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.     

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.     

Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.     

Soluble HIV-1 Envelope Immunogens Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low Potency Neutralizing Antibodies

We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses.     

Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d

DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses. Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes. In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1.     

Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.     

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.     

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 ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.     

Alterations in the Immunogenic Properties of Soluble Trimeric Human Immunodeficiency Virus Type 1 Envelope Proteins Induced by Deletion or Heterologous Substitutions of the V1 Loop

HIV-1 gp140 envelope immunogens express conserved epitopes that are targeted by broadly cross-reactive neutralizing antibodies, but they fail to elicit similar antibodies upon immunization. The poor immunogenicity of conserved epitopes on gp140 could be linked to the high immunogenicity of variable Env regions on such constructs. Previous studies have shown that the first hypervariable region (V1 loop) is immunogenic on soluble gp140s but elicits type-specific antibodies. To address issues related to the high immunogenicity of the V1 loop, two conceptually opposite approaches were tested. In the first approach, we eliminated the V1 loop from our gp140 construct and examined how V1 deletion altered the immunogenic properties of other Env regions. In the second approach, we took advantage of the high immunogenicity of the V1 loop and engrafted four diverse V1 loops onto a common gp140 Env “scaffold.” These four scaffolds were used as a cocktail of immunogens to elicit diverse anti-V1 antibodies, under the hypothesis that eliciting diverse anti-V1 antibodies would expand the neutralizing breadth of immune sera. Our study indicates that three of four heterologous V1 loops were immunogenic on the common Env backbone “scaffold,” but heterologous anti-V1 neutralizing responses were observed in only one case. Both types of V1 modification dampened the immunogenicity of the V3 loop, differentially altered the immunogenicity of the transmembrane gp41 subunit, and altered the relative immunogenicities of unknown Env regions, including potentially the CD4-binding site (CD4-bs) and trimer-specific targets, which elicited cross-reactive neutralizing antibodies but of limited breadth.An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will need to incorporate an envelope-derived immunogen capable of eliciting potent and broadly cross-reactive neutralizing antibody responses against diverse primary HIV-1 isolates. The target of anti-HIV neutralizing antibodies (NAbs), the viral envelope (Env) glycoprotein, is expressed as a single transmembrane polypeptide precursor (gp160) that is glycosylated and cleaved into an extracellular subunit (gp120) and a transmembrane subunit (gp41) during intracellular processing (10, 20, 21, 54). The functional Env form on virion surfaces is a trimer composed of three noncovalently associated gp120-gp41 heterodimers. Soluble forms of the trimeric Env have been generated by introducing stop codons immediately upstream of the transmembrane domain of gp41. These constructs are commonly referred to as gp140 proteins and have been tested extensively as immunogens to elicit anti-HIV-1 NAbs. Soluble gp140s express epitopes that are targets of NAbs, including cross-reactive NAbs such as b12, 4E10, and 2G12 (5, 17, 34, 45, 47, 49, 50, 52, 57). Immunization with gp140 immunogens nonetheless does not result in a broadly cross-reactive neutralizing antibody response (2, 3, 17, 18, 26, 56, 58).Epitope mapping analyses of the Abs elicited by soluble trimeric gp140 immunogens revealed that a large fraction of the gp140-induced neutralization response targets the first hypervariable region of gp120 (the V1 loop). In our hands, ∼40 to 70% of the neutralizing activity of sera from animals immunized with SF162 gp140 constructs is due to anti-V1 antibodies (17). In a study by Li et al. with YU2 gp140 (30) and a study by Wu et al. with HxB2/BaL gp145 (56), ∼10 to 80% of anti-YU2 neutralizing activity and 100% of anti-HxB2 neutralizing activity, respectively, were due to anti-V1 Abs. These anti-V1 Abs, however, are not cross-reactive. Previously, we also demonstrated that the diverse positionings of the V1 across heterologous strains limit access of broadly cross-reactive monoclonal antibodies (MAbs) to their targets (12).Here, taking into consideration the V1 loop''s high immunogenicity, we employed two opposing approaches aimed at the elicitation of cross-reactive neutralizing antibody responses to HIV-1. In the first approach, we deleted the V1 loop on our soluble trimeric gp140 construct (ΔV1SF162 gp140) and examined whether and how this modification altered the immunogenic properties of other Env regions. In the second approach, we substituted the V1 loop on our SF162 gp140 construct with the V1 loops from four heterologous HIV-1 viruses (89.6, YU2, JRFL, and HxB2) that differ in their amino acid compositions and in the number of potential N-linked glycosylation sites (PNGs). These four heterologous viruses display various neutralization phenotypes (7) and coreceptor utilization profiles (15, 35, 36, 48, 51). A total of four SF162 Env-based gp140 “scaffolds” expressing four different V1 loops were created and used as immunogens in a cocktail to test as a “proof of principle” the hypothesis that if diverse V1 loops are presented to the immune system simultaneously, the elicitation of anti-V1 NAbs with diverse specificities would broaden the overall neutralizing activity of immune sera. We also immunized animals with each of the four V1 chimeric scaffolds individually to ensure that all V1 loops were immunogenic when presented on the heterologous SF162 Env background.All immunogens (including wild-type [WT] SF162 gp140 and ΔV1SF162 gp140) elicited homologous anti-SF162 NAbs. All immunogens except the scaffold construct expressing the YU2 V1 also elicited heterologous NAbs against the sensitive lab-adapted strain HxB2. The heterologous YU2, 89.6, and HxB2 V1 loops, but not the JRFL V1 loop, were immunogenic on the background of the SF162 Env scaffold. However, only anti-V1 neutralizing activity against the HxB2 virus was observed. Although neither approach resulted in the development of broad anti-HIV-1 cross-neutralizing antibody responses, cross-neutralizing antibody responses of narrow breadth were elicited. These responses were not due to antibodies that target to variable regions of gp120 but were due to antibodies that target either epitopes of the CD4-binding site (CD4-bs) or epitopes that are not present on monomeric gp120. These observations have implications for guiding rational Env-based immunogen design and for potentially eliciting broadly cross-reactive NAb responses.     

Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) complex comprises three gp120 exterior glycoproteins each noncovalently linked to a gp41 transmembrane glycoprotein. Monomeric gp120 proteins can elicit antibodies capable of neutralizing atypically sensitive test viruses in vitro, but these antibodies are ineffective against representative primary isolates and the gp120 vaccines failed to provide protection against HIV-1 transmission in vivo. Alternative approaches to raising neutralizing antibodies are therefore being pursued. Here we report on the antibody responses generated in rabbits against a soluble, cleaved, trimeric form of HIV-1(JR-FL) Env. In this construct, the gp120 and gp41 moieties are covalently linked by an intermolecular disulfide bond (SOS gp140), and an I559P substitution has been added to stabilize gp41-gp41 interactions (SOSIP gp140). We investigated the value of DNA priming and compared the use of membrane-bound and soluble priming antigens and of repeat boosting with soluble and particulate protein antigen. Compared to monomeric gp120, SOSIP gp140 trimers elicited approximately threefold lower titers of anti-gp120 antibodies. Priming with DNA encoding a membrane-bound form of the SOS gp140 protein, followed by several immunizations with soluble SOSIP gp140 trimers, resulted in antibodies capable of neutralizing sensitive strains at high titers. A subset of these sera also neutralized, at lower titers, HIV-1(JR-FL) and some other primary isolates in pseudovirus and/or whole-virus assays. Neutralization of these viruses was immunoglobulin mediated and was predominantly caused by antibodies to gp120 epitopes, but not the V3 region.     

Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.     

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.     

Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.     

Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by semliki forest virus

The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.     

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.     

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.     

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.     

Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates

Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies.     

Study of the V3 Loop as a Target Epitope for Antibodies Involved in the Neutralization of Primary Isolates versus T-Cell-Line-Adapted Strains of Human Immunodeficiency Virus Type 1

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