A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.

ChAdOx1 nCoV-19 vaccine elicits monoclonal antibodies with cross-neutralizing activity against SARS-CoV-2 viral variants.

Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here, we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that, despite a relatively low serum neutralization titer, Spike-reactive IgG+ B cells are still detectable 9 months post-boost. Furthermore, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (Alpha, Gamma, Beta, Delta, and Omicron) are present. The vaccine-elicited neutralizing mAbs form eight distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222-elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post-infection. These findings provide molecular insights into the AZD1222 vaccine-elicited antibody response.

Humoral and cellular immunogenicity to a second dose of COVID-19 vaccine BNT162b2 in people receiving methotrexate or targeted immunosuppression: a longitudinal cohort study.

BACKGROUND: COVID-19 vaccines have robust immunogenicity in the general population. However, data for individuals with immune-mediated inflammatory diseases who are taking immunosuppressants remains scarce. Our previously published cohort study showed that methotrexate, but not targeted biologics, impaired functional humoral immunity to a single dose of COVID-19 vaccine BNT162b2 (Pfizer-BioNTech), whereas cellular responses were similar. Here, we aimed to assess immune responses following the second dose. METHODS: In this longitudinal cohort study, we recruited individuals with psoriasis who were receiving methotrexate or targeted biological monotherapy (ie, tumour necrosis factor [TNF] inhibitors, interleukin [IL]-17 inhibitors, or IL-23 inhibitors) from a specialist psoriasis centre serving London and South-East England. The healthy control cohort were volunteers without psoriasis, not receiving immunosuppression. Immunogenicity was evaluated immediately before, on day 28 after the first BNT162b2 vaccination and on day 14 after the second dose (administered according to an extended interval regimen). Here, we report immune responses following the second dose. The primary outcomes were humoral immunity to the SARS-CoV-2 spike glycoprotein, defined as titres of total spike-specific IgG and of neutralising antibody to wild-type, alpha (B.1.1.7), and delta (B.1.617.2) SARS-CoV-2 variants, and cellular immunity defined as spike-specific T-cell responses (including numbers of cells producing interferon-γ, IL-2, IL-21). FINDINGS: Between Jan 14 and April 4, 2021, 121 individuals were recruited, and data were available for 82 participants after the second vaccination. The study population included patients with psoriasis receiving methotrexate (n=14), TNF inhibitors (n=19), IL-17 inhibitors (n=14), IL-23 inhibitors (n=20), and 15 healthy controls, who had received both vaccine doses. The median age of the study population was 44 years (IQR 33-52), with 43 (52%) males and 71 (87%) participants of White ethnicity. All participants had detectable spike-specific antibodies following the second dose, and all groups (methotrexate, targeted biologics, and healthy controls) demonstrated similar neutralising antibody titres against wild-type, alpha, and delta variants. By contrast, a lower proportion of participants on methotrexate (eight [62%] of 13, 95% CI 32-86) and targeted biologics (37 [74%] of 50, 60-85; p=0·38) had detectable T-cell responses following the second vaccine dose, compared with controls (14 [100%] of 14, 77-100; p=0·022). There was no difference in the magnitude of T-cell responses between patients receiving methotrexate (median cytokine-secreting cells per 106 cells 160 [IQR 10-625]), targeted biologics (169 [25-503], p=0·56), and controls (185 [133-328], p=0·41). INTERPRETATION: Functional humoral immunity (ie, neutralising antibody responses) at 14 days following a second dose of BNT162b2 was not impaired by methotrexate or targeted biologics. A proportion of patients on immunosuppression did not have detectable T-cell responses following the second dose. The longevity of vaccine-elicited antibody responses is unknown in this population. FUNDING: NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London; The Psoriasis Association.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

MX2-mediated innate immunity against HIV-1 is regulated by serine phosphorylation.

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-ß (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike.

The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

HIV-1 Vpr antagonizes innate immune activation by targeting karyopherin-mediated NF-κB/IRF3 nuclear transport.

HIV-1 must replicate in cells that are equipped to defend themselves from infection through intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here, we show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr-dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import and inhibition of innate immune activation to promote HIV-1 replication and transmission.

Are Evolution and the Intracellular Innate Immune System Key Determinants in HIV Transmission?

HIV-1 is the single most important sexually transmitted disease in humans from a global health perspective. Among human lentiviruses, HIV-1 M group has uniquely achieved pandemic levels of human-to-human transmission. The requirement to transmit between hosts likely provides the strongest selective forces on a virus, as without transmission, there can be no new infections within a host population. Our perspective is that evolution of all of the virus-host interactions, which are inherited and perpetuated from host-to-host, must be consistent with transmission. For example, CXCR4 use, which often evolves late in infection, does not favor transmission and is therefore lost when a virus transmits to a new host. Thus, transmission inevitably influences all aspects of virus biology, including interactions with the innate immune system, and dictates the biological niche in which the virus exists in the host. A viable viral niche typically does not select features that disfavor transmission. The innate immune response represents a significant selective pressure during the transmission process. In fact, all viruses must antagonize and/or evade the mechanisms of the host innate and adaptive immune systems that they encounter. We believe that viewing host-virus interactions from a transmission perspective helps us understand the mechanistic details of antiviral immunity and viral escape. This is particularly true for the innate immune system, which typically acts from the very earliest stages of the host-virus interaction, and must be bypassed to achieve successful infection. With this in mind, here we review the innate sensing of HIV, the consequent downstream signaling cascades and the viral restriction that results. The centrality of these mechanisms to host defense is illustrated by the array of countermeasures that HIV deploys to escape them, despite the coding constraint of a 10 kb genome. We consider evasion strategies in detail, in particular the role of the HIV capsid and the viral accessory proteins highlighting important unanswered questions and discussing future perspectives.