Pro-inflammatory feedback loops define immune responses to pathogenic Lentivirus infection.

BACKGROUND: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. METHODS: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. RESULTS: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. CONCLUSIONS: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Pseudoviruses, a safer toolbox for vaccine development against enveloped viruses.

INTRODUCTION: Pseudoviruses are recombinant, replication-incompetent, viral particles designed to mimic the surface characteristics of native enveloped viruses. They are a safer, and cost-effective research alternative to live viruses. With the potential emergence of the next major infectious disease, more vaccine scientists must become familiar with the pseudovirus platform as a vaccine development tool to mitigate future outbreaks. AREAS COVERED: This review aims at vaccine developers to provide a basic understanding of pseudoviruses, list their production methods, and discuss their utility to assess vaccine efficacy against enveloped viral pathogens. We further illustrate their usefulness as wet-lab simulators for emerging mutant variants, and new viruses to help prepare for current and future viral outbreaks, minimizing the need for gain-of-function experiments with highly infectious or lethal enveloped viruses. EXPERT OPINION: With this platform, researchers can better understand the role of virus-receptor interactions and entry in infections, prepare for dangerous mutations, and develop effective vaccines.

Variable antiviral activity of islatravir against M184I/V mutant HIV-1 selected during antiretroviral therapy.

BACKGROUND: Islatravir is a new antiretroviral drug that inhibits the reverse transcriptase (RT) of HIV-1 through multiple mechanisms. It is proposed to be used in combination with doravirine, a new NNRTI. M184V/I mutations have been shown to reduce the in vitro antiviral activity of islatravir, but their effect when pre-selected during ART has not been investigated. METHODS: HIV-1 rt sequences were obtained from four individuals of the Garrahan HIV cohort prior to, or during virological failure to ART. HIV-1 infectious molecular clones were constructed on an NL4-3 backbone, and infectious viruses were produced by transfection of 293T cells. Fold-changes in IC50 were calculated for each mutant versus the NL4-3 WT. HIV-1 phenotypic drug resistance was tested in vitro against NRTIs and NNRTIs. RESULTS: In all the cases, M184I/V, either alone or in the presence of other mutations, was associated with reduced susceptibility to islatravir, abacavir and lamivudine. Viruses carrying M184V/I showed variable levels of resistance to islatravir (4.8 to 33.8-fold). The greatest reduction in susceptibility was observed for viruses carrying the mutations M184V + V106I (33.8-fold resistance) or M184V + I142V (25.2-fold resistance). For NNRTIs, the presence of V106I alone did not affect susceptibility to doravirine or etravirine, but showed a modest reduction in susceptibility to efavirenz (6-fold). Susceptibility to doravirine was slightly reduced only for one of the mutants carrying V106I in combination with Y181C and M184V. CONCLUSIONS: Mutations and polymorphisms selected in vivo together with M184V/I depend on the viral genetic context and on ART history, and could affect the efficacy of islatravir once available for use in the clinic.

Antiviral potency of long-acting islatravir subdermal implant in SHIV-infected macaques.

Treatment nonadherence is a pressing issue in people living with HIV (PLWH), as they require lifelong therapy to maintain viral suppression. Poor adherence leads to antiretroviral (ARV) resistance, transmission to others, AIDS progression, and increased morbidity and mortality. Long-acting (LA) ARV therapy is a promising strategy to combat the clinical drawback of user-dependent dosing. Islatravir (ISL) is a promising candidate for HIV treatment given its long half-life and high potency. Here we show constant ISL release from a subdermal LA nanofluidic implant achieves viral load reduction in SHIV-infected macaques. Specifically, a mean delivery dosage of 0.21 ± 0.07 mg/kg/day yielded a mean viral load reduction of -2.30 ± 0.53 log10 copies/mL at week 2, compared to baseline. The antiviral potency of the ISL delivered from the nanofluidic implant was higher than oral ISL dosed either daily or weekly. At week 3, viral resistance to ISL emerged in 2 out of 8 macaques, attributable to M184V mutation, supporting the need of combining ISL with other ARV for HIV treatment. The ISL implant produced moderate reactivity in the surrounding tissue, indicating tolerability. Overall, we present the ISL subdermal implant as a promising approach for LA ARV treatment in PLWH.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

A rapid, high-throughput, viral infectivity assay using automated brightfield microscopy with machine learning.

Infectivity assays are essential for the development of viral vaccines, antiviral therapies, and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated viral infectivity assay (AVIATM), using convolutional neural networks (CNNs) and high-throughput brightfield microscopy on 96-well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. CNN models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit between virus dilution curves and CNN predictions, results in sensitivity ranges comparable to or better than conventional plaque or TCID50 assays, and a precision of ∼10%, which is considerably better than conventional infectivity assays. Because this technology is based on sensitizing CNNs to specific phenotypes of infection, it has potential as a rapid, broad-spectrum tool for virus characterization, and potentially identification.

Long-acting refillable nanofluidic implant confers protection against SHIV infection in nonhuman primates.

The impact of pre-exposure prophylaxis (PrEP) on slowing the global HIV epidemic hinges on effective drugs and delivery platforms. Oral drug regimens are the pillar of HIV PrEP, but variable adherence has spurred development of long-acting delivery systems with the aim of increasing PrEP access, uptake, and persistence. We have developed a long-acting subcutaneous nanofluidic implant that can be refilled transcutaneously for sustained release of the HIV drug islatravir, a nucleoside reverse transcriptase translocation inhibitor that is used for HIV PrEP. In rhesus macaques, the islatravir-eluting implants achieved constant concentrations of islatravir in plasma (median 3.14 nM) and islatravir triphosphate in peripheral blood mononuclear cells (median 0.16 picomole per 106 cells) for more than 20 months. These drug concentrations were above the established PrEP protection threshold. In two unblinded, placebo-controlled studies, islatravir-eluting implants conferred 100% protection against infection with SHIVSF162P3 after repeated low-dose rectal or vaginal challenge in male or female rhesus macaques, respectively, compared to placebo control groups. The islatravir-eluting implants were well tolerated with mild local tissue inflammation and no signs of systemic toxicity over the 20-month study period. This refillable islatravir-eluting implant has potential as a long-acting drug delivery system for HIV PrEP.

From concept to delivery: a yeast-expressed recombinant protein-based COVID-19 vaccine technology suitable for global access.

INTRODUCTION: The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. AREAS COVERED: Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. EXPERT OPINION: Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

HIV-1 subtype F integrase polymorphisms external to the catalytic core domain contribute to severe loss of replication capacity in context of the integrase inhibitor resistance mutation Q148H.

BACKGROUND: In prior studies, HIV-1 BF recombinants with subtype F integrases failed to develop resistance to raltegravir through the Q148H mutational pathway. We aimed to determine the role of subtype-specific polymorphisms in integrase on drug susceptibility, viral replication and integration. METHODS: Integrase sequences were retrieved from the Los Alamos Database or obtained from the Garrahan HIV cohort. HIV-1 infectious molecular clones with or without Q148H (+ G140S) resistance mutations were constructed using integrases of subtype B (NL4-3) or F1(BF) ARMA159 and URTR23. Integrase chimeras were generated by reciprocal exchanges of a 200 bp fragment spanning amino acids 85-150 of the catalytic core domain (CCD) of NL4-3-Q148H and either ARMA159-Q148H or URTR23-Q148H. Viral infections were quantified by p24 ELISA and Alu-gag integration PCR assay. RESULTS: At least 18 different polymorphisms distinguish subtype B from F1(BF) recombinant integrases. In phenotypic experiments, p24 at Day 15 post-infection was high (105-106 pg/mL) for WT and NL4-3-Q148H; by contrast, it was low (102-104 pg/mL) for both F1(BF)-Q148H + G140S viruses, and undetectable for the Q148H mutants. Compared with WT viruses, integrated DNA was reduced by 5-fold for NL4-3-Q148H (P = 0.05), 9-fold for URTR23-Q148H (P = 0.01) and 16000-fold for ARMA159-Q148H (P = 0.01). Reciprocal exchange between B and F1(BF) of an integrase CCD region failed to rescue the replicative defect of F1(BF) integrase mutants. CONCLUSIONS: The functional impairment of Q148H in the context of subtype F integrases from BF recombinants explains the lack of selection of this pathway in vivo. Non-B polymorphisms external to the integrase CCD may influence the pathway to integrase strand transfer inhibitor resistance.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General ofIndia. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.

Clearance of HIV-1 or SIV reservoirs by promotion of apoptosis and inhibition of autophagy: Targeting intracellular molecules in cure-directed strategies.

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

In vivo Serial Passaging of Human-Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication.

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

Serum IgG anti-SARS-CoV-2 Binding Antibody Level Is Strongly Associated With IgA and Functional Antibody Levels in Adults Infected With SARS-CoV-2.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

Preventive efficacy of a tenofovir alafenamide fumarate nanofluidic implant in SHIV-challenged nonhuman primates.

Pre-exposure prophylaxis (PrEP) using antiretroviral oral drugs is effective at preventing HIV transmission when individuals adhere to the dosing regimen. Tenofovir alafenamide (TAF) is a potent antiretroviral drug, with numerous long-acting (LA) delivery systems under development to improve PrEP adherence. However, none has undergone preventive efficacy assessment. Here we show that LA TAF using a novel subcutaneous nanofluidic implant (nTAF) confers partial protection from HIV transmission. We demonstrate that sustained subcutaneous delivery through nTAF in rhesus macaques maintained tenofovir diphosphate concentration at a median of 390.00 fmol/106 peripheral blood mononuclear cells, 9 times above clinically protective levels. In a non-blinded, placebo-controlled rhesus macaque study with repeated low-dose rectal SHIVSF162P3 challenge, the nTAF cohort had a 62.50% reduction (95% CI: 1.72% to 85.69%; p=0.068) in risk of infection per exposure compared to the control. Our finding mirrors that of tenofovir disoproxil fumarate (TDF) monotherapy, where 60.00% protective efficacy was observed in macaques, and clinically, 67.00% reduction in risk with 86.00% preventive efficacy in individuals with detectable drug in the plasma. Overall, our nanofluidic technology shows potential as a subcutaneous delivery platform for long-term PrEP and provides insights for clinical implementation of LA TAF for HIV prevention.

SARS­CoV-2 RBD219-N1C1: A yeast-expressed SARS-CoV-2 recombinant receptor-binding domain candidate vaccine stimulates virus neutralizing antibodies and T-cell immunity in mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast ( Pichia pastoris ), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel ® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel ® containing formulation and possibly in combination with other immunostimulants.