Shelly J. Krebs

Background: Vascular activation occurs during human immunodeficiency virus (HIV) infection and is a risk factor for cardiovascular disease (CVD). This study investigates whether vascular endothelial and hemostatic perturbations emerge during acute HIV infection (AHI) and persist despite immediate antiretroviral therapy (ART) initiation. Methods: Thirteen plasma biomarkers indicative of endothelial and hemostatic perturbations were measured in 83 RV254 AHI cohort participants during pre-ART AHI and at week 288 post-ART, compared with cross-sectional measures from 32 people without HIV (PWoH). Hierarchical clustering was applied to week 288 biomarker profiles of RV254 participants and PWoH to identify distinct biomarker phenotypes. Results: During pre-ART AHI, RV254 participants exhibited higher intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, neopterin, and myeloperoxidase than PWoH (P < .05), independent of age, sex, and body mass index (BMI). These markers correlated with plasma HIV RNA or the CD4/CD8 ratio (P < .05). At week 288, significant reductions in ICAM-1, VCAM-1, vascular endothelial growth factor (VEGF)–C, VEGF-D, neopterin, and myeloperoxidase and a significant increase in tissue plasminogen activator were observed in RV254 (P < .05). However, VCAM-1 and thrombomodulin remained higher, and VEGF-A remained lower in RV254 participants compared to PWoH (P < .05). Hierarchical clustering identified 2 distinct clusters. Despite comparable 10-year CVD risk, 1 cluster exhibited concomitant elevation of ICAM-1, ICAM-2, VCAM-1, and E-selectin. Notably, 45% of RV254 participants vs 13% of PWoH were classified into this phenotype. Conclusions: Endothelial and hemostatic perturbations emerge during AHI and correlate with HIV-related parameters. Despite suppressive ART, RV254 participants remained approximately 3.5 times more likely than PWoH to exhibit an endothelial activation biomarker phenotype.

To identify strategies for augmenting vaccine immunogenicity, we compared a pox-protein prime-boost regimen comprising recombinant modified vaccinia virus Ankara and multimeric HIV-1 Env gp145, adjuvanted with Army Liposomal Formulation (ALF) either adsorbed to aluminum salt (ALFA) or formulated with the QS-21 saponin (ALFQ), in rhesus macaques. ALFQ promoted greater magnitude and more durable humoral and cellular immune responses than ALFA, which exhibits similar immunogenicity to aluminum-based adjuvants. Peak Env-specific CD4 ⁺ T cell responses assessed by intracellular cytokine staining were 10-fold greater with ALFQ, and CD8 ⁺ T cell responses were unexpectedly robust, averaging greater than 1%. ALFQ induced higher levels of several immunostimulatory cytokines in plasma, which correlated with adaptive immune responses. However, vaccination did not protect against heterologous intrarectal challenge with transmitted/founder SHIV-CH505. We provide evidence that CH505 Env may maintain a relatively closed conformation, rendering it less susceptible to targeting by Fc-mediated antibody functions. Overall, ALFQ is a promising adjuvant to improve antibody and T cell response magnitude. IMPORTANCE: An effective and durable vaccine preventing HIV-1 acquisition is urgently needed to end the HIV-1 pandemic. To date, of the nine vaccine efficacy trials conducted in humans, only the RV144 vaccine trial demonstrated efficacy in reducing infections, although immune responses waned rapidly. We evaluated a modified HIV-1 vaccine regimen incorporating next-generation adjuvants to improve immune responses and vaccine efficacy in a gold standard, pre-clinical primate model. Adjuvanted protein boosting markedly increased antibody, T cell, and pro-inflammatory response magnitude. These data, combined with the adjuvant’s strong safety and immunogenicity track record in clinical trials, indicate that novel adjuvants represent a promising strategy for improving immune responses to protein immunogens. Future vaccine regimens against HIV-1 and other pathogens for which eliciting robust immunity has been difficult may benefit from incorporating these or related next-generation adjuvants.

Zika virus (ZIKV) vaccine candidates developed through Phase I clinical trials are based on the full-length envelope glycoprotein (E), which presents both desirable and undesirable antigenic determinants. Among the latter, the conserved fusion loop epitope (FLE) within domain II is a major target for flavivirus cross-reactive and poorly neutralizing responses. To eliminate unwanted FLE targeting, we redesigned ZIKV E using a reverse vaccinology approach, excising domain II and allowing domains I and III (DI-DIII) to fold into an independent subunit harboring key neutralizing epitopes. Ifnar1 ^-/- mice vaccinated with ZIKV DI-DIII elicited high ZIKV neutralizing antibodies and were protected from weight loss and death. In addition, sera from DI-DIII vaccinated mice demonstrated a reduced capacity to enhance DENV 1-4 infection in vitro, compared to mice vaccinated with full-length E. This study identifies DI-DIII as a promising immunogen, focusing antibody responses to protective epitopes on ZIKV and minimizing the elicitation of unwanted responses.

Monoclonal antibodies to SARS-CoV-2 can offer prophylactic and therapeutic protection against severe disease, with particular utility for immunosuppressed and vulnerable populations. With the constant emergence of new variants, understanding the neutralizing potency of monoclonal antibodies to dynamic spike protein epitopes is crucial. We show that a set of VH1-24-derived N-terminal domain (NTD)-directed antibodies, isolated from a convalescent donor early in the pandemic, displayed remarkable neutralization resilience against many Omicron SARS-CoV-2 variants, including BA.2, BA.5, and BQ.1.1. Neutralization potency to these Omicron variants is associated with slower off-rates to the spike protein. Structural characterization of the most potent NTD antibody, WRAIR-2008, revealed a conserved mode of interaction shared with other antibodies of the same multi-donor class. WRAIR-2008 protected mice from weight loss following BA.5 challenge and reduced infectious viral titers in the lungs. Our study highlights the retention of neutralization activity and protection of first-generation VH1-24-derived NTD-directed antibodies to specific Omicron variants and provides valuable insights into the shifting landscape of SARS-CoV-2 variants that are vulnerable to select monoclonal antibodies. IMPORTANCE: As SARS-CoV-2 circulating variants evolve, it is important to understand the vulnerabilities of these viruses to neutralizing antibodies. Within this manuscript, we describe first-generation antibodies isolated following infection with WA-1 that retain viral neutralization to subsequent Omicron variants by targeting a site of viral vulnerability called the NTD. This work highlights the shifting landscape of SARS-CoV-2 variants and provides mechanistic insights into how antibodies from prior infections may play a role in preventing subsequent SARS-CoV-2 variant infections.

Eliminating latent HIV-1 is a major goal of AIDS research but host factors determining the size of these reservoirs are poorly understood. Here, we investigate the role of host gene expression on HIV-1 reservoir size during suppressive antiretroviral therapy (ART). Peripheral blood cells of fourteen males initiating ART during acute infection and demonstrating effective viral suppression but varying magnitudes of total HIV-1 DNA were characterized by single-cell RNA sequencing. Differential expression analysis demonstrates increased CD14+ monocyte activity in participants having undetectable HIV-1 reservoirs, with IL1B expression inversely associating with reservoir size. This is validated in another cohort of 38 males comprised of different ancestry and HIV-1 subtypes, and with intact proviral DNA assay (IPDA®) measurements. Modeling interactions show monocyte IL1B expression associates inversely with reservoir size at higher frequencies of central memory CD4+ T cells, linking monocyte IL1B expression to cell types known to be reservoirs for persistent HIV-1. Functional analyses reveal that IL1B activates NF-κB, thereby promoting productive HIV-1 infection while simultaneously suppressing viral spread, suggesting a natural latency reversing activity to deplete the reservoir in ART-treated individuals. Altogether, scRNA-seq analyses reveal that monocyte IL1B expression could decrease HIV-1 proviral reservoirs in individuals initiating ART during acute infection.

We leveraged specimens from the RV217 prospective study that enrolled participants at high risk of HIV-1 acquisition to investigate how NK cells, conventional T cells, and unconventional T cells influence HIV-1 acquisition. We observed low levels of α4β7 expression on memory CD4 ⁺ T cells and invariant NK T (iNKT) cells, 2 cell types highly susceptible to HIV-1 infection, in highly exposed seronegative (HESN) compared with highly exposed seroconverter (HESC) participants. NK cells from HESN individuals had higher levels of α4β7 than did those from HESC individuals, presented a quiescent phenotype, and had a higher capacity to respond to opsonized target cells. We also measured translocated microbial products in plasma and found differences in phylum distribution between HESN and HESC participants that were associated with the immune phenotypes affecting the risk of HIV-1 acquisition. Finally, a logistic regression model combining features of NK cell activation, α4β7 expression on memory CD4 ⁺ T cells, and T-box expressed in T cells (Tbet) expression by iNKT cells achieved the highest accuracy in identifying HESN and HESC participants. This immune signature, consisting of increased α4β7 on cells susceptible to HIV infection combined with higher NK cell activation and lower gut-homing potential, could affect the efficacy of HIV-1 prevention strategies such as vaccines.

An effective HIV-1 vaccine must elicit broadly neutralizing antibodies (bnAbs) against highly diverse Envelope glycoproteins (Env). Since Env with the longest hypervariable (HV) loops is more resistant to the cognate bnAbs than Env with shorter HV loops, we redesigned hypervariable loops for updated Env consensus sequences of subtypes B and C and CRF01_AE. Using modeling with AlphaFold2, we reduced the length of V1, V2, and V5 HV loops while maintaining the integrity of the Env structure and glycan shield, and modified the V4 HV loop. Spacers are designed to limit strain-specific targeting. All updated Env are infectious as pseudoviruses. Preliminary structural characterization suggests that the modified HV loops have a limited impact on Env’s conformation. Binding assays show improved binding to modified subtype B and CRF01_AE Env but not to subtype C Env. Neutralization assays show increases in sensitivity to bnAbs, although not always consistently across clades. Strikingly, the HV loop modification renders the resistant CRF01_AE Env sensitive to 10-1074 despite the absence of a glycan at N332.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Zika virus (ZIKV) is a significant threat to pregnant women and their fetuses as it can cause severe birth defects and congenital neurodevelopmental disorders, referred to as congenital Zika syndrome (CZS). Thus, a safe and effective ZIKV vaccine for pregnant women to prevent in utero ZIKV infection is of utmost importance. Murine models of ZIKV infection are limited by the fact that immunocompetent mice are resistant to ZIKV infection. As such, interferon-deficient mice have been used in some preclinical studies to test the efficacy of ZIKV vaccine candidates against lethal virus challenge. However, interferon-deficient mouse models have limitations in assessing the immunogenicity of vaccines, necessitating the use of immunocompetent mouse pregnancy models. Using the human stat2 knock-in (hSTAT2KI) mouse pregnancy model, we show that vaccination with a purified formalin-inactivated Zika virus (ZPIV) vaccine prior to pregnancy successfully prevented vertical transmission. In addition, maternal immunity protected offspring against postnatal challenge for up to 28 days. Furthermore, passive transfer of human IgG purified from hyper-immune sera of ZPIV vaccinees prevented maternal and fetal ZIKV infection, providing strong evidence that the neutralizing antibody response may serve as a meaningful correlate of protection.

The repeat emergence of SARS-CoV-2 variants of concern (VoC) with decreased susceptibility to vaccine-elicited antibodies highlights the need to develop next-generation vaccine candidates that confer broad protection. Here we describe the antibody response induced by the SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine candidate adjuvanted with the Army Liposomal Formulation including QS21 (ALFQ) in non-human primates. By isolating and characterizing several monoclonal antibodies directed against the Spike Receptor Binding Domain (RBD), N-Terminal Domain (NTD), or the S2 Domain, we define the molecular recognition of vaccine-elicited cross-reactive monoclonal antibodies (mAbs) elicited by SpFN. We identify six neutralizing antibodies with broad sarbecovirus cross-reactivity that recapitulate serum polyclonal antibody responses. In particular, RBD mAb WRAIR-5001 binds to the conserved cryptic region with high affinity to sarbecovirus clades 1 and 2, including Omicron variants, while mAb WRAIR-5021 offers complete protection from B.1.617.2 (Delta) in a murine challenge study. Our data further highlight the ability of SpFN vaccination to stimulate cross-reactive B cells targeting conserved regions of the Spike with activity against SARS CoV-1 and SARS-CoV-2 variants.