Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

Serological assays to measure dimeric IgA antibodies in SARS-CoV-2 infections.

Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.

Virus-Like Particles Containing the E2 Core Domain of Hepatitis C Virus Generate Broadly Neutralizing Antibodies in Guinea Pigs.

A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct-acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384 to 661) that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bNAbs) than the parental form (receptor-binding domain [RBD]). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S, RBD-S, Δ123A7-S, and RBDA7-S; in the last two, 7 cysteines were replaced with alanines. While all four E2-S variant VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bNMAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (NAbs), with three of those animals generating bNAbs against 7 genotypes. Immune serum generated by animals with NAbs mapped to major neutralization epitopes located at residues 412 to 420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease, including cirrhosis and cancer. Broadly neutralizing antibodies that recognize the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here, we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus-like particles that display epitopes recognized by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

A pan-genotype hepatitis C virus viral vector vaccine generates T cells and neutralizing antibodies in mice.

BACKGROUND AND AIMS: A prophylactic vaccine targeting multiple HCV genotypes (gt) is urgently required to meet World Health Organization elimination targets. Neutralizing antibodies (nAbs) and CD4+ and CD8+ T cells are associated with spontaneous clearance of HCV, and each may contribute to protective immunity. However, current vaccine candidates generate either nAbs or T cells targeting genetically variable epitopes and have failed to show efficacy in human trials. We have previously shown that a simian adenovirus vector (ChAdOx1) encoding conserved sequences across gt1-6 (ChAd-Gt1-6), and separately gt-1a E2 protein with variable regions deleted (E2Δ123HMW ), generates pan-genotypic T cells and nAbs, respectively. We now aim to develop a vaccine to generate both viral-specific B- and T-cell responses concurrently. APPROACH AND RESULTS: We show that vaccinating with ChAd-Gt1-6 and E2Δ123HMW sequentially in mice generates T-cell and antibody (Ab) responses comparable to either vaccine given alone. We encoded E2Δ123 in ChAdOx1 (ChAd-E2Δ123) and show that this, given with an E2Δ123HMW protein boost, induces greater CD81-E2 inhibitory and HCV-pseudoparticle nAb titers compared to the E2Δ123HMW prime boost. We developed bivalent viral vector vaccines (ChAdOx1 and modified vaccinia Ankara [MVA]) encoding both Gt1-6 and E2Δ123 immunogens (Gt1-6-E2Δ123) generating polyfunctional CD4+ and CD8+ T cells and nAb titers in prime/boost strategies. This approach generated nAb responses comparable to monovalent E2Δ123 ChAd/MVA vaccines and superior to three doses of recombinant E2Δ123HMW protein, while also generating high-magnitude T-cell responses. CONCLUSIONS: These data are an important step forward for the development of a pan-genotype HCV vaccine to elicit T cells and nAbs for future assessment in humans.

Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine.

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

HIV-1 Env- and Vpu-Specific Antibody-Dependent Cellular Cytotoxicity Responses Associated with Elite Control of HIV.

Studying HIV-infected individuals who control HIV replication (elite controllers [ECs]) enables exploration of effective anti-HIV immunity. HIV Env-specific and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection, but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses in sera were studied using a novel enzyme-linked immunosorbent assay (ELISA)-based dimeric recombinant soluble FcγRIIIa (rsFcγRIIIa)-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays, and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcγRIIIa, activating NK cells, and mediating granzyme B activity (all P < 0.01) than viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcγRIIIa binding and NK cell activation than viremic subjects (both P < 0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control.IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove to be of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines.

The core domain of hepatitis C virus glycoprotein E2 generates potent cross-neutralizing antibodies in guinea pigs.

A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form. CONCLUSION: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).

Inhibition of two temporal phases of HIV-1 transfer from primary Langerhans cells to T cells: the role of langerin.

Epidermal Langerhans cells (eLCs) uniquely express the C-type lectin receptor langerin in addition to the HIV entry receptors CD4 and CCR5. They are among the first target cells to encounter HIV in the anogenital stratified squamous mucosa during sexual transmission. Previous reports on the mechanism of HIV transfer to T cells and the role of langerin have been contradictory. In this study, we examined HIV replication and langerin-mediated viral transfer by authentic immature eLCs and model Mutz-3 LCs. eLCs were productively infected with HIV, whereas Mutz-3 LCs were not susceptible because of a lack of CCR5 expression. Two successive phases of HIV viral transfer to T cells via cave/vesicular trafficking and de novo replication were observed with eLCs as previously described in monocyte-derived or blood dendritic cells, but only first phase transfer was observed with Mutz-3 LCs. Langerin was expressed as trimers after cross-linking on the cell surface of Mutz-3 LCs and in this form preferentially bound HIV envelope protein gp140 and whole HIV particles via the carbohydrate recognition domain (CRD). Both phases of HIV transfer from eLCs to T cells were inhibited when eLCs were pretreated with a mAb to langerin CRD or when HIV was pretreated with a soluble langerin trimeric extracellular domain or by a CRD homolog. However, the langerin homolog did not inhibit direct HIV infection of T cells. These two novel soluble langerin inhibitors could be developed to prevent HIV uptake, infection, and subsequent transfer to T cells during early stages of infection.

Antibody-dependent effector functions against HIV decline in subjects receiving antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy (cART) effectively controls human immunodeficiency virus (HIV) infection but does not eliminate HIV, and lifelong treatment is therefore required. HIV-specific cytotoxic T lymphocyte (CTL) responses decline following cART initiation. Alterations in other HIV-specific immune responses that may assist in eliminating latent HIV infection, specifically antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP), are unclear. METHODS: A cohort of 49 cART-naive HIV-infected subjects from Thailand (mean baseline CD4 count, 188 cells/µL; mean viral load, 5.4 log10 copies/mL) was followed for 96 weeks after initiating cART. ADCC and ADP assays were performed using serum samples obtained at baseline and after 96 weeks of cART. RESULTS: A 35% reduction in HIV type 1 envelope (Env)-specific ADCC-mediated killing of target cells (P<.001) was observed after 96 weeks of cART. This was corroborated by a significant reduction in the ability of Env-specific ADCC antibodies to activate natural killer cells (P<.001). Significantly reduced ADP was also observed after 96 weeks of cART (P=.018). CONCLUSIONS: This longitudinal study showed that cART resulted in significant reductions of HIV-specific effector antibody responses, including ADCC and ADP. Therapeutic vaccines or other immunomodulatory approaches may be required to improve antibody-mediated control of HIV during cART.

The maturation of antibody technology for the HIV epidemic.

Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV.

Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure.

Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.

Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission.

Application of a case-control study design to investigate genotypic signatures of HIV-1 transmission.

BACKGROUND: The characterization of HIV-1 transmission strains may inform the design of an effective vaccine. Shorter variable loops with fewer predicted glycosites have been suggested as signatures enriched in envelope sequences derived during acute HIV-1 infection. Specifically, a transmission-linked lack of glycosites within the V1 and V2 loops of gp120 provides greater access to an α4ß7 binding motif, which promotes the establishment of infection. Also, a histidine at position 12 in the leader sequence of Env has been described as a transmission signature that is selected against during chronic infection. The purpose of this study is to measure the association of the presence of an α4ß7 binding motif, the number of N-linked glycosites, the length of the variable loops, and the prevalence of histidine at position 12 with HIV-1 transmission. A case-control study design was used to measure the prevalence of these variables between subtype B and C transmission sequences and frequency-matched randomly-selected sequences derived from chronically infected controls. RESULTS: Subtype B transmission strains had shorter V3 regions than chronic strains (p = 0.031); subtype C transmission strains had shorter V1 loops than chronic strains (p = 0.047); subtype B transmission strains had more V3 loop glycosites (p = 0.024) than chronic strains. Further investigation showed that these statistically significant results were unlikely to be biologically meaningful. Also, there was no difference observed in the prevalence of a histidine at position 12 among transmission strains and controls of either subtype. CONCLUSIONS: Although a genetic bottleneck is observed after HIV-1 transmission, our results indicate that summary characteristics of Env hypothesised to be important in transmission are not divergent between transmission and chronic strains of either subtype. The success of a transmission strain to initiate infection may be a random event from the divergent pool of donor viral sequences. The characteristics explored through this study are important, but may not function as genotypic signatures of transmission as previously described.

Hyperimmune bovine colostrum as a low-cost, large-scale source of antibodies with broad neutralizing activity for HIV-1 envelope with potential use in microbicides.

Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.

The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin.

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.

Rapid degranulation of NK cells following activation by HIV-specific antibodies.

Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-gamma and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-gamma by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.

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.

Dimeric IgA is a specific biomarker of recent SARS-CoV-2 infection.

Current tests for SARS-CoV-2 antibodies (IgG, IgM, IgA) cannot differentiate recent and past infections. We describe a point of care, lateral flow assay for SARS-CoV-2 dIgA based on the highly selective binding of dIgA to a chimeric form of secretory component (CSC), that distinguishes dIgA from monomeric IgA. Detection of specific dIgA uses a complex of biotinylated SARS-CoV-2 receptor binding domain and streptavidin-colloidal gold. SARS-CoV-2-specific dIgA was measured both in 112 cross-sectional samples and a longitudinal panel of 362 plasma samples from 45 patients with PCR-confirmed SARS-CoV-2 infection, and 193 discrete pre-COVID-19 or PCR-negative patient samples. The assay demonstrated 100% sensitivity from 11 days post-symptom onset, and a specificity of 98.2%. With an estimated half-life of 6.3 days, dIgA provides a unique biomarker for the detection of recent SARS-CoV-2 infections with potential to enhance diagnosis and management of COVID-19 at point-of-care.

Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites.

A highly protective vaccine will greatly facilitate achieving and sustaining malaria elimination. Understanding mechanisms of antibody-mediated immunity is crucial for developing vaccines with high efficacy. Here, we identify key roles in humoral immunity for Fcγ-receptor (FcγR) interactions and opsonic phagocytosis of sporozoites. We identify a major role for neutrophils in mediating phagocytic clearance of sporozoites in peripheral blood, whereas monocytes contribute a minor role. Antibodies also promote natural killer cell activity. Mechanistically, antibody interactions with FcγRIII appear essential, with FcγRIIa also required for maximum activity. All regions of the circumsporozoite protein are targets of functional antibodies against sporozoites, and N-terminal antibodies have more activity in some assays. Functional antibodies are slowly acquired following natural exposure to malaria, being present among some exposed adults, but uncommon among children. Our findings reveal targets and mechanisms of immunity that could be exploited in vaccine design to maximize efficacy.