The work presented herein describes the first comprehensive analysis of a partially deglycosylated HIV vaccine candidate envelope protein (Env). The Env, JRFL gp140 ΔCF, with 27 potential glycosylation sites, was partially deglycosylated with PNGase F as part of a strategy to generate a more immunogenic HIV vaccine, and the resulting protein’s glycosylation was characterized in a unique workflow using two different glycosidases, Endo H and Endo F3. This unique analysis protocol provided for coverage on 26 of the 27 glycosylation sites, and the data showed that the biochemical treatment with PNGase F resulted in a highly heterogeneous glycoprotein product that had been partially deglycosylated at most of the glycosylation sites. The protocols described in this work could be useful for characterizing the glycosylation site occupancy of other native or biochemically deglycosylated proteins.

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.

Author Summary

Characterizing early adaptive immune responses to HIV-1 can inform studies of virus persistence, pathogenesis and natural history and can guide rational vaccine design. Previous studies examined the role of neutralizing antibodies (Nab) in acute and chronic HIV-1 infection but not against the precise envelope (Env) glycoproteins of transmitted/founder (T/F) viruses and not in direct comparison with autologous cellular immune responses in the same subjects. Here, we identified T/F HIV-1 env genes and their progeny in three subjects by single genome sequencing and performed a dynamic assessment of Nab responses based on env evolution and phenotypic changes in the Env glycoprotein over time. Surprisingly, we found genetic evidence of Nab activity as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. Although delayed in comparison with autologous CD8 T-cell responses, Nabs appeared earlier in HIV-1 infection than previously recognized and impeded virus entry at low titers. This raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.

The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140JR-FL. Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW664–666 core of the 2F5 epitope and two additional upstream residues (L660,663). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5—they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Genetic factors, as well as antigenic stimuli, can influence antibody repertoire formation. Moreover, the affinity of antigen for unmutated naïve B cell receptors determines the threshold for activation of germinal center antibody responses. The gp41 2F5 broadly neutralizing antibody (bNAb) uses the VH2-5 gene, which has 10 distinct alleles that use either a heavy-chain complementarity-determining region 2 (HCDR2) aspartic acid (DH54) or an HCDR2 asparagine (NH54) residue. The 2F5 HCDR2 DH54 residue has been shown to form a salt bridge with gp41 665K; the VH2-5 germ line allele variant containing NH54 cannot do so and thus should bind less avidly to gp41. Thus, the induction of 2F5 bNAb is dependent on both genetic and structural factors that could affect antigen affinity of unmutated naïve B cell receptors. Here, we studied allelic variants of the VH2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both VH2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the NH54 variant for fusion-intermediate conformation was an order of magnitude lower than that of the DH54 VH2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design.

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens

The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens.

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.

The B cell arm of the immune response becomes activated soon after HIV-1 transmission, yet the initial antibody response does not control HIV-1 replication, and it takes months for neutralizing antibodies to develop against the autologous virus. Antibodies that can be broadly protective are made only in a minority of subjects and take years to develop—too late to affect the course of disease. New studies of the earliest stages of HIV-1 infection, new techniques to probe the human B cell repertoire, the modest degree of efficacy in a vaccine trial, and new studies of human monoclonal antibodies that represent the types of immune responses an HIV-1 vaccine should induce are collectively illuminating paths that a successful HIV-1 vaccine might take.

The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.

Monoclonal antibodies (MAbs) were developed against soluble Ebola virus (EBOV) envelope glycoprotein (GP) for the study of the diversity of EBOV envelope and development of diagnostic reagents. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan, Ivory Coast), and as well as reacted with the Reston nonhuman primate EBOV GPs. A second MAb, 6D11 recognized EBOV GP species of Sudan and Sudan-Gulu. The third MAb, 17A3, was reported originally in the same article to be EBOV GP-specific has now been found to be specific for bovine and human α-2 macroglobulin (α-2M) proteins which were contaminants in the Ebola envelope protein preparation. Thus, while MAbs 15H10 and 6D11 are indeed EBOV GP specific, MAb 17A3 is an α-2 macroglobulin MAb.

Purpose of review

This review discusses select recent data that suggest that indeed it is possible to make a clinically useful preventive vaccine for HIV-1 and outlines some of the remaining obstacles that stand in the way of success.

Recent findings

Passive protection studies, with broad neutralizing antibodies for mucosal simian-HIV challenges, in nonhuman primates have suggested that lower doses of neutralizing antibodies than previously thought may be effective in preventing HIV-1 infection. The use of recombinant antibody technology coupled with the ability to culture single memory B cells has yielded new broad neutralizing antibodies and new targets for vaccine design. The success of the RV144 Thai HIV-1 efficacy trials with a replication-defective recombinant canarypox vector (ALVAC)/gp120 prime, clade B/E recombinant gp120 protein boost showing 31% efficacy has given hope that indeed a protective HIV-1 vaccine can be made.

Summary

Recent data in the last year have provided new hope that a clinically useful preventive HIV-1 vaccine can potentially be made. The path forward will require development of improved immunogens, understanding the correlates of protection to HIV-1, and development of immunogens to induce antibodies that can prevent the initial stages of HIV-1 infection at mucosal sites, in order to improve on the RV144 trial results.

HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [Kd]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41.

Abstract

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of β-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of β-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-γ rather than to β-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including β-chemokines and IFN-γ, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.

The extensive glycosylation of HIV-1 envelope proteins (Env), gp120/gp41, is known to play an important role in evasion of host immune response by masking key neutralization epitopes and presenting the Env glycosylation as “self” to the host immune system. The Env glycosylation is mostly conserved but continues to evolve to modulate viral infectivity. Thus, profiling Env glycosylation and distinguishing interclade and intraclade glycosylation variations are necessary components in unraveling the effects of glycosylation on Env’s immunogenicity. Here, we describe a mass spectrometry-based approach to characterize the glycosylation profiles of two rVV-expressed clade C Envs by identifying the glycan motifs on each glycosylation site and determining the degree of glycosylation site occupancy. One Env is a wild-type Env, while the other is a synthetic “consensus” sequence (C.CON). The observed differences in the glycosylation profiles between the two clade C Envs show that C.CON has more unutilized sites and high levels of high mannose glycans; these features mimic the glycosylation profile of a Group M consensus immunogen, CON-S. Our results also reveal a clade-specific glycosylation pattern. Discerning interclade and intraclade glycosylation variations could provide valuable information in understanding the molecular differences among the different HIV-1 clades and in designing new Env-based immunogens.

An effective HIV vaccine must elicit immune responses that recognize genetically diverse viruses1, 2. It must generate CD8+ T lymphocytes that control HIV replication and CD4+ T lymphocytes that provide help for the generation and maintenance of both cellular and humoral immune responses against the virus3–5. Creating immunogens that can elicit cellular immune responses against the genetically varied circulating isolates of HIV presents an important challenge for creating an AIDS vaccine6, 7. Polyvalent mosaic immunogens derived by in silico recombination of natural strains of HIV are designed to induce cellular immune responses that recognize genetically diverse circulating virus isolates8. In the present study we immunized rhesus monkeys by plasmid DNA prime/ recombinant vaccinia virus boost using vaccine constructs expressing either consensus or polyvalent mosaic proteins. The mosaic immunogens elicited CD8+ T lymphocyte responses to more epitopes of each viral protein than the consensus immunogens, and to more variant sequences of CD8+ T lymphocyte epitopes. This increased breadth and depth of epitope recognition may contribute both to protection against infection by genetically diverse viruses and to the control of variant viruses that emerge as they mutate away from recognition by cytotoxic T lymphocytes.

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (VH and VL) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig VH and VL genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable VH or VL genes. The utility of these Ig gene expression cassettes was established using synthetic VH or VL genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using VH and VL genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

The binding of neutralizing antibodies 2F5 and 4E10 to human immunodeficiency virus type 1 (HIV-1) gp41 involves both the viral membrane and gp41 membrane proximal external region (MPER) epitopes. In this study, we have used several biophysical tools to examine the secondary structure, orientation, and depth of immersion of gp41 MPER peptides in liposomes and to determine how the orientation of the MPER with lipids affects the binding kinetics of monoclonal antibodies (MAbs) 2F5 and 4E10. The binding of 2F5 and 4E10 both to their respective nominal epitopes and to a biepitope (includes 2F5 and 4E10 epitopes) MPER peptide-liposome conjugate was best described by a two-step encounter-docking model. Analysis of the binding kinetics and the effect of temperature on the binding stability of 2F5 and 4E10 to MPER peptide-liposome conjugates revealed that the docking of 4E10 was relatively slower and thermodynamically less favorable. The results of fluorescence-quenching and fluorescence resonance energy transfer experiments showed that the 2F5 epitope was more solvent exposed, whereas the 4E10 epitope was immersed in the polar-apolar interfacial region of the lipid bilayer. A circular dichroism spectroscopic study demonstrated that the nominal epitope and biepitope MPER peptides adopted ordered structures with differing helical contents when anchored to liposomes. Furthermore, anchoring of MPER peptides to the membrane via a hydrophobic anchor sequence was required for efficient MAb docking. These results support the model that the ability of 2F5 and 4E10 to bind to membrane lipid is required for stable docking to membrane-embedded MPER residues. These data have important implications for the design and use of peptide-liposome conjugates as immunogens for the induction of MPER-neutralizing antibodies.

The prevention of infectious disease via prophylactic immunization is a mainstay of global public health efforts. Vaccine design would be facilitated by a better understanding of the type and durability of immune responses generated by different vaccine vectors. We report here the results of a comparative immunogenicity trial of six different vaccine vectors expressing the same insert antigen, cowpox virus B5 (CPXV-B5). Of those vectors tested, recombinant adenovirus (rAd5) was the most immunogenic, inducing the highest titer anti-B5 antibodies and conferring protection from sublethal vaccinia virus challenge in mice after a single immunization. We tested select heterologous prime-boost combinations and identified recombinant vesicular stomatitis virus (rVSV) and recombinant Venezuelan equine encephalitis virus replicons (VRP) as the most synergistic regimen. Comparative data such as those presented here are critical to efforts to generate protective vaccines for emerging infectious diseases as well as for biothreat agents.

Functional human immunodeficiency virus type -1 env clones have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The CMV promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δenv backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions, this allows for quick analysis of multiple env genes from HIV-1 infected individuals.

An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity, and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wildtype (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4+, although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.

The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic group M consensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different strains of mice were immunized with CON6 as well as subtype A, B, or C env immunogens, using a DNA prime-recombinant vaccinia virus boost strategy. T-cell epitopes were mapped by gamma interferon enzyme-linked immunospot analysis using five overlapping Env peptide sets from heterologous subtype A, B, and C viruses. The CON6-derived vaccine was immunogenic and induced a greater number of T-cell epitope responses than any single wild-type subtype A, B, and C env immunogen and similar T-cell responses to a polyvalent vaccine. The responses were comparable to within-clade responses but significantly more than between-clade responses. The magnitude of the T-cell responses induced by CON6 (measured by individual epitope peptides) was also greater than the magnitude of responses induced by individual wild-type env immunogens. Though the limited major histocompatibility complex repertoire in inbred mice does not necessarily predict responses in nonhuman primates and humans, these results suggest that synthetic centralized env immunogens represent a promising approach for HIV-1 vaccine design that merits further characterization.

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated “consensus” env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.