mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Systems vaccinology of the BNT162b2 mRNA vaccine in humans.

The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) a greater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults.

BACKGROUND: Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been associated with an older age. METHODS: We conducted a phase 1, dose-escalation, open-label trial of a messenger RNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or ≥71 years). All the participants were assigned sequentially to receive two doses of either 25 µg or 100 µg of vaccine administered 28 days apart. RESULTS: Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-µg dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-µg dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, respectively. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Binding- and neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. CONCLUSIONS: In this small study involving older adults, adverse events associated with the mRNA-1273 vaccine were mainly mild or moderate. The 100-µg dose induced higher binding- and neutralizing-antibody titers than the 25-µg dose, which supports the use of the 100-µg dose in a phase 3 vaccine trial. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 Study ClinicalTrials.gov number, NCT04283461.).

Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrates the Functional Importance of the Receptor-Binding Domain.

The factors that control the development of an effective immune response to the recently emerged SARS-CoV-2 virus are poorly understood. In this study, we provide a cross-sectional analysis of the dynamics of B cell responses to SARS-CoV-2 infection in hospitalized COVID-19 patients. We observe changes in B cell subsets consistent with a robust humoral immune response, including significant expansion of plasmablasts and activated receptor-binding domain (RBD)-specific memory B cell populations. We observe elevated titers of Abs to SARS-CoV-2 RBD, full-length Spike, and nucleoprotein over the course of infection, with higher levels of RBD-specific IgG correlating with increased serum neutralization. Depletion of RBD-specific Abs from serum removed a major portion of neutralizing activity in most individuals. Some donors did retain significant residual neutralization activity, suggesting a potential Ab subset targeting non-RBD epitopes. Taken together, these findings are instructive for future vaccine design and mAb strategies.

Rapid Detection and Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Variant in a Returning Traveler.

We describe rapid detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant using targeted spike single-nucleotide polymorphism polymerase chain reaction and viral genome sequencing. This case occurred in a fully vaccinated and boosted returning traveler with mild symptoms who was identified through community surveillance rather than clinical care.

Insights into the Gene Expression Profiles of Active and Restricted Red/Green-HIV+ Human Astrocytes: Implications for Shock or Lock Therapies in the Brain.

A significant number of people living with human immunodeficiency virus type 1 (HIV-1) suffer from HIV-associated neurocognitive disorders (HAND). Many previous studies investigating HIV in astrocytes as a heterogenous population have established the relevance of astrocytes to HIV-associated neuropathogenesis. However, these studies were unable to differentiate the state of infection, i.e., active or latent, or to evaluate how this affects astrocyte biology. In this study, the pseudotyped doubly labeled fluorescent reporter red/green (R/G)-HIV-1 was used to identify and enrich restricted and active populations of HIV+ astrocytes based on the viral promoter activity. Here, we report that the majority of human astrocytes restricted R/G-HIV-1 gene expression early during infection and were resistant to reactivation by vorinostat and interleukin 1ß. However, actively infected astrocytes were inducible, leading to increased expression of viral proteins upon reactivation. R/G-HIV-1 infection also significantly decreased the cell proliferation and glutamate clearance ability of astrocytes, which may contribute to excitotoxicity. Moreover, transcriptome analyses to compare gene expression patterns of astrocyte harboring active versus restricted long terminal repeats (LTRs) revealed that the gene expression patterns were similar and that the active population demonstrated more widespread and robust changes. Our data suggest that harboring the HIV genome profoundly alters astrocyte biology and that strategies that keep the virus latent (e.g., block and lock) or those that reactivate the latent virus (e.g., shock and kill) would be detrimental to astrocyte function and possibly augment their contributions to HAND.IMPORTANCE More than 36 million people are living with HIV-1 worldwide, and despite antiretroviral therapy, 30 to 50% of the people living with HIV-1 suffer from mild to moderate neurocognitive disorders. HIV-1 reservoirs in the central nervous system (CNS) are challenging to address due to low penetration of antiretroviral drugs, lack of resident T cells, and permanent integration of provirus into neural cells such as microglia and astrocytes. Several studies have shown astrocyte dysfunction during HIV-1 infection. However, little is known about how HIV-1 latency affects their function. The significance of our research is in identifying that the majority of HIV+ astrocytes restrict HIV expression and were resistant to reactivation. Further, simply harboring the HIV genome profoundly altered astrocyte biology, resulting in a proinflammatory phenotype and functional changes. In this context, therapeutic strategies to reactivate or silence astrocyte HIV reservoirs, without excising proviral DNA, will likely lead to detrimental neuropathological outcomes during HIV CNS infection.

Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response.

Understanding the molecular features of neutralizing epitopes is important for developing vaccines/therapeutics against emerging SARS-CoV-2 variants. We describe three monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during the first wave of the pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, poorly neutralized Beta, and failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these mAbs in complex with trimeric spike protein showed that all three mAbs bivalently bind spike with two mAbs targeting class 1 and one targeting a class 4 receptor binding domain epitope. The immunogenetic makeup, structure, and function of these mAbs revealed specific molecular interactions associated with the potent multi-variant binding/neutralization efficacy. This knowledge shows how mutational combinations can affect the binding or neutralization of an antibody, which in turn relates to the efficacy of immune responses to emerging SARS-CoV-2 escape variants.

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.

Pre-existing SARS-CoV-2 immunity influences potency, breadth, and durability of the humoral response to SARS-CoV-2 vaccination.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic highlights the importance of determining the breadth and durability of humoral immunity to SARS-CoV-2 mRNA vaccination. Herein, we characterize the humoral response in 27 naive and 40 recovered vaccinees. SARS-CoV-2-specific antibody and memory B cell (MBC) responses are durable up to 6 months, although antibody half-lives are shorter for naive recipients. The magnitude of the humoral responses to vaccination strongly correlates with responses to initial SARS-CoV-2 infection. Neutralization titers are lower against SARS-CoV-2 variants in both recovered and naive vaccinees, with titers more reduced in naive recipients. While the receptor-binding domain (RBD) is the main neutralizing target of circulating antibodies, Moderna-vaccinated naives show a lesser reliance on RBDs, with >25% neutralization remaining after depletion of RBD-binding antibodies. Overall, we observe that vaccination induces higher peak titers and improves durability in recovered compared with naive vaccinees. These findings have broad implications for current vaccine strategies deployed against the SARS-CoV-2 pandemic.