Heterologous humoral immunity to human and zoonotic coronaviruses: Aiming for the achilles heel.

Coronaviruses are evolutionarily successful RNA viruses, common to multiple avian, amphibian and mammalian hosts. Despite their ubiquity and potential impact, knowledge of host immunity to coronaviruses remains incomplete, partly owing to the lack of overt pathogenicity of endemic human coronaviruses (HCoVs), which typically cause common colds. However, the need for deeper understanding became pressing with the zoonotic introduction of three novel coronaviruses in the past two decades, causing severe acute respiratory syndromes in humans, and the unfolding pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This renewed interest not only triggered the discovery of two of the four HCoVs, but also uncovered substantial cellular and humoral cross-reactivity with shared or related coronaviral antigens. Here, we review the evidence for cross-reactive B cell memory elicited by HCoVs and its potential impact on the puzzlingly variable outcome of SARS-CoV-2 infection. The available data indicate targeting of highly conserved regions primarily in the S2 subunits of the spike glycoproteins of HCoVs and SARS-CoV-2 by cross-reactive B cells and antibodies. Rare monoclonal antibodies reactive with conserved S2 epitopes and with potent virus neutralising activity have been cloned, underscoring the potential functional relevance of cross-reactivity. We discuss B cell and antibody cross-reactivity in the broader context of heterologous humoral immunity to coronaviruses, as well as the limits of protective immune memory against homologous re-infection. Given the bidirectional nature of cross-reactivity, the unprecedented current vaccination campaign against SARS-CoV-2 is expected to impact HCoVs, as well as future zoonotic coronaviruses attempting to cross the species barrier. However, emerging SARS-CoV-2 variants with resistance to neutralisation by vaccine-induced antibodies highlight a need for targeting more constrained, less mutable parts of the spike. The delineation of such cross-reactive areas, which humoral immunity can be trained to attack, may offer the key to permanently shifting the balance of our interaction with current and future coronaviruses in our favour.

Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis.

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.

Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

SARS-CoV-2 S2-targeted vaccination elicits broadly neutralizing antibodies.

Several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current coronavirus disease 2019 (COVID-19) pandemic. Although antibody cross-reactivity with the spike glycoproteins (S) of diverse coronaviruses, including endemic common cold coronaviruses (HCoVs), has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to protection when induced by infection or through vaccination. Using a mouse model, we found that prior HCoV-OC43 S-targeted immunity primes neutralizing antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, vaccination with SARS-CoV-2 S2 elicited antibodies in mice that neutralized diverse animal and human alphacoronaviruses and betacoronaviruses in vitro and provided a degree of protection against SARS-CoV-2 challenge in vivo. Last, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induced broader neutralizing antibody response than booster Wuhan S vaccination, suggesting that it may prevent repertoire focusing caused by repeated homologous vaccination. These data establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern, as well as to future coronavirus zoonoses.

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains.

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric. Funding: This work was supported by the Francis Crick Institute and the Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg.

Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases.

BACKGROUND: Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained, and the effect of underlying immune dysfunction or suppression is unknown. Here, we sought to examine the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM), and juvenile systemic lupus erythematosus (JSLE) against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. METHODS: Sera were collected from JIA (n = 118), JDM (n = 49), and JSLE (n = 30) patients and from healthy control (n = 54) children and adolescents prior to the coronavirus disease 19 (COVID-19) pandemic. We used sensitive flow-cytometry-based assays to determine titers of antibodies that reacted with the spike and nucleoprotein of HCoV-OC43 and cross-reacted with the spike and nucleoprotein of SARS-CoV-2, and we compared them with respective titers in sera from patients with multisystem inflammatory syndrome in children and adolescents (MIS-C). FINDINGS: Despite immune dysfunction and immunosuppressive treatment, JIA, JDM, and JSLE patients maintained comparable or stronger humoral responses than healthier peers, which was dominated by immunoglobulin G (IgG) antibodies to HCoV-OC43 spike, and harbored IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM, and JSLE patients, which argues against increased exposure. CONCLUSIONS: Consequently, autoimmune rheumatic diseases and their treatment were associated with a favorable ratio of spike to nucleoprotein antibodies. FUNDING: This work was supported by a Centre of Excellence Centre for Adolescent Rheumatology Versus Arthritis grant, 21593, UKRI funding reference MR/R013926/1, the Great Ormond Street Children's Charity, Cure JM Foundation, Myositis UK, Lupus UK, and the NIHR Biomedical Research Centres at GOSH and UCLH. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Influenza Virus between Mammals.

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.

Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Changes in the hemagglutinin of H5N1 viruses during human infection--influence on receptor binding.

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.