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

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

BST-2 expression does not modulate IAV susceptibility of murine epithelial cells and macrophages to IAV infection.

<p>(A) Primary AEC or (B) macrophages isolated from BST-2 wild type (WT) and BST-2-deficient (BST-2^-/-) mice were incubated with the indicated MOI and strain of IAV for 1 hour at 37°C, washed to remove excess virus and cultured as indicated. Monolayers were fixed at 8 hours post-infection before staining by immunofluorescence to detect newly synthesized viral NP. Data show the mean (± 1 SD) pooled from 3 independent experiments. n.s. = no significant difference, <i>p</i> = > 0.05, two-way ANOVA followed by Bonferroni analysis.</p

BST-2 expression does not modulate IAV release from murine epithelial cells and macrophages following IAV infection.

<p>(A) Primary AEC or (B) macrophages isolated from BST-2 wild type (WT) and BST-2-deficient (BST-2^-/-) mice were incubated with the indicated strain of IAV for 1 hour at 37°C, washed to remove excess virus and cultured. AEC were infected at a MOI of 1 of HKx31, Brazil/78 and Sol Is/06 and macrophages infected at a MOI 5 for HKx31 and a MOI of 50 for Brazil/78. Culture supernatants were removed at 2 hours, or at 8, 24 or 48 hours as indicated, clarified by centrifugation and titres of infectious virus were determined by plaque assay on MDCK cells. Data is displayed as viral titer (log₁₀PFU/ml) and represent the mean (± 1 SD) from triplicate samples. Data is representative of 2 independent experiments. n.s. = no significant difference, *** <i>p</i> < 0.001, two-way ANOVA followed by Bonferroni analysis.</p

BST-2 expression is upregulated on murine macrophages but not alveolar epithelial cells in response to influenza A virus.

<p>Monolayers of (A) the LA-4 AEC line and primary AEC, or (B) RAW264.7 macrophages and primary macrophages were incubated (i) with a MOI of 5 (HKx31) for 1 hour at 37°C and washed to remove excess virus (IAV infection, solid black line), (ii) in 1000 IU/ml recombinant mouse IFNα (dashed line) or (iii) in media alone (no infection, grey histogram). Cells were then incubated at 37°C for a total of 4 or 24 hours and levels of cell-surface BST-2 determined by flow cytometry. For each cell type, the isotype control (solid black histograms) is shown for ‘no infection’ cells only but is representative of profiles obtained using IAV-infected and IFNα-treated cells. Data are representative of 3 independent experiments.</p

Lack of BST-2 does not alter the susceptibility of mice to IAV infection or the ability of IAV to replicate in the airways.

<p>(A) Peripheral blood from wild type and BST-2^-/- mice was screened by the Advia2120 automated hematology analyzer. Each symbol represents an individual mouse and the bar indicates the mean. n.s. = no significant difference, <i>p</i> = > 0.05, two way ANOVA followed by Bonferroni analysis. (B) Wild type and BST-2^-/- mice were infected with either 10² PFU or 10⁴ PFU of HKx31 via the intranasal route. Mice were weighed daily and the results expressed as the mean percentage weight change per group (± 1 SEM) relative to original body weight. (C) Virus titres were determined in clarified homogenates prepared from lungs and nasal tissues using a standard plaque assay on MDCK cells. Symbols show titres from individual animals and horizontal bars represent the mean virus titre. n.s. = no significant difference, <i>p</i> = > 0.05, Student’s <i>t</i>-test; two-tailed.</p

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

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 &Aring;, 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

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

Summary: 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