HIV-1 Envgp140 trimers elicit neutralizing antibodies without efficient induction of conformational antibodies

Currently, no vaccine for human immunodeficiency virus (HIV-1) provides protection from virus infection. One reason for these disappointing results has been the difficulty of current vaccine candidates to elicit high-titer, broadly reactive immunity to a large number of viral proteins. Recently, our laboratory demonstrated that the coupling of C3d to a soluble trimerized HIV-1 envelope (Env(gp140(FT))) elicited higher titers of neutralizing antibodies than monomers of Env(gp120) coupled to C3d [Bower JF, Yang X, Sodroski J, Ross TM. Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d. J Virol 2004;78(9):4710-9]. To determine if the induction of conformational antibodies correlated with neutralization, mice (BALB/c) were primed (2x) with DNA plasmids expressing monomeric Env(gp120) or trimeric Env(gp140) alone or fused to mC3d(3) at one of two doses (2.0microg or 0.2microg), followed by a boost of recombinant uncleaved, trimeric Env(gp140). Regardless of the priming dose of DNA, all mice had high-titer anti-Env IgG antibodies. Interestingly, Env(gp140) trimers did not elicit higher titers of antibodies that recognized conformational Env epitopes compared to monomers of Env(gp120). Therefore, additional parameters were examined for correlation with neutralization. For neutralization-resistant HIV-1 isolates, ADA and YU-2, neutralization correlated with high-titer, high avidity antibodies, with Env(gp140) eliciting slightly higher neutralization titers than Env(gp120). In contrast, none of the measured parameters correlated with neutralization for the more neutralization-sensitive isolates, MN or 89.6. Therefore, even though soluble, uncleaved Env(gp140) trimers may be marginally more effective at eliciting neutralizing antibodies than Env(gp120), neutralization does not appear to correlate with the elicitation of conformationally dependent antibodies.     

Safety and immunogenicity of an HIV-1 gp120-CD4 chimeric subunit vaccine in a phase 1a randomized controlled trial

A major challenge for HIV vaccine development is to raise anti-envelope antibodies capable of recognizing and neutralizing diverse strains of HIV-1. Accordingly, a full length single chain (FLSC) of gp120-CD4 chimeric vaccine construct was designed to present a highly conserved CD4-induced (CD4i) HIV-1 envelope structure that elicits cross-reactive anti-envelope humoral responses and protective immunity in animal models of HIV infection. IHV01 is the FLSC formulated in aluminum phosphate adjuvant. We enrolled 65 healthy adult volunteers in this first-in-human phase 1a randomized, double-blind, placebo-controlled study with three dose-escalating cohorts (75 µg, 150 µg, and 300 µg doses). Intramuscular injections were given on weeks 0, 4, 8, and 24. Participants were followed for an additional 24 weeks after the last immunization. The overall incidence of adverse events (AEs) was not significantly different between vaccinees and controls. The majority (89%) of vaccine-related AE were mild. The most common vaccine-related adverse event was injection site pain. There were no vaccine-related serious AE, discontinuation due to AE, intercurrent HIV infection, or significant decreases in CD4 count. By the final vaccination, all vaccine recipients developed antibodies against IHV01 and demonstrated anti-CD4i epitope antibodies. The elicited antibodies reacted with CD4 non-liganded Env antigens from diverse HIV-1 strains. Antibody-dependent cell-mediated cytotoxicity against heterologous infected cells or gp120 bound to CD4+ cells was evident in all cohorts as were anti-gp120 T-cell responses. IHV01 vaccine was safe, well tolerated, and immunogenic at all doses tested. The vaccine raised broadly reactive humoral responses against conserved CD4i epitopes on gp120 that mediates antiviral functions.     

Elicitation of broadly reactive antibodies against glycan-modulated neutralizing V3 epitopes of HIV-1 by immune complex vaccines

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab′)₂ was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.     

Trimeric HIV-1 gp140 fused with APRIL,BAFF, and CD40L on the mucosal gp140-specific antibody responses in mice

HIV-1 envelope (Env)-specific antibody present at mucosal surfaces can block entry of HIV-1 into these portals and thus should be elicited by an HIV-1 preventive vaccine. Since three molecules of tumor necrosis factor superfamily (TNFSF), APRIL, BAFF, and CD40L, could promote mucosal antibody responses, we made fusion constructs of them with an HIV-1 gp140 trimer and tested the mucosal gp140-specific antibody elicited by the fusion constructs in mice using a DNA prime-protein boost vaccination regimen. The fusion constructs formed trimers and displayed both broadly neutralizing antibody epitopes and non-broadly neutralizing antibody epitopes. Compared with the control construct, trimeric gp140, trimeric gp140-APRIL and gp140-BAFF fusion proteins mildly promoted B cell proliferation in vitro, enhanced HIV-1 gp140-binding IgG responses in vaginal lavage or fecal pellets, respectively, and decreased HIV-1 gp140-binding IgA in sera. Gp140-APRIL also augmented HIV-1 gp140-binding IgG in sera. Surprisingly, gp140-CD40L did not promote B cell proliferation in vitro and inhibited mucosal and systemic HIV-1 gp140-binding IgG or IgA. These results suggest that APRIL and BAFF should be further explored as molecular adjuvants for HIV-1 vaccines to enhance mucosal antibody responses, but covalent fusion of TNFSFs to gp140 may hinder their adjuvancy due to steric interactions.     

Structural and immunogenicity studies of a cleaved,stabilized envelope trimer derived from subtype A HIV-1

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.     

Envelope proteins of two HIV-1 clades induced different epitope-specific antibody response

Using HIV-1 envelope protein (Env)-based immunogens that closely mimic the conformation of functional HIV-1 Envs and represent the isolates prevalent in relevant geographical region is considered a rational approach towards developing HIV vaccine. We recently reported that like clade B Env, JRFL, membrane bound Indian clade C Env, 4-2.J41 is also efficiently cleaved and displays desirable antigenic properties for plasmid DNA immunization. Here, we evaluated the immune response in rabbit by injecting the animals with plasmid expressing membrane bound efficiently cleaved 4-2.J41 Env followed by its gp140-foldon (gp140-fd) protein boost. The purified 4-2.J41-gp140-fd protein is recognized by a wide panel of broadly neutralizing antibodies (bNAbs) including the quaternary conformation-dependent antibody, PGT145 with high affinity. We have also evaluated and compared the quality of antibody response elicited in rabbits after immunizing with plasmid DNA expressing the membrane bound efficiently cleaved Env followed by gp140-fd proteins boost with either of clade C Env, 4-2.J41 or clade B Env, JRFL or in combination. In comparison to JRFL group, 4-2.J41 group elicited autologous as well as limited low level cross clade neutralizing antibody response. Preliminary epitope-mapping of sera from animals show that in contrast to JRFL group, no reactivity to either linear peptides or V3-loop is detected in 4-2.J41 group. Furthermore, the presence of conformation-specific antibody in sera from animals immunized with 4-2.J41 Env is observed. However, unlike JRFL group, in 4-2.J41 group of animals, CD4-binding site-directed antibodies cannot be detected. Additionally, we have demonstrated that the quality of antibody response in combination group is guided by JRFL Env-based immunogen suggesting that the selection and the quality of Envs in multicade candidate vaccine are important factors to elicit desirable response.     

Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state

The conserved membrane proximal external region (MPER) of the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 is the target of two broadly neutralizing antibodies, 2F5 and 4E10. However, no neutralizing antibodies have been elicited against immunogens bearing these epitopes. Given that structural and biochemical studies suggest that the lipid membrane of the virion is involved in their proper configuration, HIV-1 gp41 derivatives in a pre-fusion state were expressed on the surface of immature virus like particles (VLP) derived from Sf9 cells. Guinea pigs were immunized with three doses of VLPs or Sf9 cells presenting gp41 derivatives with or without E. coli heat-labile enterotoxin (LT) as an adjuvant. While immune sera contained high titer anti-VLP antibodies, the specific anti-gp41 antibody responses were low with no neutralizing antibodies detected. An explanation for this absence may be the low level of gp41 expression relative to the many other proteins derived from host cells which are incorporated onto the VLP surface. In addition, the anti-gp41 immune response was preferentially directed to the C-helical domain, away from the MPER. Future vaccine design needs to contend with the complexity of epitope display as well as immunodominance.     

Analysis of the immunogenic properties of a single-chain polypeptide analogue of the HIV-1 gp120-CD4 complex in transgenic mice that produce human immunoglobulins

The potential utility of gp120 complexed to CD4 in HIV-1 vaccine development has been shown by studies in which such complexes were able to induce antibodies to cryptic gp120 epitopes and to generate broadly neutralizing humoral responses. Recently, a full-length single-chain (FLSC) analogue of the gp120-CD4 receptor complex, consisting of HIV-1 Bal gp120 joined to the D1D2 domains of CD4 by a 20-amino-acid linker, has been described. We tested the immunogenicity of this protein in transgenic XMG2 XenoMouse) mice that express human IgG2 with kappa light chain loci and that model human humoral immune responses. Six mice immunized with purified FLSC all developed high antibody titers for the immunogen, but none of the sera possessed neutralizing activities against HIV(BaL) or HIV(SF162) virus. A panel of 39 human monoclonal antibodies (mAbs) were generated from an immunized mouse. Only three of these mAbs recognized linear epitopes. One of these mapped to the V3 region and two to the C-terminus of gp120. The majority of the mAbs (36/39) were directed against one of two distinct conformational epitopes specific for FLSC. Binding of representative mAbs to these epitopes was not blocked by antibodies to a number of known targets on gp120, but was enhanced by binding of 17b, directed to a CD4-induced epitope on gp120. None of the FLSC-induced mAbs possessed neutralization activity against either HIV-1 BaL or SF162. These results suggest that a major portion of the antibody response against the FLSC protein may be directed against immunodominant conformational epitopes unique to the fusion protein that do not mediate viral neutralization. This property may limit the utility of this chimeric molecule as an HIV-1 vaccine candidate.     

Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation

HIV-1 envelope (Env) gp120 is an important target for neutralizing antibody (Ab) responses against the virus; however, developing gp120 vaccines that elicit potent and broad neutralizing Abs has proven to be a formidable challenge. Previously, removal of an N-linked glycan at residue 448 by an N to Q mutation (N448Q) has been found to enhance the in vitro antigenicity of neutralizing epitopes in the V3 loop. In this study the mutated gp120 was first compared with wild type gp120 for immunogenicity in mice using a DNA prime and protein boost immunization regimen. The N448Q mutant did not elicit higher titers of anti-gp120 serum Abs and failed to generate anti-V3 Abs. The sera also had no virus-neutralizing activity, even though the mutant induced higher levels of lymphoproliferation and cytokine production. Subsequently, the N448Q mutant was used to construct an immune complex vaccine with the anti-CD4 binding site monoclonal antibody (mAb) 654. The N448Q/654 complex stimulated comparably high levels of serum Abs to gp120 and V3 as the wild type complex. However, Abs against the C1 and C2 regions in the gp120 core were more elevated. Importantly, the mutant complex also elicited higher titers of neutralizing Abs activity than the wild type counterpart. Similar results were achieved with a complex made with gp120 bearing an N448E mutation, confirming the importance of the N448-linked glycan in modulating gp120 immunogenicity. Neutralizing activity was directed to V3 and other undefined neutralizing epitopes. Improved immunogenicity of the immune complexes correlated with alterations in exposure of V3 and other Ab epitopes and their stability against proteases. These data demonstrate the advantage of combining site-specific N-glycan removal and immune complex formation as a novel vaccine strategy to improve immunogenicity of targeted Ab epitopes on critical regions of HIV-1 gp120.     

Live rubella vectors can express native HIV envelope glycoproteins targeted by broadly neutralizing antibodies and prime the immune response to an envelope protein boost

Following HIV infection, most people make antibodies to gp120 and gp41, yet only a few make broadly neutralizing antibodies that target key antigenic sites on the envelope glycoproteins. The induction of broadly neutralizing antibodies by immunization remains a major challenge of HIV vaccine research. Difficulties include: variable protein sequence, epitopes that depend on the native conformation, glycosylation that conceals key antigenic determinants, and the assembly of Env trimers that mimic viral spikes. In addition, more potent immunogens may be needed to initiate the response of germline antibody precursors and drive B cell maturation toward antibodies with broad neutralizing activity.We have expressed HIV Env glycoproteins by incorporation into live attenuated rubella viral vectors. The rubella vaccine strain RA27/3 has demonstrated its safety and potency in millions of children. As a vector, it has elicited potent and durable immune responses in macaques to SIV Gag vaccine inserts. We now find that rubella/env vectors can stably express Env core derived glycoproteins ranging in size up to 363 amino acids from HIV clade C strain 426c. The expressed Env glycoproteins bind broadly neutralizing antibodies that target the native CD4 binding site. The vectors grew well in rhesus macaques, and they elicited a vaccine “take” in all animals, as measured by anti-rubella antibodies. By themselves, the vectors elicited modest antibody titers to the Env insert. But the combination of rubella/env prime followed by a homologous protein boost gave a strong response. Neutralizing antibodies appeared gradually after multiple vaccine doses. The vectors will be useful for testing new vaccine inserts and immunization strategies under optimized conditions of vector growth and protein expression.     

Comparison of multiple adjuvants on the stability and immunogenicity of a clade C HIV-1 gp140 trimer

Immunogens based on the human immunodeficiency virus type-1 (HIV-1) Envelope (Env) glycoprotein have to date failed to elicit potent and broadly neutralizing antibodies against diverse HIV-1 strains. An understudied area in the development of HIV-1 Env-based vaccines is the impact of various adjuvants on the stability of the Env immunogen and the magnitude of the induced humoral immune response. We hypothesize that optimal adjuvants for HIV-1 gp140 Env trimers will be those with high potency but also those that preserve structural integrity of the immunogen and those that have a straightforward path to clinical testing. In this report, we systematically evaluate the impact of 12 adjuvants on the stability and immunogenicity of a clade C (CZA97.012) HIV-1 gp140 trimer in guinea pigs and a subset in non-human primates. Oil-in-water emulsions (GLA-emulsion, Ribi, Emulsigen) resulted in partial aggregation and loss of structural integrity of the gp140 trimer. In contrast, alum (GLA-alum, Adju-Phos, Alhydrogel), TLR (GLA-aqueous, CpG, MPLA), ISCOM (Matrix M) and liposomal (GLA-liposomes, virosomes) adjuvants appeared to preserve trimer integrity as measured by size exclusion chromatography. However, multiple classes of adjuvants similarly augmented Env-specific binding and neutralizing antibody responses in guinea pigs and non-human primates.     

Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1

The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described. Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography. A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding. Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits. The C clade gp140 was incorrectly folded and poorly antigenic. Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41. Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration. The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation. The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation. These reagents have potential as immunogens for use in vaccine development.     

Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein.

We report on the properties of a set of HIV-1 IIIB Env mutants carrying a linear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-1 neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants were properly expressed, retained reactivity to conformation-dependent monoclonal antibodies and exhibited patterns similar to the parental Env molecule. However, insertion of this epitope in the V3 loop was associated with drastically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased binding to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs were immunized with plasmid expression vectors for the mutant proteins. For both mice and guinea pigs, all four mutants elicited anti-gp120 antibody responses. In mice the V1 and V3 insertion mutants, but neither the V2 or V4 insertion mutant nor the parental env, elicited significant titers against the epitope peptide, whereas in guinea pigs, V2 insertion mutant was most effective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody responses. This indicates that the precise position at which the epitope insertion takes place dictates the ability of the mutant to induce the epitope-specific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody failed to enhance the virus neutralizing activity, suggesting that the configuration of 2F5 epitope plays a critical role in inducing neutralizing antibody responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-1 Env immunogenicity.     

A recombinant live attenuated measles vaccine vector primes effective HLA-A0201-restricted cytotoxic T lymphocytes and broadly neutralizing antibodies against HIV-1 conserved epitopes

Live attenuated measles vaccine (MV) could provide a safe and efficient pediatric vaccination vector to immunize children simultaneously against measles and human immunodeficiency virus type 1 (HIV-1). To evaluate the capacity of a vector derived from the certified Schwarz measles vaccine (MVSchw) to prime effective cytotoxic T cells (CTL) and broad neutralizing antibodies against HIV-1 conserved epitopes, we generated recombinant MVSchw viruses expressing HIV-1 antigens. We demonstrate that a recombinant MVSchw virus expressing an HIV-1-derived CTL polyepitope primes effective HLA-A0201-restricted CTLs against multiple conserved HIV-1 epitopes in mice susceptible to measles and humanized for the major histocompatibility complex (MHC) class-I molecule HLA-A0201. Additionally, we show that a recombinant MVSchw virus expressing an HIV-1(89.6) gp140 glycoprotein whose hyper variable V1, V2 and V3 loops were deleted (DeltaV1V2V3gp140), induces broadly neutralizing antibodies against HIV-1 primary isolates. These results show that the MVSchw pediatric vaccination vector induces efficient cellular and humoral HIV-specific immune responses.     

Dense display of HIV-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to HIV-1 Env

The generation of strong, virus-neutralizing antibody responses to the HIV-1 envelope spike (Env) is a major goal in HIV-1 vaccine research. To try to enhance the Env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. To do this, an in vitro complementation system was used to “decorate” phage particles with glycosylated, mammalian cell-derived envelope oligomers. We compared the immune response to lambda phage particles displaying HIV-1 Env to that elicited by soluble oligomeric gp140 in rabbits. Env-binding antibody titers were higher in animals that received oligomeric gp140 as compared to Env decorated phage particles, as were virus neutralizing antibody responses. The Env decorated phage particles were, however, able to efficiently boost a protein-primed humoral response to levels equivalent to those elicited by high-dose adjuvanted Env oligomers. These results show that display of HIV-1 envelope spikes on the bacteriophage lambda capsid does not result in an improved, Env-specific humoral immune response.     

Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits

The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine.     

Antigenicity and immunogenicity of HIV-1 gp140 with different combinations of glycan mutation and V1/V2 region or V3 crown deletion

The carbohydrate moieties on HIV-1 envelope glycoprotein (Env) act as shields to mask conserved neutralizing epitopes, while the hyperimmunogenic variable regions are immunodominant in inducing non-neutralizing antibodies, representing the major challenge for using Env as a vaccine candidate to induce broadly neutralizing antibodies (bNAbs). In this study, we designed a series of HIV-1 gp140 constructs with the removal of N276/N463 glycans, deletion of the V1/V2 region and the V3 crown, alone or in combination. We first demonstrated that all the constructs had a comparable level of expression and were mainly expressed as trimers. Following purification of gp140s from mammalian cells, we measured their binding to bNAbs and non-NAbs in vitro and capability in inducing bNAbs in vivo. Antibody binding assay showed that removal of N276/N463 glycans together with the deletion of V1/V2 region enhanced the binding of gp140s to CD4-binding site-targeting bNAbs VRC01 and 3BNC117, and CD4-induced epitopes-targeting non-NAbs A32, 17b and F425 A1g8, whereas further deletion of V3 crown in the gp140 mutants demonstrated slightly compromised binding capability to these Abs. Immunogenicity study showed that the above mutations did not lead to the induction of a higher Env-specific IgG response via either DNA-DNA or DNA-protein prime-boost strategies in mice, while neutralization assay did not show an apparent difference between wild type and mutated gp140s. Taken together, our results indicate that removal of glycans at N276/N463 and deletion of the V1/V2 region can expose the CD4-binding site and CD4-induced epitopes, but such exposure alone appears incapable of enhancing the induction of bNAbs in mice, informing that additional modification or/and immunization strategies are needed. In addition, the strategies which we established for producing gp140 proteins and for analyzing the antigenicity and immunogenicity of gp140 provide useful means for further vaccine design and assessment.     

Env–2dCD4S60C complexes act as super immunogens and elicit potent,broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1)

The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4^S60C are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env–2dCD4^S60C complexes described here are “super” immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4^60C. Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env–2dCD4^S60C complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.     

Design,display and immunogenicity of HIV1 gp120 fragment immunogens on virus-like particles

The broadly neutralizing antibody against HIV-1, b12, binds to the CD4 binding site (CD4bs) on the outer domain (OD) of the gp120 subunit of HIV-1 Env. We have previously reported the design of an E. coli expressed fragment of HIV-1 gp120, b122a, containing about 70% of the b12 epitope with the idea of focusing the immune response to this structure. Since the b122a structure was found to be only partially folded, as assessed by circular dichroism and protease resistance, we attempted to stabilize it by the introduction of additional disulfide bonds. One such mutant, b122a1-b showed increased stability and bound b12 with 30-fold greater affinity as compared to b122a. Various b122a and OD fragment proteins were displayed on the surface of Qβ virus-like particles. Sera raised against these particles in six-month long rabbit immunization studies could neutralize Tier1 viruses across different subtypes with the best results observed with b122a1-b displayed particles. Significantly higher amounts of antibodies directed towards the CD4bs were also elicited by particles displaying b122a1-b. This study highlights the ability of fragment immunogens to focus the antibody response to the conserved CD4bs of HIV-1.     

Cross-reactive HIV-1-neutralizing activity of serum IgG from a rabbit immunized with gp41 fused to IgG1 Fc: Possible role of the prolonged half-life of the immunogen

The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp41_89.6 was fused to a human IgG₁ Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp140_89.6. Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.