Harnessing the power of IFN for therapeutic approaches to COVID-19.

Interferons (IFNs) are essential for defense against viral infections but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, we explore the complexity of the IFN response in COVID-19, examine the effects of manipulating IFN on SARS-CoV-2 viral replication and pathogenesis, and highlight pre-clinical and clinical studies evaluating the therapeutic efficacy of IFN in limiting COVID-19 severity.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals. It remains unclear if exposures to antigenically distant SARS-CoV-2 variants can overcome memory B cell biases established by initial SARS-CoV-2 encounters. We determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that targeted epitopes conserved between the BA.5 and ancestral spike. XBB exposures also elicited antibody responses that primarily targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low frequencies of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

A non-human primate model for human norovirus infection.

Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection. We show that rhesus macaques are susceptible to oral infection with human noroviruses from two different genogroups. Variation in duration of virus shedding (days to weeks) between animals, evolution of the virus over the time of infection, induction of virus-specific adaptive immune responses, susceptibility to reinfection and preferential replication of norovirus in the jejunum of rhesus macaques was similar to infection reported in humans. We found minor pathological signs and changes in epithelial cell surface glycosylation patterns in the small intestine during infection. Detection of viral protein and RNA in intestinal biopsies confirmed the presence of the virus in chromogranin A-expressing epithelial cells, as it does in humans. Thus, rhesus macaques are a promising non-human primate model to evaluate vaccines and therapeutics against norovirus disease.

A family of olfactory receptors uniquely expanded in marsupial and monotreme genomes are expressed by a T cell subset also unique to marsupials and monotremes.

Olfactory receptors (OR), expressed on olfactory neurons, mediate the sense of smell. Recently, OR have also been shown to be expressed in non-olfactory tissues, including cells of the immune system. An analysis of single-cell transcriptomes of splenocytes of the grey short-tailed opossum (Monodelphis domestica) found OR are expressed on a subset of T cells, the γµ T cells, that are unique to marsupials and monotremes. A majority of opossum γµ T cells transcriptomes contain OR family 14 transcripts, specifically, from the OR14C subfamily. Amongst the mammals, the OR14 gene family is expanded in the genomes of marsupials and monotremes, and rarer or absent in placental mammals. In summary, here we demonstrate the intriguing correlation that a family of OR genes, abundant in the genomes of marsupials and monotremes, are ectopically expressed in a particular subset of T cells unique to the marsupials and monotremes.

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants.

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination (∼1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans.

Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals and prompting the development of updated booster vaccines. Here, we determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that primarily targeted epitopes conserved between the BA.5 and ancestral spike, with poor reactivity to the XBB.1.5 variant. XBB exposures also elicited antibody responses that targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low levels of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

Adjuvanted SARS-CoV-2 spike protein vaccination elicits long-lived plasma cells in nonhuman primates.

Durable humoral immunity is mediated by long-lived plasma cells (LLPCs) that reside in the bone marrow. It remains unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein vaccination is able to elicit and maintain LLPCs. Here, we describe a sensitive method to identify and isolate antigen-specific LLPCs by tethering antibodies secreted by these cells onto the cell surface. Using this method, we found that two doses of adjuvanted SARS-CoV-2 spike protein vaccination are able to induce spike protein-specific LLPC reservoirs enriched for receptor binding domain specificities in the bone marrow of nonhuman primates that are detectable for several months after vaccination. Immunoglobulin gene sequencing confirmed that several of these LLPCs were clones of memory B cells elicited 2 weeks after boost that had undergone further somatic hypermutation. Many of the antibodies secreted by these LLPCs also exhibited improved neutralization and cross-reactivity compared with earlier time points. These findings establish our method as a means to sensitively and reliably detect rare antigen-specific LLPCs and demonstrate that adjuvanted SARS-CoV-2 spike protein vaccination establishes spike protein-specific LLPC reservoirs.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Predicting the impact of COVID-19 non-pharmaceutical intervention on short- and medium-term dynamics of enterovirus D68 in the US.

Recent outbreaks of enterovirus D68 (EV-D68) infections, and their causal linkage with acute flaccid myelitis (AFM), continue to pose a serious public health concern. During 2020 and 2021, the dynamics of EV-D68 and other pathogens have been significantly perturbed by non-pharmaceutical interventions against COVID-19; this perturbation presents a powerful natural experiment for exploring the dynamics of these endemic infections. In this study, we analyzed publicly available data on EV-D68 infections, originally collected through the New Vaccine Surveillance Network, to predict their short- and long-term dynamics following the COVID-19 interventions. Although long-term predictions are sensitive to our assumptions about underlying dynamics and changes in contact rates during the NPI periods, the likelihood of a large outbreak in 2023 appears to be low. Comprehensive surveillance data are needed to accurately characterize future dynamics of EV-D68. The limited incidence of AFM cases in 2022, despite large EV-D68 outbreaks, poses further questions for the timing of the next AFM outbreaks.

Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates.

As SARS-CoV-2 variants continue evolving, testing updated vaccines in non-human primates remains important for guiding human clinical practice. To date, such studies have focused on antibody titers and antigen-specific B and T cell frequencies. Here, we extend our understanding by integrating innate and adaptive immune responses to mRNA-1273 vaccination in rhesus macaques. We sorted innate immune cells from a pre-vaccine time point, as well as innate immune cells and antigen-specific peripheral B and T cells two weeks after each of two vaccine doses and used single-cell sequencing to assess the transcriptomes and adaptive immune receptors of each cell. We show that a subset of S-specific T cells expresses cytokines critical for activating innate responses, with a concomitant increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more cytotoxic phenotype. The second vaccine dose, administered 4 weeks after the first, elicits an increase in circulating germinal center-like B cells 2 weeks later, which are more clonally expanded and enriched for epitopes in the receptor binding domain. Both doses stimulate inflammatory response genes associated with elevated antibody production. Overall, we provide a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggest potential mechanisms for the enhanced response to secondary exposure.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

High frequency of HIV precursor-target-specific B cells in sub-Saharan populations.

HIV gp120 engineered outer domain germline-targeting version 8 (eOD-GT8) was designed specifically to engage naive B cell precursors of VRC01-class antibodies. However, the frequency and affinity of naive B cell precursors able to recognize eOD-GT8 have been evaluated only in U.S. populations. HIV infection is disproportionally concentrated in sub-Saharan Africa, so we seek to characterize naive B cells able to recognize eOD-GT8 in sub-Saharan cohorts. We demonstrate that people from sub-Saharan Africa have a higher or equivalent frequency of naive B cells able to engage eOD-GT8 compared with people from the U.S. Genetically, the higher frequency of eOD-GT8-positive cells is accompanied by a higher level of naive B cells with gene signatures characteristic of the VRC01 class, as well as other CD4bs-directed antibodies. Our study demonstrates that vaccination with eOD-GT8 in sub-Saharan Africa could be successful at expanding and establishing a pool of CD4bs-directed memory B cells from naive precursors.

Mucosal Adenoviral-vectored Vaccine Boosting Durably Prevents XBB.1.16 Infection in Nonhuman Primates.

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera.

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways.

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163+ MRC1- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-ß pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

Investigation of the impact of clonal hematopoiesis on severity and pathophysiology of COVID-19 in rhesus macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection.

HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide.

Elicitation of antibodies that neutralize the tier-2 neutralization-resistant isolates that typify HIV-1 transmission has been a long-sought goal. Success with prefusion-stabilized envelope trimers eliciting autologous neutralizing antibodies has been reported in multiple vaccine-test species, though not in humans. To investigate elicitation of HIV-1 neutralizing antibodies in humans, here, we analyze B cells from a phase I clinical trial of the "DS-SOSIP"-stabilized envelope trimer from strain BG505, identifying two antibodies, N751-2C06.01 and N751-2C09.01 (named for donor-lineage.clone), that neutralize the autologous tier-2 strain, BG505. Though derived from distinct lineages, these antibodies form a reproducible antibody class that targets the HIV-1 fusion peptide. Both antibodies are highly strain specific, which we attribute to their partial recognition of a BG505-specific glycan hole and to their binding requirements for a few BG505-specific residues. Prefusion-stabilized envelope trimers can thus elicit autologous tier-2 neutralizing antibodies in humans, with initially identified neutralizing antibodies recognizing the fusion-peptide site of vulnerability.