Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate

The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162DeltaV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV(SF162P4) virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R(h)) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M(w) of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162DeltaV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162DeltaV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162DeltaV2 Env immunogen in a successful HIV vaccine.     

Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region

Immunization of macaques with the soluble oligomeric gp140 form of the SF162 envelope (SF162gp140) or with an SF162gp140-derived construct lacking the central region of the V2 loop (DeltaV2gp140) results in the generation of high titers of antibodies capable of neutralizing the homologous human immunodeficiency virus type 1 (HIV-1), SF162 virus (Barnett et al. J. Virol. 75:5526-5540, 2001). However, the DeltaV2gp140 immunogen is more effective than the SF162gp140 immunogen in eliciting the generation of antibodies capable of neutralizing heterologous HIV-1 isolates. This indicates that deletion of the V2 loop alters the immunogenicity of the SF162gp140 protein. The present studies were aimed at identifying the envelope regions whose immunogenicity is altered following V2 loop deletion. We report that the antibodies elicited by the SF162gp140 immunogen recognize elements of the V1, V2, and V3 loops, the CD4-binding site, and the C1 and C2 regions on the homologous SF162 gp120. With the exception of the V1 and V2 loops, the same regions are recognized on heterologous gp120 proteins. Surprisingly, although a minority of the SF162gp140-elicited antibodies target the V3 loop on the homologous gp120, the majority of the antibodies elicited by this immunogen that are capable of binding to the heterologous gp120s tested recognize their V3 loops. Deletion of the V2 loop has two effects. First, it alters the immunogenicity of the V3 and V1 loops, and second, it renders the C5 region immunogenic. Although deletion of the V2 loop does not result in an increase in the immunogenicity of the CD4-binding site per se, the relative ratio of anti-CD4-binding site to anti-V3 loop antibodies that bind to the heterologous gp120s tested is higher in sera collected from the DeltaV2gp140-immunized animals than in the SF162gp140-immunized animals. Overall, our studies indicate that it is possible to alter the immunogenic structure of the HIV envelope by introducing specific modifications.     

The first hypervariable region of the gp120 Env glycoprotein defines the neutralizing susceptibility of heterologous human immunodeficiency virus type 1 isolates to neutralizing antibodies elicited by the SF162gp140 immunogen

Current vaccine efforts to elicit cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus (HIV) focus on the engineering of soluble mimetics of the trimeric HIV Env glycoprotein (commonly termed gp140 immunogens). Such immunogens are thought to be more effective than previously tested monomeric gp120 immunogens at eliciting cross-reactive NAbs. Still, the breadth of neutralizing antibody responses elicited by gp140 immunogens is narrow. Understanding why antibodies elicited by gp140 immunogens fail to neutralize a wide range of heterologous primary HIV isolates is necessary for improving the design of such immunogens. We previously reported that antibodies elicited in macaques by SF162 Env-derived gp140 immunogens fail to neutralize several heterologous “neutralization-resistant” primary HIV type 1 isolates, such as JRFL, ADA, and YU2. Here we show that by replacing the V1 region of Env on these heterologous viruses with that of SF162, we render them highly susceptible to neutralization by the SF162gp140-elicited antibodies. We observed that viral neutralization was mediated not only by vaccine-elicited anti-V1 but also by anti-V3 antibodies and antibodies directed against as yet unidentified Env regions, depending on the heterologous Env background. Our study indicates that common neutralization epitopes are differentially exposed on diverse primary HIV isolates and that the V1 loop contributes to this differential exposure. Therefore, the antibody responses elicited by soluble gp140 immunogens will have to overcome several distinct obstacles in order to neutralize diverse primary HIV isolates.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants

We previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1 isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140 immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A, and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140 trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response, sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as determined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutralizing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by the trimeric immunogens.     

Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.     

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.     

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.     

DNA vaccination with the human immunodeficiency virus type 1 SF162DeltaV2 envelope elicits immune responses that offer partial protection from simian/human immunodeficiency virus infection to CD8(+) T-cell-depleted rhesus macaques

DNA immunization of macaques with the SF162DeltaV2 envelope elicited lymphoproliferative responses and potent neutralizing antibodies. The animals were depleted of their CD8(+) T lymphocytes and then challenged intravenously with SHIV162P4. Compared to unvaccinated animals, the vaccinated macaques had lower peak viremia levels, rapidly cleared plasma virus, and showed delayed seroconversion.     

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.     

Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.     

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140_SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140_SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.     

An Envelope Modification That Renders a Primary, Neutralization-Resistant Clade B Human Immunodeficiency Virus Type 1 Isolate Highly Susceptible to Neutralization by Sera from Other Clades

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162ΔV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162ΔV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162ΔV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.     

Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers

Human immunodeficiency virus (HIV-1) envelope glycoprotein subunits, such as the gp120 exterior glycoprotein, typically elicit antibodies that neutralize T-cell-line-adapted (TCLA), but not primary, clinical isolates of HIV-1. Here we compare the immunogenicity of gp120 and soluble stabilized trimers, which were designed to resemble the functional envelope glycoprotein oligomers of primary and TCLA HIV-1 strains. For both primary and TCLA virus proteins, soluble stabilized trimers generated neutralizing antibody responses more efficiently than gp120 did. Trimers derived from a primary isolate elicited antibodies that neutralized primary and TCLA HIV-1 strains. By contrast, trimers derived from a TCLA isolate generated antibodies that neutralized only the homologous TCLA virus. Thus, soluble stabilized envelope glycoprotein trimers derived from primary HIV-1 isolates represent defined immunogens capable of eliciting neutralizing antibodies that are active against clinically relevant HIV-1 strains.     

Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIV(SF162) resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIV(SF162) strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.     

Two closely related Env antigens from the same patient elicited different spectra of neutralizing antibodies against heterologous HIV-1 isolates

Identification of immunogens capable of eliciting broadly neutralizing antibody (NAb) responses against HIV-1 is a major goal toward the development of an AIDS vaccine. Despite significant progress in understanding the structural features of the HIV-1 envelope glycoprotein (Env) and the discovery of multiple broadly neutralizing monoclonal antibodies with defined antigenic structures, the design of optimal Env immunogens to elicit broad NAbs remains a major challenge. As the structural determinants of Env immunogenicity remain unclear, we assessed two closely related Env antigens isolated from the same HIV-1-infected patient with different phenotypic features to identify what may result in a favorable immunogenic profile. One Env, B33, isolated from brain, was highly macrophage tropic with a high CD4 affinity, while the other, LN40, isolated from the lymph nodes, was poorly macrophage tropic with a low CD4 affinity. Using a DNA prime-protein boost approach, rabbits primed with LN40 Env antigen had a NAb response against heterologous primary isolates, while B33 Env antigens were capable of eliciting NAbs against only homologous and sensitive viral isolates. Further analysis revealed that the specificity of NAbs elicited by the LN40 antigen mapped to limited residues within or flanking the CD4 binding site. Certain key structural determinants were identified that could differentiate primary Env immunogens based on their potential to elicit broader NAbs. This progress will facilitate the rational design of effective HIV-1 vaccine formulations with optimal Env antigens.     

In Vitro Neutralization of Low Dose Inocula at Physiological Concentrations of a Monoclonal Antibody Which Protects Macaques against SHIV Challenge

Background

Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV) – rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) in vitro and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus.

Methods And Findings

We have used GHOST cell assays to quantify individual infectious events with HIV-1_SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIV_SF162P4 isolate and the more resistant SHIV_SF162P3. A plot of the number of fluorescent GHOST cells with increasing HIV-1_SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIV_SF162P3 than the sensitive SHIV_SF162P4. In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization.

Conclusions

Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV _SF162P3. Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of human trials.     

Quaternary Epitope Specificities of Anti-HIV-1 Neutralizing Antibodies Generated in Rhesus Macaques Infected by the Simian/Human Immunodeficiency Virus SHIVSF162P4

Monoclonal antibodies (MAbs) that neutralize human immunodeficiency virus type 1 (HIV-1) have been isolated from HIV-1-infected individuals or animals immunized with recombinant HIV-1 envelope (Env) glycoprotein constructs. The epitopes of these neutralizing antibodies (NAbs) were shown to be located on either the variable or conserved regions of the HIV-1 Env and to be linear or conformational. However, one neutralizing MAb, 2909, which was isolated from an HIV-1-infected subject, recognizes a more complex, quaternary epitope that is present on the virion-associated functional trimeric Env spike of the SF162 HIV-1 isolate. Here, we discuss the isolation of 11 anti-HIV NAbs that were isolated from three rhesus macaques infected with the simian/human immunodeficiency virus SHIV_SF162P4 and that also recognize quaternary epitopes. A detailed epitope mapping analysis of three of these rhesus antibodies revealed that their epitopes overlap that of the human MAb 2909. Despite this overall similarity in binding, however, differences in specific amino acid and glycosylation pattern requirements for MAb 2909 and the rhesus MAbs were identified. These results highlight similarities in the B-cell responses of humans and macaques to structurally complex neutralization epitopes on related viruses, HIV-1 and SHIV.HIV-1 infection typically elicits high levels of antibodies directed against the viral surface envelope (Env) glycoprotein, gp160. The initial anti-Env antibody response is nonneutralizing (28), but within 1 or 2 months after infection, neutralizing antibodies (NAbs) emerge which tend to be highly strain specific for the autologous virus and exhibit little or no neutralizing activity against heterologous HIV-1 strains (10, 22). However, several recent reports have indicated that approximately 25% of HIV-1-infected, antiretroviral-naïve patients develop broad cross-neutralizing antibody responses (5, 23, 26). In some cases, these broad neutralizing antibody responses can be mapped to the CD4-binding site of Env while in most cases a single epitope specificity cannot be identified to recapitulate the neutralizing breadth of the corresponding plasma (1, 4, 14, 15, 23, 25). Detailed analyses of the epitope specificities of broad plasma neutralizing antibody responses performed by several groups revealed the presence in HIV-positive (HIV⁺) plasmas of NAbs with as yet undefined epitope specificities (1, 15, 18, 23). It is possible that these undefined specificities include quaternary neutralizing epitopes (QNEs) and/or sugar molecules which coat the HIV Env spike expressed on the surface of viral particles.The human monoclonal antibody (MAb) 2909 recognizes a QNE present on the oligomeric Env spike present on the surface of HIV-1 SF162 virions (8). MAb 2909 can bind and neutralize SF162 virions but does not bind to the corresponding soluble SF162 Env. The binding of MAb 2909 to its QNE depends on the presence of the second and third variable regions of gp120 (the V2 and V3 loops, respectively). One particular amino acid at the amino terminal side of the V2 loop (K at position 158, based on the SF162 numbering, or position 160, based on the strain HxB2 numbering) appears to be critical for its binding (11). MAb 2909 was isolated from a person who was not infected with SF162, but a virus isolated from the donor of MAb 2909 bears a V2 loop with similarities to that of SF162 and, in particular, possesses the same K158 residue (M. K. Gorny, unpublished data). More recently, two additional human MAbs, PG9 and PG16, were isolated from a subject infected with clade A HIV-1 and were shown to bind to a QNE that also includes the V2 and V3 loops (30). In contrast, however, to the narrow neutralizing potential of MAb 2909, MAbs PG9 and PG16 display far broader neutralizing abilities.Similar to the infection of humans by HIV-1, chronic infection of rhesus macaques by simian/human immunodeficiency viruses (SHIVs) or chimpanzees by HIV-1 also results in the elicitation of potent NAbs against the autologous virus and, to a much lesser extent, against heterologous SHIV isolates or HIV-1 viruses (3, 6, 12, 17). Here, we describe a panel of MAbs from SHIV_SF162P4-infected rhesus macaques that demonstrates extremely potent neutralization against the homologous virus (that expresses the same Env as HIV-1 SF162) and that recognizes QNEs present on the surface of intact virions. Similar to the human MAbs 2909, PG9, and PG16, these rhesus macaque monoclonal antibodies (RhMAbs) recognize QNEs that include the V2 and V3 loops. Also, similar to MAb 2909, the RhMAbs neutralize only viruses expressing the SF162 Env. Consequently, we compared the fine epitope specificities of these RhMAbs to the epitope specificity of the human MAb 2909. Our detailed epitope mapping analysis reveals that although the human MAb 2909 and the RhMAbs recognize that same overall Env complex region, their specific requirements for binding differ. Thus, these studies of human and rhesus MAbs indicate that infection of humans and rhesus macaques with viruses expressing distinct Envs can result in the elicitation of antibodies that bind to overlapping conserved quaternary epitopes.     

Protection in macaques immunized with HIV-1 candidate vaccines can be predicted using the kinetics of their neutralizing antibodies

Background

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.

Methods and Findings

We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV_SF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.

Conclusions

Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses.