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.     

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.     

Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.     

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.     

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.     

Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.     

Conserved structures exposed in HIV-1 envelope glycoproteins stabilized by flexible linkers as potent entry inhibitors and potential immunogens

The HIV-1 envelope glycoprotein (Env) undergoes conformational changes while driving entry. We hypothesized that some of the intermediate Env conformations could be represented in tethered constructs where gp120 and the ectodomain of gp41 are joined by flexible linkers. Tethered Envs with long linkers (gp140-14 with 15 aa and gp140-24 with 26 aa) were stable and recognized by conformationally dependent anti-gp120 and anti-gp41 monoclonal antibodies (mAbs). Surprisingly, these proteins potently inhibited membrane fusion mediated by R5, X4, and R5X4 Envs with 5-100-fold lower IC50 than a tethered Env with short linker (gp140-4 with 4 aa), gp120, gp140, soluble CD4, or DP178 (T20). Compared to gp140, gp140-14,24 exhibited increased binding to anti-gp41 cluster II mAbs but not to cluster I mAbs. Cluster II mAbs but not cluster I, IV, or V mAbs reversed the inhibitory effect of gp140-14,24 suggesting a role of exposed conserved gp41 structures for the mechanism of inhibition. These findings suggest the existence of conserved gp41 structures that are important for HIV-1 entry and can be stably exposed in the native environment of the Env even in the absence of receptor-mediated activation. Thus, tethered Envs with long linkers may not only be important as HIV-1 inhibitors but also for elucidation of viral entry mechanisms and development of novel vaccine immunogens.     

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.     

Immune Focusing and Enhanced Neutralization Induced by HIV-1 gp140 Chemical Cross-Linking

Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.     

Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16

PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.     

Neutralizing Antibody Responses in Macaques Induced by Human Immunodeficiency Virus Type 1 Monovalent or Trivalent Envelope Glycoproteins

A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb). In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs) would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q), was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B) and 14/00/4 (subtype F), both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q). The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.     

Elicitation of neutralizing antibodies directed against CD4-induced epitope(s) using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.     

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.     

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.     

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.     

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.     

Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1_AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.     

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.     

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.     

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.