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The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10−5 at week 10 that declined to 10−3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10−5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

High Performance Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering detection (HPSEC-MALS) is an important tool to provide a reliable molecular weight measurement for a large complex biomolecule. A recent HIV-1 soluble envelope trimer vaccine candidate, BG505 DS-SOSIP.664, is among the most glycosylated proteins to enter a clinical trial to date, and determination of its protein and glycan molecular weight is one of the key attributes in pre-clinical characterization. However, protein and glycans possess disparate dndcvalues making molecular weight measurement inaccurate in conventional SEC-MALS. To overcome these challenges, a simple mathematically guided experiment was explored, and a composite dndcvalue was established by utilizing protein and glycan mass contributions for the HIV-1 envelope trimer. This establishment was further verified by an orthogonal mass spectrometry analysis. This innovative, simple, and quick analytical approach can be applied broadly to measuring the molecular weight of various composite molecular structures, such as complex glycoconjugates.

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.

Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (T(FH)) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the T(FH)-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of T(FH) cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only mice that received the combination ADA-1 adjuvanted vaccine had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.

Highlights • K155M stabilizes V1V2 β-strand conformation relevant to broad antibody recognition. • β-strand stabilized V1V2 immunogens elicit antibody responses with greater cross-reactivity. • Fine-tuning immunogen conformation modulates immunogenicity., Antibodies against the HIV-1 V1V2 loops were the only correlate of reduced infection risk in the RV144 vaccine trial, highlighting the V1V2 loops as promising targets for vaccine design. The V1V2 loops are structurally plastic, exhibiting either an α-helix-coil or β-strand conformation. V1V2-specific antibodies may thus recognize distinct conformations, and an antibody’s conformational specificity can be an important determinant of breadth and function. Restricting V1V2 conformational plasticity in an immunogen may thus provide control over the conformational specificity and quality of a vaccine-elicited antibody response. Previously, we identified a V1V2 sequence variant (K155M) that results in enhanced recognition by cross-reactive antibodies recognizing the β-strand conformation. Here, we relate V1V2 antigenicity to immunogenicity by comparing the immunogenicity profiles of wildtype and K155M immunogens in two mouse models. In one model, immunization with gp70 V1V2 K155M but not wildtype elicited antibody responses that were cross-reactive to a panel of heterologous gp120 and gp140 antigens. In a second model, we compared the effect of K155M on immunogenicity in the context of gp70 V1V2, gD V1V2 and gp120, examining the effects of scaffold, epitope-focusing and immunization regimen. K155M variants, especially in the context of a gp120 immunogen, resulted in more robust, durable and cross-reactive antibody responses than wildtype immunogens. Restriction of the β-stranded V1V2 conformation in K155M immunogens may thus be associated with the induction of cross-reactive antibody responses thought to be required of a protective HIV-1 vaccine.

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.

In studies on monoclonal IgG antibodies (mAbs) from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated Abs against a complex conformational epitope with contributions from both gp41 the N terminal and C terminal heptad repeat helices. Despite using the VH1-2 gene segment, known to contribute to some of the broadest neutralizing Abs against HIV, members of these Abs, termed group 76C Abs, did not exhibit broad neutralization. Because of the high number of mutations and use of VH1-2, our goal was to characterize the non-neutralizing functions of Abs of group 76C, to assess if targeting of the epitope correlates with LTNP, and to assess the maturation of these Abs by comparison to their predicted common ancestor. Serum competition assays showed group 76C Abs were enriched in LTNPs, in comparison to VRC-01. Specific group 76C clones 6F5 and 6F11, expressed as recombinant Abs, both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. Competition with group 76C recombinant Ab 6F5 confirms 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc demonstrates comparable ADCC to 6F5 and 6F11 when using gp41 constructs of both clade B and clade C. The functional capability of Abs utilizing germline VH1-2 has implications for disease control and vaccine development.

Induction of antibodies targeting viral glycoproteins is a key for the development of a vaccine against enveloped virus infection. Glycoproteins on the virion exhibiting native multimer structure may be a good immunogen to present antibody epitopes, but it is often difficult to prepare immunogenic inactivated virions. Preparation of soluble glycoprotein multimers has been attempted, while virus-like particles carrying target glycoproteins can be a more immunogenic antigen. In the present study, a target glycoprotein-embedded Sendai virus (SeV) particle was developed for induction of anti-virus antibodies. We constructed a chimeric antigen, HIV-1 EnvF, consisting of HIV-1 Env ectodomain and SeV F transmembrane-cytoplasmic domain, which was shown to be efficiently incorporated into the SeV virion. EnvF was recognized by anti-HIV-1 broadly-neutralizing monoclonal antibodies (bnAbs) including 35O22 that targets an Env trimer-dependent epitope. Analysis revealed that HIV-1

Induction of a sustained and broad antibody (Ab) response is a major goal in developing a protective HIV-1 vaccine. DNA priming alone shows reduced levels of immunogenicity; however, when combined with protein boosting is an attractive vaccination strategy for induction of humoral responses. Using the VC10014 DNA and protein-based vaccine consisting of HIV-1 envelope (Env) gp160 plasmids and trimeric gp140 proteins derived from an HIV-1 clade B infected subject who developed broadly neutralizing serum Abs, and which has been previously demonstrated to induce Tier 2 heterologous neutralizing Abs in rhesus macaques, we evaluated whether MPLA and IL-33 when administered during the DNA priming phase enhances the humoral response in mice. The addition of IL-33 during the gp160 DNA priming phase resulted in high titer gp120-specific plasma IgG after the first immunization. The IL-33 treated mice had higher plasma IgG Ab avidity, breadth, and durability after DNA and protein co-immunization with alum adjuvant as compared to MPLA and alum only treated mice. IL-33 was also associated with a significant IgM Env-specific response and expansion of peritoneal and splenic B-1b B cells. These results indicate that DNA priming in the presence of exogenous IL-33 qualitatively alters the HIV-1 Env-specific humoral response, improving the kinetics and breadth of potentially protective Ab.

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13–15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

To date, inducing the production of broadly neutralizing antibodies (bnAbs) against HIV-1 in humans has been unsuccessful. Several studies have explored the coevolution of HIV-1 and neutralizing antibodies (nAbs), but little is known about what affects the lack of bnAbs after long-term infection. A better understanding of the coevolution of the virus and nAbs in cases involving no bnAb production will help in the design of an effective HIV-1 vaccine. An individual with acute CRF01_AE HIV-1 infection who lacked bnAbs at just over 2 years post-infection (p.i.) was identified from a cohort of HIV negative men who have sex with men. The coevolution of the viral envelope gene and nAbs was studied over 741 days p.i. Strain-specific antibodies (ss-Abs) to the transmitted/founder (T/F) virus developed within 54 days p.i., but plasma collected at subsequent time points could not neutralize synchronous viruses until 557 days p.i., when the plasma acquired low-level synchronous but not heterologous neutralizing activity. The V4 region of envelope gene mutated firstly and continually evolve up to 2 years p.i. Multiple variations in the V4 region, including substitutions, deletions and glycosylation mutations, were driven by ss-Abs and mediated immune escape partially by impacting the binding of nAbs to the virus. The remarkable variations in the V4 region mediated immune escape from ss-Abs and contributed to the affinity maturation of ss-Abs against the T/F virus but may not promote the development of bnAbs. Thus, the V4 region might not be a good target for an HIV-1 vaccine.

Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product. Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products. The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility. This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications. Clinical trial identifier. NCT03220724, NCT03856996.

Background Eliciting durable humoral immunity with sufficient breadth and magnitude is important for HIV-1 vaccine design. The HVTN 114 vaccine trial evaluated different boost regimens administered after a 7-year rest period in participants previously enrolled in HVTN 205, who received either three MVA/HIV62B (MMM) or two DNA and two MVA/HIV62B (DDMM) injections; both vaccines expressed multiple HIV-1 antigens in non-infectious virus-like-particles. The primary objective of HVTN 114 was to assess the impact of a heterologous gp120 protein AIDSVAX B/E boost on the magnitude, breadth and durability of vaccine-induced immune responses. Methods We enrolled 27 participants from HVTN 205 into five groups. Eight participants who previously received MMM were randomized and boosted with either MVA/HIV62B alone (T1; n = 4) or MVA/HIV62B and AIDSVAX B/E (T2; n = 4). Nineteen participants who received DDMM were randomized and boosted with MVA/HIV62B alone (T3; n = 6), MVA/HIV62B and AIDSVAX B/E (T4; n = 6), or AIDSVAX B/E alone (T5; n = 7). Boosts were at months 0 and 4. Participants were followed for safety and immunogenicity for 10 months and were pooled for analysis based on the regimen: MVA-only (T1 + T3), MVA + AIDSVAX (T2 + T4), and AIDSVAX-only (T5). Results All regimens were safe and well-tolerated. Prior to the boost vaccination, binding antibody and CD4+T-cell responses were observed 7 years after HVTN 205 vaccinations. Late boosting with AIDSVAX, with or without MVA, resulted in high binding antibody responses to gp120 and V1V2 epitopes, with increased magnitude and breadth compared to those observed in HVTN 205. Late boosting with MVA, with or without AIDSVAX, resulted in increased gp140 and gp41 antibody responses and higher CD4+T-cell responses to Env and Gag. Conclusions Late boosting with AIDSVAX, alone or in combination with MVA, can broaden binding antibody responses and increase T-cell responses even years following the original MVA/HIV62B with or without DNA-priming vaccine.

Metastable glycosylated immunogens present challenges for GMP manufacturing. The HIV-1 envelope (Env) glycoprotein trimer is covered by N-linked glycan comprising half its mass and requires both trimer assembly and subunit cleavage to fold into a prefusion-closed conformation. This conformation, the vaccine-desired antigenic state, is both metastable to structural rearrangement and labile to subunit dissociation. Prior reported GMP manufacturing for a soluble trimer stabilized in a near-native state by disulfide (SOS) and Ile-to-Pro (IP) mutations has employed affinity methods based on antibody 2G12, which recognizes only ~30% of circulating HIV strains. Here, we develop a scalable manufacturing process based on commercially available, non-affinity resins, and we apply the process to current GMP (cGMP) production of trimers from clades A and C, which have been found to boost cross-clade neutralizing responses in vaccine-test species. The clade A trimer, which we named “BG505 DS-SOSIP.664”, contained an engineered disulfide (201C-433C; DS) within gp120, which further stabilized this trimer in a prefusion-closed conformation resistant to CD4-induced triggering. BG505 DS-SOSIP.664 was expressed in a CHO-DG44 stable cell line and purified with initial and final tangential flow filtration steps, three commercially available resin-based chromatography steps, and two orthogonal viral clearance steps. The non-affinity purification enabled efficient scale-up, with a 250 L-scale cGMP run yielding 9.6 g of purified BG505 DS-SOSIP.664. Antigenic analysis indicated retention of a prefusion-closed conformation, including recognition by apex-directed and fusion peptide-directed antibodies. The developed manufacturing process was suitable for 50 L-scale production of a second prefusion-stabilized Env trimer vaccine candidate, ConC-FP8v2 RnS-3mut-2G-SOSIP.664, yielding 7.8 g of this consensus clade C trimer. The successful process development and purification scale-up of HIV-1 Env trimers from different clades by using commercially available materials provide experimental demonstration for cGMP manufacturing of trimeric HIV-Env vaccine immunogens, in an antigenically desired conformation, without the use of costly affinity resins.

Two HIV-1 vaccines (SAAVI DNA-C2 and SAAVI MVA-C) were previously developed in South Africa and tested in preclinical studies and Phase 1 clinical trials. Here we report on improvements made to the SAAVI MVA-C vaccine design which include: the use of different promoters for both the Gag and Env genes, replacement of the native Gag gene with an in silico designed subtype C mosaic Gag antigen which forms virus-like particles and the modification of Env by sequence changes to improve stability and transport to the cell surface. A head-to-head comparison of the newly conceived MVAGD5 candidate vaccine with SAAVI MVA-C showed increased in vitro expression of both Env and Gag, and superior immunogenicity in rabbits. MVAGD5 induced high levels of binding antibodies to Env and Tier 1A and 1B neutralizing antibodies, neither of which were induced by SAAVI MVA-C.

Recent studies have revealed that the interface of gp120 and gp41 and some parts of gp41 are also critical epitopes for elicitation of broadly neutralizing antibodies. Therefore, potential trimeric gp41 or gp140 immunogen candidates are needed. Previously, we developed a trimer motif MTQ and demonstrated that it could help formation of trimeric gp120 and gp140 proteins. In the present study, we immunized Balb/c mice using trimeric gp41-expressing plasmid for prime and monomeric gp41 or trimeric gp140 protein as well as a mutant (Q577A) for boost. The antibody responses in the context of regimens with various immunization intervals and DNA adjuvants including praziquantel (PZQ), cimetidine (CIM), and amiloride (AML) were evaluated. We found that these three adjuvants were not enough to elicit remarkable specific Abs after gp41 DNA immunization, while AML could significantly promote humoral immune responses after protein boosts. Long immunization interval could induce the specific binding Abs earlier and higher and maintain a high level of Abs in the following 27 weeks after final protein boost. Moreover, two times of protein boosts with DNA adjuvant and a longer time interval achieved a higher titer of specific Abs than three times of protein boosts with a shorter time interval. Q577A mutant was benefit for trimeric gp140 boost in the production of binding Abs but harmful to inducing neutralizing Abs, while this mutant in monomeric gp41 presented the opposite trend which may be associated with the immunogen structures. This study highlights the significance of DNA adjuvant Amiloride and long immunization interval in promoting antibody responses and provides new insights into effective HIV immunization regimen design in the future.

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.

Background Superinfection of individuals already infected with HIV-1 suggests that pre-existing immune responses may not adequately protect against re-infection. We assessed high-risk female sex workers initially infected with HIV-1 clades A, D or A/D recombinants, to determine if HIV-1 broadly neutralizing antibodies were lacking prior to superinfection. Methods Six superinfected female sex workers previously stratified by HIV-1 high-risk behavior, infecting virus clade and volunteer CD4 counts were evaluated at baseline (n = 5) and at 350 days post-superinfection (n = 6); one superinfected volunteer lacked pre-superinfection plasma. Retrospective plasmas were assessed for neutralization of a multi-clade panel of 12 HIV-1 viruses before superinfection, and then at quarterly intervals thereafter. Similarly stratified singly infected female sex workers were correspondingly assessed at baseline (n = 19) and 350 days after superinfection (n = 24). Neutralization of at least 50% of the 12 viruses (broad neutralization), and geometric means of the neutralization titers (IC50) were compared before and after superinfection; and were correlated with the volunteer HIV-1 superinfection status, CD4 counts, and pseudovirus clade. Results Preexisting broad neutralization occurred in 80% (4/5) of the superinfected subjects with no further broadening by 350 days after superinfection. In one of the five subjects, HIV-1 superinfection occurred when broad neutralization was lacking; with subsequent broadening of neutralizing antibodies occuring within 9 months and plateauing by 30 months after detection of superinfection. Clade B and C pseudoviruses were more sensitive to neutralization (13; [87%]); and (12; [80%]) than the locally circulating clades A (10; [67%]) and D (6; [40%]), respectively (p = 0.025). Low antibody titers correlated with clade D viruses and with >500 CD4 T cell counts, but not with the superinfection status. Conclusion These data demonstrate that HIV-1 superinfection can occur both in the presence, and in the absence of broadly neutralizing antibodies.

Human Immunodeficiency virus (HIV) virus-like Particles (VLPs) composed of HIV(IIIB) Gag and HIV(BaL) gp120/gp41 envelope are a pseudovirus vaccine capable of presenting antigens in their native conformations. To enhance the immunogenicity of the HIV Env antigen, VLPs were coupled to VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) containing one of four toll-like-receptor (TLR) ligands, each activating a receptor with distinct cellular localization and downstream pathways. C57Bl/6 mice were vaccinated by intranasal prime followed by two sub-cheek boosts and their sera immunoglobulin and neutralizing potency were measured over a duration of 3 months after vaccination. PBS control, VLPs alone, CALV + VLPs, and VLPs complexed with CALV and ligands for TLR2 (PAM3CAG), TLR3 (dsRNA), TLR4 (MPLA), and TLR7/8 (resiquimod) were evaluated based on antibody titer, IgG1 and IgG2c class switching, germinal center formation, T follicular cells and potency of neutralizing antibodies. Consistently, the TLR3 ligand dsRNA complexed to CALV and in combination with VLPs (CALV(dsRNA)+VLPs) induced the strongest response. CALV(dsRNA)+VLPs induced the highest titers against the recombinant vaccine antigens clade B Bal gp120 and pr55 Gag. Additionally, CALV(dsRNA)+VLPs induced cross-clade antibodies, represented by high titers of antibody to clade c 96ZM651 gp120. CALV(dsRNA)+VLPs induced predominantly IgG2c over IgG1, a response associated with T helper type 1 (Th1)-like cytokines. In turn, CALV(dsRNA)+VLP immunized mice generated the most potent neutralizing antibodies against HIV strain MN.3. Finally, at time of sacrifice, a significant increase in germinal center B cells and T follicular cells was detected in mice which received CALV(dsRNA)+VLPs compared to PBS. Our results indicate that CALV(dsRNA) is a superior adjuvant for HIV VLPs in generating a Th1-like immunoglobulin profile, while prolonging lymph node germinal centers, T follicular cells and generating neutralizing antibodies to a highly sensitive tier 1A variant of HIV.

Vaccines eliciting protective and persistent immune responses against multiple human immunodeficiency virus type 1 (HIV-1) clades are needed. This study evaluated the persistence of immune responses induced by an investigational, AS01-adjuvanted HIV-1 vaccine as long as 14 years after vaccination.This phase I, open-label, descriptive, mono-centric, extension study with a single group (NCT03368053) was conducted in adults who received ≥3 doses of the clade B gp120-NefTat/AS01At Year 14, positive IgG binding antibody responses were detected in 15 out of the 16 antigens from the BAMA V1V2 breadth panel, with positive response rates ranging from 7.1% to 60.7%. The highest response rates were observed for clade B strain V1V2 antigens, with some level of binding antibodies against clade C strains. Anti-V1V2 IgG3 response magnitude breadth, which correlated with decreased risk of infection in a previous efficacy trial, was of limited amplitude. Response rates to the antigens from the gp120 and gp140 breadth panels ranged from 7.7% to 94.1% and from 15.4% to 96.2% at Year 14, respectively. Following stimulation with gp120 peptide pool, highly polyfunctional gp120-specific CD4+ T-cells persisted up to Year 14, with high frequencies of CD40L tumor necrosis factor alpha (TNF-α), CD40L interleukin-2 (IL-2), CD40L TNF-α IL-2 and CD40L interferon gamma (IFN-γ) TNF-α IL-2 CD4+ T-cells, but no CD8+ T-cells detected.Persistent antibodies binding to HIV-1 envelope glycoproteins, including the V1V2-scaffold, and gp120-specific cellular immunity were observed in volunteers vaccinated 14 years earlier with the gp120-NefTat/AS01

The potential advantages and unique challenges of the early life immune system for the development of HIV-specific broadly neutralizing antibodies were discussed during a workshop entitled “Immunological Mechanisms of Inducing HIV Immunity in Infants” sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) in conjunction with the 2018 HIVR4P Conference held in Madrid, Spain. A safe and effective HIV vaccine remains a critical need in the fight against the HIV pandemic, especially to prevent emerging infections in infants, adolescents, and young adults. To successfully target these populations, a vaccine should ideally induce protective immune responses during childhood. Interestingly, several recent studies highlighting differences in immune responses between adults and children have suggested that the early life immune system could present advantages for the elicitation of broadly neutralizing antibodies (bnAbs), a response highly desired for an HIV vaccine. Notably, HIV-infected children develop bnAbs responses earlier and more frequently than infected adults; with emerging evidence that the pathways of elicitation of bnAb lineages may differ between adults and children. Moreover, there is precedent for the prevention of lifelong infections with pediatric immunization, and early life provides a unique window of opportunity for the administration of a multi-dose HIV vaccine that will likely be needed to achieve protective immunity. Further understanding of how the distinct early life immune system can be harnessed to trigger bnAb lineages for induction of durable and polyfunctional HIV-specific immunity is warranted. This strategy will include testing promising HIV vaccine candidates in pediatric populations in preclinical and clinical studies. Novel approaches to identify molecular markers of protection are also key to guide and accelerate pediatric HIV vaccine development., SCOPUS: cp.j, info:eu-repo/semantics/published

An effective prophylactic vaccine targeting HIV must induce a robust humoral response and must direct the bulk of this response to the mucosa-the primary site of HIV transmission. The chemokine, CCL28, is secreted by epithelial cells at mucosal surfaces and recruits′ cells expressing its receptor CCR10. CCR10 is predominantly expressed by IgA + ASCs. We hypothesized that co-immunization with plasmid DNA encoding consensus envelope antigens with plasmid-encoded CCL28 would enhance anti-HIV IgA responses at mucosal surfaces. Indeed, animals receiving pCCL28 and pEnvA/C had significantly increased HIV-specific IgA in fecal extract. Surprisingly, CCL28 co-immunization induced a significant increase in anti-HIV IgG in the serum in mice compared to those receiving pEnvA/C alone. These robust antibody responses were not associated with changes in the frequency of germinal center B cells but depended upon the expression of CCR10, as these responses we abolished in CCR10-deficient animals. Finally, immunization with CCL28 led to increased frequencies in HIV-specific CCR10 + and CCR10 + IgA + B cells in the small intestine and Peyer’s patches of vaccinated animals as compared to those receiving pEnvA/C alone. These data indicate that CCL28 administration can enhance antigen-specific humoral responses systemically and at mucosal surfaces.

The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env. Truncated Env overexpressed VRC01 and 17b binding antigen on the surface of transduced cells while the full length Env vaccine presented more and similar amounts of antigen binding to the trimer conformation sensitive antibodies PGT151 and PGT145, respectively. The adenoviral vectors were used to prime Balb/c mice followed by sequential boosting with chimpanzee type 63, and chimpanzee type 3 adenoviral vectors encoding SIVmac239 Gag and full length consensus Env. Both vaccine regimens induced increasing titers of binding antibody responses after each immunization, and significant differences in immune responses between the two groups were observed after the final immunization. Full length Env priming skewed antibody responses towards gp41, while truncated Env priming induced responses primarily targeting gp120 containing and derived antigens. Importantly, no differences in neutralizing antibody responses were found between the different priming regimens as both induced high titered tier 1 neutralizing antibodies, but no tier 2 antibodies, possibly reflecting the similar presentation of trimer specific antibody epitopes. The described vaccine regimens provide insight into the effects of the HIV-1 Env cytoplasmic tail on epitope presentation and subsequent immune responses, which is relevant for the interpretation of current clinical trials that are using truncated Env as an immunogen. The regimens described here provide similar neutralization titers, and thus are useful for investigating the importance of specificity in non-neutralizing antibody mediated protection against viral challenge.

The 2015 Cent Gardes Conference on HIV vaccines took place on October 25-27 at the Merieux Foundation Conference Center in Veyrier du Lac, near Annecy, France. The meeting reviewed progress in the development of HIV vaccines and identified new directions of future research. The field has advanced incrementally over the past year but major progress will require additional information from new clinical trials. In this article, we review the presentations on humoral immune responses to HIV, and highlight the difficulty of eliciting broadly neutralizing antibodies by vaccination. Advances in cellular immunity for HIV prevention will be reviewed separately, in a following article.

RV144 vaccinees with low HIV-1 Envelope-specific IgA antibodies (Abs) also had Abs directed to the hypervariable region 3 (V3) that inversely correlated with infection risk. Thus, anti-V3 HIV-1 Abs may contribute to protection from HIV-1 infection. The V3 region contains two dominant clusters of epitopes; one is preferentially recognized by mAbs encoded by VH5-51 and VL lambda genes, while the second one is recognized by mAbs encoded by other VH genes. We designed a study in rhesus macaques to induce anti-V3 Abs specific to each of these two dominant clusters of V3 epitopes to test whether the usage of the VH5-51 gene results in different characteristics of antibodies. The two C4-V3 immunogens used for immunization were each comprised of a fusion of the C4 peptide containing the T cell epitope and a V3 mimotope peptide mimicking the V3 epitope. The C4-447 peptide was designed to target B cells with several VH1-VH4 genes, the C4-VH5-51 peptide was designed to specifically target B cells with the VH5-51 gene. Six animals in two groups were immunized five times with these two immunogens, and screening of 10 sequential plasma samples post immunization demonstrated that C4-447 induced higher titers of plasma anti-V3 Abs and significantly more potent neutralizing activities against tier 1 and some tier 2 pseudoviruses than C4-VH5-51. Levels of anti-V3 Abs in buccal secretions were significantly higher in sequential samples derived from C4-447- than from C4-VH5-51-immunized animals. The titers of anti-V3 Abs in plasma strongly correlated with their levels in mucosal secretions. The results show that high titers of vaccine-induced anti-V3 Abs in plasma determine the potency and breadth of neutralization, as well as the rate of transduction of Abs to mucosal tissues, where they can play a role in preventing HIV-1 infection.

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.

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.

Phase 1 clinical studies will soon evaluate novel HIV-1 envelope immunogens targeting distinct 'germline' and memory B cell receptors to ultimately elicit HIV-1 broadly neutralizing antibodies (bNAbs). The National Institute of Allergy and Infectious Diseases (NIAID) recently convened a panel of US-based expert scientists, clinicians, sponsors and ethicists to discuss the role of sampling draining lymph nodes within preventive HIV vaccine trials. The meeting addressed the importance of evaluating germinal center (GC) responses following immunization to predict bNAb potency and breadth, and reviewed key aspects of this procedure within the clinical research setting, including informed consent, adverse event monitoring, study participant acceptability, medical expertise and training. We review highlights from the meeting and discuss the advantages and disadvantages of sampling lymph nodes by excisional biopsies compared to fine needle aspirations (FNA) in the context of prophylactic HIV vaccine trials.

Liposomes have shown promise as constituents of adjuvant formulations in vaccines to parasitic and viral diseases. A particular type of liposomal construct, referred to as Army Liposome Formulation (ALF), containing neutral and anionic saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA), has been used as an adjuvant for many years. Here we investigated the effects of physical and chemical changes of ALF liposomes on adjuvanted immune responses to CN54 gp140, a recombinant HIV-1 envelope protein. While holding the total amounts of liposomal MPLA and the gp140 antigen constant, different liposome sizes and liposomal MPLA:phospholipid molar ratios, and the effect of adding QS21 to the liposomes were compared for inducing immune responses to the gp140. For liposomes lacking QS21, higher titers of IgG binding antibodies to gp140 were induced by small unilamellar vesicle (SUV) rather than by large multilamellar vesicle (MLV) liposomes, and the highest titers were obtained with SUV having the MPLA:phospholipid ratio of 1:5.6. ALF plus QS21 (ALFQ) liposomes induced the same maximal binding antibody titers regardless of the MPLA:phospholipid ratio. ALF MLV liposomes induced mainly IgG1 and very low IgG2a antibodies, while ALF SUV liposomes induced IgG1≥IgG2a>IgG2b antibodies. Liposomes containing QS21 induced IgG1>IgG2a>IgG2b>IgG3 antibodies. ELISPOT analysis of splenocytes from immunized mice revealed that ALF liposomes induced low levels of IFN-γ, but ALFQ induced high levels. ALF and ALFQ liposomes each induced approximately equivalent high levels of IL-4. Based on antibody subtypes and cytokine secretion, we conclude that ALF liposomes predominantly stimulate Th2, while ALFQ strongly induces both Th1 and Th2 immunity. When CN54 gp140 was adjuvanted with either ALF or ALFQ liposomes, antibodies were induced that neutralized two HIV-1 tier 1 clade C strain pseudoviruses.

Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation.

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1 + dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline.

The vast majority of HIV-1 infections occur at mucosa during sexual contact. It may therefore be advantageous to provide mucosal barrier protection against this entry by mucosal vaccination. While a number of mucosal routes of vaccination are possible, many like enteric oral vaccines or intranasal vaccines have significant impediments that limit vaccine efficacy or pose safety risks. In contrast, immunogens applied to the sublingual region of the mouth could provide a simple route for mucosal vaccination. While sublingual immunization is appealing, this site does not always drive strong immune responses, particularly when using protein antigens. To address this issue, we have tested the ability of two mucosal adjuvants: alpha-galactosylceramide (αGalCer) that is a potent stimulator of natural killer T cells and CpG-oligodeoxynucleotide (CpG-ODN) a TLR9 agonist for their ability to amplify immune responses against clade C gp140 HIV-1 envelope protein antigen. Immunization with envelope protein alone resulted in a weak T cell and antibody responses. In contrast, CD4(+) and CD8(+) T cells responses in systemic and mucosal tissues were significantly higher in mice immunized with gp140 in the presence of either αGalCer or CpG-ODN and these responses were further augmented when the two adjuvants were used together. While both the adjuvants effectively increased gp140-specific serum IgG and vaginal IgA antibody levels, combining both significantly improved these responses. Memory T cell responses 60 days after immunization revealed αGalCer to be more potent than CpG-ODN and the combination of the αGalCer and CpG-ODN adjuvants was more effective than either alone. Serum and vaginal washes collected 60 days after immunization with gp140 with both αGalCer and CpG-ODN adjuvants had significant neutralization activity against Tier 1 and Tier 2 SHIVs. These data support the utility of the sublingual route for mucosal vaccination particularly in combination with αGalCer and CpG-ODN adjuvants.

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses.