from the NH2 terminus, whereas there was at least one distinct epitope on each 15- and 17-kDa peptide, which were recognized by lupus autoantibodies.     

Candidate multi-epitope vaccines in aluminium adjuvant induce high levels of antibodies with predefined multi-epitope specificity against HIV-1

Some neutralizing epitopes on HIV-1 envelope proteins were identified to induce antibodies which could effectively inhibit the infection of different strains in vitro. But only very low levels of these antibodies were determined in the HIV-1 infected individuals. To increase the levels of protective antibodies in vivo, we suggested multi-epitope vaccine as a new strategy to induce high level of neutralization antibodies with predefined multi-epitope specificity. A synthesized epitope peptide MP (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) containing three neutralizing epitopes (GPGRAFY, ELDKWA, RILAVERYLKD) was conjugated to carrier protein KLH, and then used for immunization in mouse together with aluminium adjuvant or Freund's adjuvant (FA). The candidate MP-KLH multi-epitope vaccine in aluminium adjuvant could induce antibody response very strongly to the epitope peptide C-(RILAVERYLKD-G)2 and the immunosuppressive peptide (P1) (LQARILAVERYLKDQQL) (antibody titer: 1:51200), strongly to the epitope peptide C-(ELDKWA-G)4 and the C-domain peptide (P2) (1:12800), and moderately to the epitope peptide C-(GPGRAFY)4 and the V3 loop peptide (1:1600). The immunoblotting analysis demonstrated that the antibodies in sera could recognize P1, P2, V3 loop peptides and rsgp41 (aa 539-684). These results are similar with that in the case of PI-BSA in FA, and suggest that the multi-epitope vaccine in aluminium could induce high levels of antibodies of predefined multi-epitope specificity, which provides experimental evidence for the new strategy to develop an effective neutralizing antibody-based multi-epitope vaccine against HIV-1.     

Continuous epitopes of the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates.

Immunoreactive regions of human immunodeficiency virus type 1 (HIV-1) gp41 were mapped by reacting HIV-1 antibody-positive human sera with overlapping synthetic peptides which covered the transmembrane protein. Three immunoreactive domains were identified, and five different and partially overlapping epitopes recognized by HIV-1-positive human sera were found within one immunodominant region. The effect on antibody recognition after single amino acid substitutions within one defined epitope was also studied. The reactivity of various HIV-1-positive sera to synthetic peptides with amino acid substitutions representing known isolates suggests an important substitution in the major epitope of African HIV-1 strains.     

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.     

Murine monoclonal antibodies biologically active against the amino region of HIV-1 gp120: isolation and characterization

The human immunodeficiency virus (HIV)-1 envelope glycoprotein is synthesized as a precursor (gp160) and subsequently cleaved to generate the external gp120 and transmembrane gp41 glycoproteins. Both gp120 and gp41 have been demonstrated to mediate critical functions of HIV, including viral attachment and fusion with the cell membrane. The antigenic variability of the HIV-1 envelope glycoprotein has presented a significant problem in the design of appropriate and successful vaccines and offers one explanation for the ability of HIV to evade immune surveillance. Therefore, the development and characterization of functional antibodies against conserved regions of the envelope glycoprotein is needed. Because of this need, we generated a panel of murine monoclonal antibodies (MuMabs) against the HIV-1 envelope glycoprotein. To accomplish this, we immunized Balb/C mice with a recombinant glycoprotein 160 (gp160) that was synthesized in a baculovirus expression system. From the growth-positive hybridomas, three MuMabs were generated that demonstrated significant reactivity with recombinant gp120 but failed to show reactivity against HIV-1 gp41, as determined by enzyme-linked immunosorbent assay (ELISA). Using vaccinia constructs that synthesize variant truncated subunits of gp160, we were able to map reactivity of all three of the Mabs (ID6, AC4, and AD3) to the first 204 residues of gp120 (i.e., the N terminus of gp120) via Western blot analysis. Elucidation of the epitopes for these Mabs may have important implications for inhibition of infection by HIV-1. Our initial attempts to map these Mabs with linear epitopes have not elucidated a specific antigenic determinant; however, several physical characteristics have been determined that suggest a continuous surface epitope. Although these antibodies failed to neutralize cell-free or cell-associated infection by HIV-1, they did mediate significant antibody-dependent cellular cytotoxicity (ADCC) activity, indicating potential therapeutic utility. In summary, these data suggest the identification of a potentially novel site in the first 200 aa of gp120 that mediates ADCC.     

Characterization of the Borna disease virus phosphoprotein, p23.

Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.     

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).     

Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2.

Nine murine monoclonal antibodies directed to the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) were obtained and then tested by using an epitope mapping system (Pepscan) covering the whole p24HIV1 protein to characterize antigenic domains. Four different linear epitopes were identified. Monoclonal antibodies recognizing three of these epitopes also reacted to p26HIV2 in Western blotting (immunoblotting). A monoclonal antibody specific for the fourth epitope, located at position 179 to 188 of the gag polyprotein p55HIV1 (human T-cell lymphotropic virus type 3B strain), did not react with HIV type 2 (HIV-2) core proteins. The corresponding sequence is constant in all known HIV-2 and simian immunodeficiency virus (SIV) isolates, including a very divergent SIV strain from African green monkeys (SIVagm/tyo). This observation may be relevant to the phylogeny of primate lentiviruses. Two of the conserved epitopes might be immunogenic during natural infection and could therefore be used for diagnosis and prognosis purposes. These two epitopes are AAEWDRVHP and EIYKRWII, starting at positions 209 and 260 of the polyprotein p55HIV1, respectively.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.