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 presence of B cells in tumors is correlated with favorable prognosis and efficient response to immunotherapy. While tumor-reactive antibodies have been detected in several cancer types, identifying antibodies that specifically target tumor-associated antigens remains a challenge. Here, we investigated the antibodies spontaneously elicited during breast and lung cancer that bind the cancer-associated antigen MET. We screened patients with lung (n = 25) and breast (n = 75) cancer and found that 13% had antibodies binding to both the recombinant ectodomain of MET, and the ligand binding part of MET, SEMA. MET binding in the breast cancer cohort was significantly correlated with hormone receptor-positive status. We further conducted immunoglobulin sequencing of peripheral MET-enriched B cells from 6 MET-reactive patients. The MET-enriched B cell repertoire was found to be polyclonal and prone to non-IgG1 subclass. Nine monoclonal antibodies were cloned and analyzed, and these exhibited MET binding, low thermostability, and high polyreactivity. Among these, antibodies 87B156 and 69B287 effectively bound to tumor cells and inhibited MET-expressing breast cancer cell lines. Overall, our data demonstrate that some patients with breast and lung cancer develop polyreactive antibodies that cross-react with MET. These autoantibodies have a potential contribution to immune responses against tumors.

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.

Vaccines are pivotal for control of the coronavirus disease (COVID-19) pandemic. Patients with inflammatory bowel diseases (IBDs) treated with antitumor necrosis factor (TNF)-α have lower serologic response after two COVID-19 vaccine doses. Data regarding a third vaccine dose are scarce. An Israeli multicenter prospective observational study recruited 319 subjects: 220 with IBD (79 treated with anti-TNFα) and 99 healthy control (HC) participants. All patients received two mRNA-BNT162b2 vaccines (Pfizer/BioNTech), 80% of whom received a third vaccine dose. Evaluation included disease activity, anti-spike (S) and nucleocapsid (N) antibody levels, anti-TNFα drug levels, and adverse events (AEs). All participants showed significant serologic response one month after receiving a third dose. However, three months later, the anti-S levels decreased significantly in patients treated with anti-TNFα compared with the non-anti-TNFα and HC groups. A correlation between serologic response to the third vaccine dose and anti-TNF drug levels was not found. No significant AE or IBD exacerbation was observed. Importantly, lower serologic response after the third vaccine dose predicted infection. A third dose of BNT162b2 is effective and safe in patients with IBD. Lower serologic response predicted infection, even in seropositive subjects. Lower serologic responses and their rapid decline suggest a fourth vaccine dose in this patient population.

Monkeypox virus (MPXV) resides in two forms; mature and enveloped, and depending on it, distinct proteins are displayed on the viral surface. Here, we expressed two MPXV antigens from the mature, and one from the enveloped form, and tested their reactivity to sera of 11 MPXV recoverees while comparing to sera from recently and past vaccinated individuals. 8 out of 11 recoverees exhibited detectable neutralization levels against Vaccinia Lister. Sera from all recoverees bound strongly to A35R and H3L antigens. Moreover, the responses to A35R were significantly higher within the recoverees compared to both recently and past vaccinated donors. Lastly, A35R- and H3L-specific IgG(+) B cells ranging from 0.03-0.46% and 0.11-0.36%, respectively, were detected in all recoverees (A35R), and in 9 out of 11 recoverees (H3L). Therefore, A35R and H3L represent MPXV immune targets and could be used in a heat-inactivated serological ELISA for the identification of recent MPXV infection.

Abstract Background Vaccines are pivotal for control of the ongoing coronavirus disease (COVID-19) pandemic. Patients with inflammatory bowel diseases (IBD) treated with anti-tumor necrosis factor (TNF)-α have lower serologic response after two COVID-19 vaccine doses. Data regarding a 3rd vaccine are scarce. Methods Aim: To assess immune responses to, and safety of COVID-19 vaccines in patients with IBD, stratified according to therapy, and compared to healthy controls (HC). Subjects were recruited before the 1st vaccine (BNT162b2, Pfizer) and prospectively evaluated after the 2nd and 3rd vaccine doses. Evaluation included: disease activity, anti-spike (S) and nucleocapsid (N), anti-TNFα drug levels and adverse events (AE) Results Of 198 subjects having the 3rd vaccine dose, 125 had IBD: average age: 39.1±14.8 years; 40.8% females; 82- Crohn’s disease (CD), 33 ulcerative colitis (UC), 6 pouch, 3 IBD-U. There were 73 HC: average age 39.4±12.5 years, 69.9% females. Among patients with IBD: 51 and 74 (40.8%, 59.2%)) were treated or not with anti-TNFα, respectively. A month after the 3rd vaccine dose IBD activity was comparable in all patients regardless of treatment, and no increase in C-reactive protein or white blood cells was observed. Higher but not significant AE rate was registered in all subjects after the 3rd compared to 2nd vaccine dose (81% vs. 76%, respectively). AE rate in IBD and HC was comparable. No serious AE detected. There was a significant increase in anti-S levels one month after compared to pre 3rd vaccine dose in all participants. Furthermore, increase was 2-3 folds higher than that observed one month after the 2nd dose. Importantly, patients treated with anti-TNFα compared to non-anti-TNFα treated had significantly lower responses: 9219 (6347-13390) vs 16955 (13721-20951) (GMC (95%CI)), p<0.05. Serologic response did not correlate with anti-TNFα drug levels, antibodies or interval between drug and vaccine administration. During extended follow-up post 3rd dose, we found that lower serologic response predicts infection over time. Conclusion This prospective study shows that a 3rd dose of BNT162b2 vaccine is effective and safe in patients with IBD. Furthermore, patients treated with anti-TNFα had significantly lower serologic responses compared to anti-TNFα untreated ones. Lack of correlation between anti-TNFα drug levels and immune responses suggests there is no need to modify vaccination timing relatively to anti-TNFα administration. The significantly steeper increase in anti-S levels between 2nd and 3rd doses, suggests the 3rd dose is crucial in anti-TNFα treated patients, specifically due to the fact that higher serologic response predicts better defense from infection.

Stress-induced β2-adrenergic receptor (β2AR) activation in B cells increases IgG secretion; however, the impact of this activation on antibody affinity and the underlying mechanisms remains unclear. In the current study, we demonstrate that stress in mice following ovalbumin (OVA) or SARS-CoV-2 RBD immunization significantly increases both serum and surface-expressed IgG binding to the immunogen, while concurrently reducing surface IgG expression and B cell clonal expansion. These effects were abolished by pharmacological β2AR blocking or when the experiments were conducted in β2AR -/- mice. In the second part of our study, we used single B cell sorting to characterize the monoclonal antibodies (mAbs) generated following β2AR activation in cultured RBD-stimulated B cells from convalescent SARS-CoV-2 donors. Ex vivo β2AR activation increased the affinities of the produced anti-RBD mAbs by 100-fold compared to mAbs produced by the same donor control cultures. Consistent with the mouse experiments, β2AR activation reduced both surface IgG levels and the frequency of expanded clones. mRNA sequencing revealed a β2AR-dependent upregulation of the PI3K pathway and B cell receptor (BCR) signaling through AKT phosphorylation, as well as an increased B cell motility. Overall, our study demonstrates that stress-mediated β2AR activation drives changes in B cells associated with BCR activation and higher affinity antibodies.

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.

Monkeypox virus (MPXV) resides in two forms, mature and enveloped virions, and depending on it, distinct proteins are displayed on the viral surface. We expressed in mammalian cells two MPXV antigens from the mature form and two MPXV antigens from the enveloped form and tested their reactivity to sera of 11 MPXV convalescent donors diagnosed in Israel during May-June 2022 and collected 33-62 days post infection. While only 4 out of 11 donors neutralized the related Vaccinia Lister strain, all MPXV recoverees demonstrated a strong serological response to a 124-amino acid truncation of the A35R antigen, and to a 276-amino acid truncation of the H3L antigen. Moreover, A35R- and H3L-specific B cells ranging from 0.03-0.5% and 0.01-0.35% of IgG+CD19+ cells, respectively, were detected in all 11 MPXV donors (A35R), and in 8 out of 11 donors (H3L). Therefore, A35R and H3L represent MPXV immune targets, and can be used in a simple heat-inactivated serological enzyme-linked immunosorbent assay for the identification of recent MPXV infection.

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 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.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Adrenergic signaling plays a central role in physiological regulation, including modulation of processes in the immune system. However, the effects of adrenergic activation on antibody-mediated immune response remain unknown. Here, we investigate the effects of stress - induced β-adrenergic receptor activation on the B cell response at molecular and the systemic levels. We find that β-adrenergic agonist treatment of B cells from three convalescent SARS coronavirus-2 donors, reduced both membrane IgG expression and clonal expansion when the cells were stimulated ex vivo with spike receptor binding domain (RBD). Interestingly, monoclonal anti-RBD antibodies cloned from B cells cultured in the presence of β-adrenergic agonist, exhibited higher affinity for RBD compared to antibodies cloned from control cultures, suggesting that clones under stress exhibit higher antigen affinity. As a corollary, following ovalbumin immunization in mice, physiological stress during germinal center reaction phase increased the levels of antigen specific serum IgG. At the same time, B cell clonal expansion and membrane IgG expression were reduced, along with an increase in MHC class II expression. These effects were abolished by treatment with the non-selective β-adrenergic antagonist, propranolol. Our study suggests that under stress conditions selection of high affinity variants comes in the expense of clonal expansion.

Despite ongoing efforts to characterize the host response toward SARS-CoV-2, a major gap in our knowledge still exists regarding the magnitude and duration of the humoral response. Analysis of the antibody response in mild versus moderate/severe patients, using our new developed quantitative electrochemiluminescent assay for detecting IgM/IgA/IgG antibodies toward SARS-CoV-2 antigens, revealed a rapid onset of IgG/IgA antibodies, specifically in moderate/severe patients. IgM antibodies against the viral receptor binding domain, but not against nucleocapsid protein, were detected at early stages of the disease. Furthermore, we observed a marked reduction in IgM/IgA antibodies over-time. Adapting our assay for ACE2 binding-competition, demonstrated that the presence of potentially neutralizing antibodies is corelated with IgG/IgA. Finally, analysis of the cytokine profile in COVID-19 patients revealed unique correlation of an IL-12p70/IL33 and IgG seroconversion, which correlated with disease severity. In summary, our comprehensive analysis has major implications on the understanding and monitoring of SARS-CoV-2 infections.

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 reduce Mycobacterium 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.

AbstractDespite ongoing efforts to characterize the host response toward SARS-CoV-2, a major gap in our knowledge still exists regarding the magnitude and duration of the humoral response. We report the development of a rapid, highly specific and sensitive electrochemiluminescent assay for detecting IgM, IgA, and IgG antibodies toward two distinct SARS-CoV-2 antigens namely, the receptor binding domain (RBD) and the nuclear protein (NP). Whereas IgM antibodies toward RBD were detected at early stages of the disease, IgM antibodies against NP did not develop. Analysis of the antibody response in mild versus moderate/severe patients revealed a rapid onset of IgG and IgA antibodies, specifically in moderate/severe patients. Finally, we observed a marked reduction in IgM/IgA antibodies and to lesser extent, IgG, over time. We provide a comprehensive analysis of the human antibody response, and has major implications on our understanding and monitoring of SARS-CoV-2 infections, as well as finding effective vaccines.One Sentence SummaryUsing a newly developed assay to detect anti-SARS-Cov-2 IgM, IgG and IgA antibodies we reveal a rapid onset of IgG and IgA antibodies towards distinct viral antigens, specifically in moderate/severe COVID-19 patients,