Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.     

Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.     

Antibodies from HIV-positive and AIDS patients bind to an HIV envelope multivalent vaccine

A major problem impeding development of an effective HIV vaccine is the rapid antigenic variability that is characteristic of several envelope glycoprotein epitopes. Frequent mutations alter the composition of the most immunogenic regions of the envelope glycoprotein. We have prepared a synthetic immunogen representing the evolution of the major hypervariable epitopes on the envelope glycoprotein (gp120) of HIV-1. Five synthetic constructs, representing each of the HIV-1 gp120 hypervariable epitopes were tested for recognition by antibodies from patients infected with HIV-1 from different geographic regions worldwide. An HIV-1 human plasma panel provided a representation of the antibodies recognizing subtype-specific epitope sequences prevalent at different parts of the world. The vaccine construct was recognized by antibodies from HIV-1-positive individuals infected with subtypes A, B, C, D, E, and F. Antibodies in pooled HIV-1 patient sera from San Francisco also recognized all five constructs. This complex immunogen was recognized by antibodies in sera from individual HIV-1-positive and AIDS patients from Puerto Rico and Canada, with a strong binding to the complete vaccine and the V3 component. Altogether, our results demonstrate that antibodies from seropositive patients infected with different HIV-1 clades recognize and bind to the HIV hypervariable epitope construct vaccine preparation and its individual components.     

Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies

It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.     

Characterization of a novel human immunodeficiency virus type 1 neutralizable epitope within the immunodominant region of gp41

Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.     

Selective recognition of oligomeric HIV-1 primary isolate envelope glycoproteins by potently neutralizing ligands requires efficient precursor cleavage

A critical component of an effective HIV vaccine will be the induction of broadly neutralizing antibodies. Comprising the HIV spike, the exterior envelope glycoprotein gp120 and the transmembrane glycoprotein gp41 mediate receptor binding, viral entry, and are the targets for neutralizing antibodies. The gp120 and gp41 glycoproteins are derived from the gp160 precursor glycoprotein and following gp160 glycosylation, oligomerization and cleavage in the endoplasmic reticulum and Golgi, remain as non-covalently associated trimers of heterodimers. Previously, using cell-surface envelope glycoproteins derived from infection of a laboratory-adapted HIV-1 strain, a correlation had been established between the binding of gp120-directed antibodies to the viral glycoprotein and the ability of the antibodies to neutralize laboratory-adapted isolates. However, this has been more difficult to demonstrate for glycoproteins derived from primary patient isolates. Here, using a FACS-based method, we report that only gp120-directed neutralizing antibodies and the neutralizing ligand soluble CD4 efficiently bind to glycoproteins derived from the JR-FL primary isolate provided that the gp160 precursor protein is efficiently cleaved. Precursor cleavage was demonstrated by cell-surface biotinylation and Western blotting. In stark contrast, both non-neutralizing and neutralizing antibodies bind non-cleaved envelope glycoproteins from JR-FL and YU2 isolates. These data imply that significant changes in Env spike structure are dependent upon precursor gp160 cleavage and are consistent with a restricted-binding-to-Env model of neutralization. The data also have implications in regards to the use and design of non-cleaved envelope glycoprotein trimeric immunogens as a means to selectively and preferentially present neutralizing epitopes to the host immune system.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.     

Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.     

Specific amino acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency virus type 1

The HIV-1 envelope glycoprotein is expressed on the viral membrane as a trimeric complex, formed by three gp120 surface glycoproteins non-covalently associated with three membrane-anchored gp41 subunits. The labile nature of the association between gp120 and gp41 hinders the expression of soluble, fully cleaved, trimeric gp140 proteins for structural and immunization studies. Disruption of the primary cleavage site within gp160 allows the production of stable gp140 trimers, but cleavage-defective trimers are antigenically dissimilar from their cleaved counterparts. Soluble, stabilized, proteolytically cleaved, trimeric gp140 proteins can be generated by engineering an intermolecular disulfide bond between gp120 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP). We have found that SOSIP gp140 proteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared to a prototypic strain (JR-FL, subtype B). We now show that the determinants of this enhanced stability are located in the N-terminal region of KNH11144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability. The stabilized trimers retain the epitopes for several neutralizing antibodies (b12, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. The ability to increase the stability of gp140 trimers might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.     

Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates

The HIV-1 gp120 V3 loop is a potent inducer of neutralizing antibodies for T cell line adapted-HIV-1, but less so for primary isolates. We hypothesized that peptides representative of the diversity of natural HIV-1 V3 loop variants might capture elements of conserved higher order structures and so stimulate broadly reactive neutralizing antibodies. We designed a panel of 29 subtype B V3 sequences postulated to reflect the range of V3 diversity. These peptides were used to immunize guinea pigs. The most effective peptide (62.19) clustered around the subtype B consensus sequence and induced antibodies that reproducibly neutralized 31% of the subtype B HIV-1 primary isolates evaluated, but exhibited limited cross-neutralization of non-subtype B HIV-1 strains. Taken together, these data demonstrated that the limited neutralization profile of antibodies induced by optimal subtype B V3 motifs likely represents the maximum breadth of neutralization of subtype B HIV-1 primary isolates attainable by anti-V3 peptide antibodies.     

CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins.

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.     

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.     

Fine specificity of the humoral immune response to HIV-1 GP160 in HIV-1 infected individuals from Tanzania.

A total of 160 sera from HIV-1 infected individuals from Tanzania were examined for their fine specificity characteristics relative to 9 synthetic peptides that define HIV-1 gp160 epitopes. Immunorecessive and immunodominant epitopes were identified in both gp120 and gp41 based on serologic reactivity of these HIV-1 infected sera. A significant difference in fine specificity among HIV-1 infected individuals from Tanzania and the United States was observed for an immunodominant gp41 epitope. No significant differences in reactivity among asymptomatic vs. symptomatic HIV-1 infected individuals were detected for the selected HIV-1 gp160 epitopes defined by these peptides. The majority of sera from HIV-1 infected Tanzanians contained antibodies that recognized a peptide corresponding to the V3 region of gp120 from the HIV-1 MN isolate. These data suggest that regional isolates of HIV-1 may exist in Tanzania that differ from HIV-1 isolated in the United States. However, based on serology, HIV-1 isolates exhibiting sequences with HIV-1 MN V3 similarity may also be prevalent in Tanzania. The results of this study may be useful for the design of more effective AIDS diagnostic and therapeutic products for use worldwide.     

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins

To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were "oligomer sensitive," whereas mAbs to V2 and the distal epitope of C5 tended to be "monomer sensitive" (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.     

Selection of a novel gp41-specific HIV-1 neutralizing human antibody by competitive antigen panning

The HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against Env-based antigens has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a novel gp41-specific human monoclonal antibody (hmAb), m48, from an immune library derived from long-term nonprogressors with high titers of broadly cross-reactive neutralizing antibodies (bcnAbs). Selection of m48 was only successful using CAP and not through the conventional pre-incubation methodology. In assays based on spreading infection in peripheral blood mononuclear cells (PBMCs) m48 neutralized a panel of HIV-1 primary isolates from different clades more potently than the well-characterized broadly cross-reactive HIV-1-neutralizing antibodies IgG1 4E10 and Fab Z13. These results may have implications for the selection of novel gp41-specific bcnAbs and other antibodies, and for the development of HIV-1 inhibitors and vaccine immunogens.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

Induction of high levels of antibodies recognizing the neutralizing epitope ELDKWA and the D- or K-position-mutated epitopes by candidate epitope vaccines against HIV-1

Monoclonal antibody 2F5 recognizing the ELDKWA epitope on HIV-1 gp41 has a significant neutralization potency against 90% of the investigated viruses of African, Asian, American, and European strains, but the antibody responses to the epitope 2F5 in HIV-1-infected individuals were very low. We attempted to induce high levels of epitope-specific antibodies to ELDKWA and its three mutated epitopes by candidate epitope vaccines. The four candidate epitope vaccines all induced strong antibody responses at dilutions from about 1:6,400 to 1:25,600. We tested the cross-reactions between these antisera and four epitope peptides. The ELDKWA-specific antisera showed strong cross-reactivity with three neutralizing-resistant mutated epitopes which contain changes in the D or K positions of the epitope sequence. Virus variants containing these changes could escape neutralization by monoclonal antibody 2F5. In immunoblotting analysis, the ELDKWA, ELDEWA, and ELEKWA epitope specific antibodies all recognized rsgp41 which confirms that the antibodies against both mutated epitopes, ELDEWA and ELEKWA, could cross-react with the native epitope on rsgp41. Although it is not clear whether the polyclonal antibodies induced by the ELDKWA epitope vaccine could neutralize the mutated viruses containing these mutated epitopes, it is conceivable that epitope vaccines based on mutated epitopes could induce strong antibody responses with predefined epitope specificity to neutralize mutated viruse containing the mutated epitope. An epitope vaccine, using different epitopes including mutated epitopes, could provide a new concept for developing a new vaccine against HIV-1.     

Monoclonal antibodies to an HIV-1 group O envelope recombinant

Monoclonal antibodies were developed to a recombinant HIV-I group O envelope protein derived from the isolate HAM112. These monoclonal antibodies were characterized for reactivity to a series of overlapping synthetic peptides (29-30 mers) covering gp120 C-terminal and gp41 ectodomain regions of the HIV-1 group O envelope protein. Most of these monoclonal antibodies reacted with peptides spanning sequences analogous to HIV-1 group M epitopes identified from studies in mice and humans. However, several of the antibodies that were nonreactive to individual peptides did react to a mixture of longer peptides from the N-terminal and C-terminal helical regions of the gp41 ectodomain. The monoclonal antibodies described in this study are valuable tools for characterization of antigenic differences between HIV-1 group O and group M viruses.