Michael Mor

The COVID-19 pandemic, which has resulted in over seven million global fatalities, poses a substantial threat to public health and precipitated a global economic crisis. Emerging variants of concern (VOCs) with enhanced transmissibility and improved immune evasion may compromise the efficacy of current antiviral and immunotherapies, necessitating comprehensive investigations into the immune response to SARS-CoV-2. The conformational dynamics of the receptor binding domain in SARS-CoV-2 spike and the presentation of neutralizing antibody epitopes influence viral transmission and infection rates. In this study, we have identified highly conserved non-receptor-binding motif epitopes for two potent monoclonal antibodies (mAbs), TAU-1109 and TAU-2310, isolated from convalescent human patients, which contribute to the broad neutralizing activity of these mAbs against all the circulating VOCs, including the recently emerged Omicron subvariants. We employed high-resolution structural data in conjunction with systematic biochemical investigation to elucidate the neutralization mechanism of TAU-1109 and TAU-2310. The mechanism involves antibody-mediated destabilization of the spike trimer, resulting in the premature shedding of the S1 subunit and rendering the spike incapable of mediating host cell entry. The identification of conserved cryptic epitopes in our study advances the mechanistic understanding of immune response against SARS-CoV-2, providing alternative avenues for the development of universal therapeutic antibodies and vaccines to combat COVID-19.

The COVID-19 pandemic, which has resulted in over seven million global fatalities, poses a substantial threat to public health and precipitated a global economic crisis. Emerging variants of concern (VOCs) with enhanced transmissibility and improved immune evasion may compromise the efficacy of current antiviral and immunotherapies, necessitating comprehensive investigations into the immune response to SARS-CoV-2. The conformational dynamics of the receptor binding domain (RBD) in SARS-CoV-2 spike and the presentation of neutralizing antibody epitopes influence viral transmission and infection rates. In this study, we have identified highly conserved non-RBM epitopes for two potent monoclonal antibodies (mAbs), TAU-1109 and TAU-2310, isolated from convalescent human patients, which contribute to the broad neutralizing activity of these mAbs against all the circulating VOCs, including the recently emerged Omicron subvariants. We employed high- resolution structural data in conjunction with systematic biochemical investigation to elucidate the neutralization mechanism of TAU-1109 and TAU-2310. The mechanism involves antibody-mediated destabilization of the spike trimer, resulting in the premature shedding of the S1 subunit and rendering the spike incapable of mediating host cell entry. The identification of conserved cryptic epitopes in our study advances the mechanistic understanding of immune response against SARS-CoV-2, providing novel avenues for the development of universal therapeutic antibodies and vaccines to combat COVID-19.

Human cytomegalovirus can cause severe birth defects upon infection in pregnant women and complications in immunocompromised patients. A major challenge for vaccine design is our incomplete understanding of the diverse protein complexes this virus uses to infect cells. In Herpesviridae, glycoproteins H and L (gH and gL) form complexes with other viral proteins that bind receptors to mediate cell-type-specific entry. Here we identify a distinct gH complex that is abundant on human cytomegalovirus virions and enhances infection of endothelial cells. In this complex, gH associates with UL116 and UL141 (an immunoevasin previously known to function intracellularly) but not with gL. We term this the gH-associated tropism and entry (GATE) complex and provide the cryo-electron microscopy structure at ~3.5 Å. The structure shows gH-only scaffolding, UL141-mediated dimerization and a heavily glycosylated UL116 cap. These findings identify a third virion surface complex that promotes cell entry and may represent a new target for vaccines or antiviral therapies.

The first approved vaccines for human use against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are nanotechnology‐based. Although they are modular, rapidly produced, and can reduce disease severity, the currently available vaccines are restricted in preventing infection, stressing the global demand for novel preventive vaccine technologies. Bearing this in mind, we set out to develop a flexible nanovaccine platform for nasal administration to induce mucosal immunity, which is fundamental for optimal protection against respiratory virus infection. The next‐generation multiepitope nanovaccines co‐deliver immunogenic peptides, selected by an immunoinformatic workflow, along with adjuvants and regulators of the PD‐L1 expression. As a case study, we focused on SARS‐CoV‐2 peptides as relevant antigens to validate the approach. This platform can evoke both local and systemic cellular‐ and humoral‐specific responses against SARS‐CoV‐2. This led to the secretion of immunoglobulin A (IgA), capable of neutralizing SARS‐CoV‐2, including variants of concern, following a heterologous immunization strategy. Considering the limitations of the required cold chain distribution for current nanotechnology‐based vaccines, it is shown that the lyophilized nanovaccine is stable for long‐term at room temperature and retains its in vivo efficacy upon reconstitution. This makes it particularly relevant for developing countries and offers a modular system adaptable to future viral threats.

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19–recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.

Patients with inflammatory bowel disease (IBD) treated with anti-tumor-necrosis factor-alpha (TNFα) exhibited lower serologic responses one-month following the second dose of the COVID-19 BNT162b2 vaccine compared to those not treated with anti-TNFα (non-anti-TNFα) or to healthy controls (HCs). We comprehensively analyzed long-term humoral responses, including anti-spike (S) antibodies, serum inhibition, neutralization, cross-reactivity and circulating B cell six months post BNT162b2, in patients with IBD stratified by therapy compared to HCs. Subjects enrolled in a prospective, controlled, multi-center Israeli study received two BNT162b2 doses. Anti-S levels, functional activity, specific B cells, antigen cross-reactivity, anti-nucleocapsid levels, adverse events and IBD disease score were detected longitudinally. In total, 240 subjects, 151 with IBD (94 not treated with anti-TNFα and 57 treated with anti-TNFα) and 89 HCs participated. Six months after vaccination, patients with IBD treated with anti-TNFα had significantly impaired BNT162b2 responses, specifically, more seronegativity, decreased specific circulating B cells and cross-reactivity compared to patients untreated with anti-TNFα. Importantly, all seronegative subjects were patients with IBD; of those, >90% were treated with anti-TNFα. Finally, IBD activity was unaffected by BNT162b2. Altogether these data support the earlier booster dose administration in these patients.

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å, respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 Å, 7.76 Å, 6.47 Å, 3.45 Å, and 7.32 Å) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Mycobacterium tuberculosis(Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduceMycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.