Long trimer-immunization interval and appropriate adjuvant reduce immune responses to the soluble HIV-1-envelope trimer base.

Soluble 'SOSIP'-stabilized HIV-1 envelope glycoprotein (Env) trimers elicit dominant antibody responses targeting their glycan-free base regions, potentially diminishing neutralizing responses. Previously, using a nonhuman primate model, we demonstrated that priming with fusion peptide (FP)-carrier conjugate immunogens followed by boosting with Env trimers reduced the anti-base response. Further, we demonstrated that longer immunization intervals further reduced anti-base responses and increased neutralization breadth. Here, we demonstrate that long trimer-boosting intervals, but not long FP immunization intervals, reduce the anti-base response. Additionally, we identify that FP priming before trimer immunization enhances antibody avidity to the Env trimer. We also establish that adjuvants Matrix M and Adjuplex further reduce anti-base responses and increase neutralizing titers. FP priming, long trimer-immunization interval, and an appropriate adjuvant can thus reduce anti-base antibody responses and improve Env-directed vaccine outcomes.

Soluble prefusion-closed HIV-envelope trimers with glycan-covered bases.

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response.

Assessment of Crosslinkers between Peptide Antigen and Carrier Protein for Fusion Peptide-Directed Vaccines against HIV-1.

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

Broad and ultra-potent cross-clade neutralization of HIV-1 by a vaccine-induced CD4 binding site bovine antibody.

Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC50]: 0.009 µg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC50 of 0.090 µg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs.

Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env.

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

Enhancement of Antibody-Dependent Cellular Cytotoxicity and Phagocytosis in Anti-HIV-1 Human-Bovine Chimeric Broadly Neutralizing Antibodies.

No prophylactic vaccine has provided robust protection against human immunodeficiency virus type 1 (HIV-1). Vaccine-induced broadly neutralizing antibodies (bNAbs) have not been achieved in humans and most animals; however, cows vaccinated with HIV-1 envelope trimers produce bNAbs with unusually long third heavy complementarity-determining regions (CDRH3s). Alongside neutralization, Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP), may be critical for in vivo bNAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bNAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bNAbs by using an exceptionally long 60-amino acid (aa) CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create the following three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding, namely, G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human natural killer (NK) cell activation, and mediated higher levels of ADCC and ADP activity than the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity than GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultralong CDRH3s could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, human immunodeficiency virus (HIV) is still a lifelong persistent virus, and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 broadly neutralizing antibodies (bNAbs) and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bNAbs (with higher potency and breadth than most human bNAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high-potency bovine variable region bNAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

Fusion peptide priming reduces immune responses to HIV-1 envelope trimer base.

Soluble "SOSIP"-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome.

Conjugation of an scFab domain to the oligomeric HIV envelope protein for use in immune targeting.

A promising strategy for the enhancement of vaccine-mediated immune responses is by directly targeting protein antigens to immune cells. Targeting of antigens to the dendritic cell (DC) molecule Clec9A has been shown to enhance antibody affinity and titers for model antigens, and influenza and enterovirus antigens, and may be advantageous for immunogens that otherwise fail to elicit antibodies with sufficient titers and breadth for broad protection, such as the envelope protein (Env) of HIV. Previously employed targeting strategies often utilize receptor-specific antibodies, however it is impractical to conjugate a bivalent IgG antibody to oligomeric antigens, including HIV Env trimers. Here we designed single chain variable fragment (scFv) and single chain Fab (scFab) constructs of a Clec9A-targeting antibody, expressed as genetically fused conjugates with the soluble ectodomain of Env, gp140. This conjugation did not affect the presentation of Env neutralising antibody epitopes. The scFab moiety was shown to be more stable than scFv, and in the context of gp140 fusions, was able to mediate better binding to recombinant and cell surface-expressed Clec9A, although the level of binding to cell-surface Clec9A was lower than that of the anti-Clec9A IgG. However, binding to Clec9A on the surface of DCs was not detected. Mouse immunization experiments suggested that the Clec9A-binding activity of the scFab-gp140 conjugate was insufficient to enhance Env-specific antibody responses. This is an important first proof of principle study demonstrating the conjugation of a scFab to an oligomeric protein antigen, and that an scFab displays better antigen binding than the corresponding scFv. Future developments of this technique that increase the scFab affinity will provide a valuable means to target oligomeric proteins to cell surface antigens of interest, improving vaccine-generated immune responses.

HIV-1-based Virus-like Particles that Morphologically Resemble Mature, Infectious HIV-1 Virions.

A prophylactic vaccine eliciting both broad neutralizing antibodies (bNAbs) to the HIV-1 envelope glycoprotein (Env) and strong T cell responses would be optimal for preventing HIV-1 transmissions. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present authentic-structured, virion-associated Env to elicit bNAbs, and also stimulate T cell responses. Here, we optimize our DNA vaccine plasmids as VLP expression vectors for efficient Env incorporation and budding. The original vector that was used in human trials inefficiently produced VLPs, but maximized safety by inactivating RNA genome packaging, enzyme functions that are required for integration into the host genome, and deleting accessory proteins Vif, Vpr, and Nef. These original DNA vaccine vectors generated VLPs with incomplete protease-mediated cleavage of Gag and were irregularly sized. Mutations to restore function within the defective genes revealed that several of the reverse transcriptase (RT) deletions mediated this immature phenotype. Here, we made efficient budding, protease-processed, and mature-form VLPs that resembled infectious virions by introducing alternative mutations that completely removed the RT domain, but preserved most other safety mutations. These VLPs, either expressed from DNA vectors in vivo or purified after expression in vitro, are potentially useful immunogens that can be used to elicit antibody responses that target Env on fully infectious HIV-1 virions.

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

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

Repeated Vaccination of Cows with HIV Env gp140 during Subsequent Pregnancies Elicits and Sustains an Enduring Strong Env-Binding and Neutralising Antibody Response.

An important feature of a potential vaccine against HIV is the production of broadly neutralising antibodies (BrNAbs) capable of potentially blocking infectivity of a diverse array of HIV strains. BrNAbs naturally arise in some HIV infected individuals after several years of infection and their serum IgG can neutralise various HIV strains across different subtypes. We previously showed that vaccination of cows with HIV gp140 AD8 trimers resulted in a high titre of serum IgG against HIV envelope (Env) that had strong BrNAb activity. These polyclonal BrNAbs concentrated into the colostrum during the late stage of pregnancy and can be harvested in vast quantities immediately after calving. In this study, we investigated the effect of prolonged HIV gp140 vaccination on bovine colostrum IgG HIV Env-binding and BrNAb activity over subsequent pregnancies. Repeated immunisation led to a maintained high titre of HIV Env specific IgG in the colostrum batches, but this did not increase through repeated cycles. Colostrum IgG from all batches also strongly competed with sCD4 binding to gp140 Env trimer and with human-derived monoclonal VRC01 and b12 BrNAbs that bind the CD4 binding site (CD4bs). Furthermore, competition neutralisation assays using RSC3 Env gp120 protein core and a derivative CD4bs mutant, RSC3 Δ371I/P363N, showed that CD4bs neutralising antibodies contribute to the neutralising activity of all batches of purified bovine colostrum IgG. This result indicates that the high IgG titre/avidity of anti-CD4bs antibodies with BrNAb activity was achieved during the first year of vaccination and was sustained throughout the years of repeated vaccinations in the cow tested. Although IgG of subsequent colostrum batches may have a higher avidity towards the CD4bs, the overall breadth in neutralisation was not enhanced. This implies that the boosting vaccinations over 4 years elicited a polyclonal antibody response that maintained the proportion of both neutralising and non-neutralising CD4bs antibodies.

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection.

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.

Breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity: relevance to global HIV vaccine design.

OBJECTIVE: The objective of this study is to determine the breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity (ADCC) in HIV controllers and HIV progressors with a view to design globally relevant HIV vaccines. DESIGN: The breadth of ADCC towards four major HIV-1 Env subtypes was measured in vitro for 11 HIV controllers and 11 HIV progressors. METHODS: Plasma from 11 HIV controllers (including long-term slow progressors, viremic controllers, elite controller and posttreatment controller) and 11 HIV progressors, mostly infected with HIV-1 subtype B, was analysed for ADCC responses. ADCC assays were performed against 10 HIV-1 gp120 and 8 gp140 proteins from four major HIV-1 subtypes (A, B, C and E) and 3 glycosylation-mutant gp140 proteins. RESULTS: ADCC-mediated natural killer cell activation was significantly broader (P = 0.02) and of higher magnitude (P < 0.001) in HIV controllers than in HIV progressors. HIV controllers also showed significantly higher magnitude of ADCC-mediated killing of Env-coated target cells than HIV progressors to both HIV-1 subtype B and the heterologous subtype E gp140 (P = 0.001). We found good ADCC reactivity to subtype B and E Envs, less cross-reactivity to subtype A and minimal cross-reactivity to subtype C Envs. Glycosylation-dependent ADCC epitopes comprise a significant proportion of the total Env-specific ADCC response, as evident from the reduction in ADCC to nonglycosylated form of HIV-1 gp140 (P = 0.004). CONCLUSION: HIV controllers have robust ADCC responses that recognize a broad range of HIV-1 Env. Glycosylation of Env was found to be important for recognition of ADCC epitopes. Identifying conserved ADCC epitopes will assist in designing globally relevant ADCC-based HIV vaccines.