Membrane bound modified form of clade B Env,JRCSF is suitable for immunogen design as it is efficiently cleaved and displays all the broadly neutralizing epitopes including V2 and C2 domain-dependent conformational epitopes

Background

Antigenicity of HIV-1 envelope proteins (Envs) of both lab-adapted and primary isolates expressed on the cell surface rarely match with in vitro neutralization of viruses, pseudo-typed with corresponding Envs. Often, both neutralizing and non-neutralizing antibodies bind to Envs expressed on the cell membrane. This could be due to the lack of efficient cleavage of Env expressed on the cell surface. Naturally occurring, efficiently cleaved Envs with appropriate antigenic properties are relatively rare. Given viral diversity it is essential to increase the pool of candidate Envs suitable for immunogen design. Previously, it has been reported that JRFL Env is the only clade B Env, which is efficiently cleaved on the cell surface and retains desirable antigenic properties. JRCSF is a clade B Env isolated from the same patient as JRFL. JRCSF Env has not been explored aggressively for designing immunogen as the binding characteristics of JRCSF Env to broadly neutralizing antibodies on the cell surface and its cleavage status are unknown.

Results

Although JRCSF preferentially binds to most of the other gp120-directed neutralizing antibodies and cleavage dependent antibody, PGT151 efficiently, it binds poorly to CD4-binding-site-directed (CD4-bs-directed) neutralizing antibodies on cell surface. Membrane bound form of modified JRCSF Env containing the N197D mutation binds to CD4-bs-directed neutralizing antibodies better than JRFL, without debilitating its ability to bind quaternary epitope-directed neutralizing antibodies or exposing the CD4i antibody epitopes. In comparison to JRFL (E168K), JRCSF Env binds more efficiently to PG9/PGT145 class of V1/V2-directed conformational antibodies. Biochemical, cell surface staining and gp120 shedding experiments suggest that JRCSF is efficiently cleaved on the cell surface.

Conclusions

Binding of JRCSF Env expressed on cell surface to the various HIV-1 Env-directed antibodies has not been reported earlier. Here, for the first time, we report that compared to JRFL, JRCSF displays epitopes for a larger number of broadly neutralizing antibodies and is also efficiently cleaved when expressed on the cell surface. Thus, considering the diversity of viral Envs and the discovery of conformation dependent glycan-directed antibodies in HIV-1 infected individuals, an innately cleaved JRCSF Env as present on the viral membrane and displaying those distinct epitopes may be an important candidate for immunogen design.

     

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.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.     

Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.     

Structural Characterization of Cleaved,Soluble HIV-1 Envelope Glycoprotein Trimers

Human immunodeficiency virus type 1 (HIV-1) infection is a significant global public health problem for which development of an effective prophylactic vaccine remains a high scientific priority. Many concepts for a vaccine are focused on induction of appropriate titers of broadly neutralizing antibodies (bNAbs) against the viral envelope (Env) glycoproteins gp120 and gp41, but no immunogen has yet accomplished this goal in animals or humans. One approach to induction of bNAbs is to design soluble, trimeric mimics of the native viral Env trimer. Here, we describe structural studies by negative-stain electron microscopy of several variants of soluble Env trimers based on the KNH1144 subtype A sequence. These Env trimers are fully cleaved between the gp120 and gp41 components and stabilized by specific amino acid substitutions. We also illustrate the structural consequences of deletion of the V1/V2 and V3 variable loops from gp120 and the membrane-proximal external region (MPER) from gp41. All of these variants adopt a trimeric configuration that appropriately mimics native Env spikes, including the CD4 receptor-binding site and the epitope for the VRC PG04 bNAb. These cleaved, soluble trimer designs can be adapted for use with multiple different env genes for both vaccine and structural studies.     

Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is composed of two noncovalently associated subunits: an extracellular subunit (gp120) and a transmembrane subunit (gp41). The functional unit of Env on the surface of infectious virions is a trimer of gp120/gp41 heterodimers. Env is the target of anti-HIV neutralizing antibodies. A considerable effort has been invested in the engineering of recombinant soluble forms of the virion-associated Env trimer as vaccine candidates to elicit anti-HIV neutralizing antibody responses. These soluble constructs contain three gp120 subunits and the extracellular segments of the corresponding gp41 subunits. The individual gp120/gp41 protomers on these soluble trimers are identical in amino acid sequence (homotrimers). Here, we engineered novel soluble trimeric gp140 proteins that are formed by the association of gp140 protomers that differ in amino acid sequence and glycosylation patterns (heterotrimers). Specifically, we engineered soluble heterotrimeric proteins composed of clade A and clade B Env protomers. The clade A gp140 protomers were derived from viruses isolated during acute infection (Q168a2, Q259d2.17, and Q461e2), whereas the clade B gp140 protomers were derived from a virus isolated during chronic infection (SF162). The amino acid sequence divergence between the clade A and the clade B Envs is approximately 24%. Neutralization epitopes in the CD4 binding sites and coreceptor binding sites, as well as the membrane-proximal external region (MPER), were differentially expressed on the heterotrimeric and homotrimeric proteins. The heterotrimeric gp140s elicited broader anti-tier 1 isolate neutralizing antibody responses than did the homotrimeric gp140s.     

Design and characterization of a germ-line targeting soluble,native-like,trimeric HIV-1 Env lacking key glycans from the V1V2-loop

BackgroundThe HIV-1 envelope glycoprotein (Env) is the primary target for broadly neutralizing antibodies (bNAbs) which can block infection. The current design strategy of soluble forms of Env in native-like trimeric conformation induces neutralizing antibodies with minimal breadth and potency. Extensive shielding by N-glycans on the surface of the HIV-1 Env acts as an immune evasion mechanism by restricting B cell recognition of conserved neutralizing determinants. An alternate approach is to design Env protein with glycan deletion to expose the protein surface.MethodsA stable native-like trimeric Env with glycan holes at potentially immunogenic locations is expected to elicit better induction of germ-line B-cells due to exposure of the immunogenic regions. However, the extent and consequences of glycan removal from the trimer apex that form an important epitope is not explored. In this work, we have designed a construct with glycans deleted from the trimer apex of an Indian clade C origin Env that has previously been characterized for immunogenicity, to understand the impact of deglycosylation on the structural and functional integrity as well as on the antibody binding properties.ResultsThe V1V2 glycan-deleted protein maintains native-like trimeric conformation with improved accessibility of the V1V2-directed germ-line antibodies. Furthermore, we showed that the protein binds specifically to quaternary conformation-dependent bnAbs but minimally to non-neutralizing antibodies.ConclusionsThis study provide an important design aspect of HIV-1 Env-based immunogens with glycan holes in the apex region that could be useful in eliciting apex directed antibodies in immunization studies.     

HIV-1 Env三聚体抗原改造研究进展

HIV-1疫苗研发是当前艾滋病研究的一大热点,其病毒表面包膜糖蛋白Env三聚体介导病毒与细胞融合,是HIV-1疫苗研究的重要靶点。因此,设计并在体外表达类天然Env三聚体对HIV-1疫苗的研发具有重要的意义。近年来,Env三聚体研究取得了显著的进展。SOSIP、NFL2P、UFO等抗原改造方法实现了类天然Env三聚体的体外表达,逐步解决了改造抗原产量低、结构不稳定等问题,且表达的Env三聚体抗原能在动物免疫中诱导机体产生较高的中和抗体水平。Env三聚体改造方法促进了HIV-1疫苗的研究。文中综述了SOSIP、NFL2P、UFO三种HIV-1 Env三聚体抗原改造方法,对比各个改造方法优缺点,并结合自身工作提出相关建议,为后续HIV-1抗原的相关设计提供指导。     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

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

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

HIV-1 Envelope Glycoprotein Trimers Display Open Quaternary Conformation When Bound to the gp41 Membrane-Proximal External-Region-Directed Broadly Neutralizing Antibody Z13e1

We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.     

Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques

Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs) of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.     

Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion

We have engineered two soluble, covalently linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 (HIV-1) envelope (Env)-mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35CCG-N13 and N34CCG are approximately 15 and approximately 95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV. Polyclonal antibodies were raised against N35CCG-N13 and a tightly binding fraction of anti-N35CCG-N13 inhibits T cell and macrophage tropic HIV-1 Env-mediated cell fusion with respective IC50 values of approximately 0.5 and approximately 1.5 microg/ml at 37 degrees C. The tightly binding anti-N35CCG-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the prehairpin intermediate state of gp41 in a manner analogous to peptides derived from the C region of the gp41 ectodomain. The potency of the tightly binding anti-N35CCG-N13 antibody fraction in the fusion assay is comparable with that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35CCG-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35CCG-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the prehairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier.     

Human Immunodeficiency Virus Type 2 (HIV-2)/HIV-1 Envelope Chimeras Detect High Titers of Broadly Reactive HIV-1 V3-Specific Antibodies in Human Plasma

Deciphering antibody specificities that constrain human immunodeficiency virus type 1 (HIV-1) envelope (Env) diversity, limit virus replication, and contribute to neutralization breadth and potency is an important goal of current HIV/AIDS vaccine research. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 scaffolds may provide a sensitive, biologically functional context by which to quantify specific antibody reactivities even in complex sera. Here, we describe a novel HIV-2 proviral scaffold (pHIV-2_KR.X7) into which we substituted the complete variable region 3 (V3) of the env gene of HIV-1_YU2 or HIV-1_Ccon to yield the chimeric proviruses pHIV-2_KR.X7 YU2 V3 and pHIV-2_KR.X7 Ccon V3. These HIV-2/HIV-1 chimeras were replication competent and sensitive to selective pharmacological inhibitors of virus entry. V3 chimeric viruses were resistant to neutralization by HIV-1 monoclonal antibodies directed against the CD4 binding site, coreceptor binding site, and gp41 membrane proximal external region but exhibited striking sensitivity to HIV-1 V3-specific monoclonal antibodies, 447-52D and F425 B4e8 (50% inhibitory concentration of [IC₅₀] <0.005 μg/ml for each). Plasma specimens from 11 HIV-1 clade B- and 10 HIV-1 clade C-infected subjects showed no neutralizing activity against HIV-2 but exhibited high-titer V3-specific neutralization against both HIV-2/HIV-1 V3 chimeras with IC₅₀ measurements ranging from 1:50 to greater than 1:40,000. Neutralization titers of B clade plasmas were as much as 1,000-fold lower when tested against the primary HIV-1_YU2 virus than with the HIV-2_KR.X7 YU2 V3 chimera, demonstrating highly effective shielding of V3 epitopes in the native Env trimer. This finding was replicated using a second primary HIV-1 strain (HIV-1_BORI) and the corresponding HIV-2_KR.X7 BORI V3 chimera. We conclude that V3 is highly immunogenic in vivo, eliciting antibodies with substantial breadth of reactivity and neutralizing potential. These antibodies constrain HIV-1 Env to a structure(s) in which V3 epitopes are concealed prior to CD4 engagement but do not otherwise contribute to neutralization breadth and potency against most primary virus strains. Triggering of the viral spike to reveal V3 epitopes may be required if V3 immunogens are to be components of an effective HIV-1 vaccine.     

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.     

Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from Env_SF162 into the Env_HXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the Env_HXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.