The role of antibodies in the host response against Mycobacterium tuberculosis (M. tb) bacteria is still poorly understood. We previously isolated two monoclonal antibodies (mAbs), p4-36 and p4-163, from an M. tb infected donor that target two non-overlapping epitopes on PstS1, a subunit of the M. tb phosphate transporter. Although these antibodies reduced lung bacterial burden in mice (30-40% reduction in CFU), their efficacy remained modest for therapeutic application. Here, we employed a rational antibody engineering approach to further enhance their anti-M. tb potency. Affinity maturation of p4-163 yielded p4-163LR, a variant with superior binding to PstS1 and improved recognition of live, attenuated M. tb. Surprisingly, p4-163LR alone did not confer enhanced protection against virulent M. tb in vivo. However, the generation of a bispecific antibody combining p4-36 and p4-163LR (Bi-S 36/163LR) significantly improved bacterial binding and antibody-dependent cellular phagocytosis (ADCP). Notably, prophylactic administration of Bi-S 36/163LR led to a ~ 1 log reduction in lung bacterial burden compared to control animals treated with isotype control. These findings define a novel, structure-guided strategy to amplify the functional capacity of natural anti-M. tb antibodies and highlight bispecific antibody platforms as promising candidates for host-directed tuberculosis immunotherapy.

The human immune system employs both innate and adaptive mechanisms to control pathogens, with antibodies playing a pivotal role in immune memory and defense, in particular against viral infections. In tuberculosis, antibody titers have long been used to assess immune responses, but their presence alone fails to predict protective efficacy. Recent studies highlight that antibody functionality is critical for effective immune activity. Despite widespread detection of Mycobacterium tuberculosis (Mtb)-reactive antibodies in individuals with active disease, Mtb infection, and even in healthy controls, their potential to control Mtb growth is variable and only detected in a proportion of individuals. This perspective emphasizes the need for robust functional assessment of antibodies to better understand their role in mycobacterial control and inform vaccine development. Notably, antibodies binding to purified protein derivative of Mtb, a mixture of degraded antigens from Mtb cultures, are widespread but not universally functional, underscoring the importance of Fc characteristics and epitope specificity. Initial high-throughput screening using phagocytosis and direct mycobacterial binding assays is an active indicator of antibody function. By refining and combining existing assays, as recommended in this perspective, we can better characterize antibody contributions, particularly their immunomodulatory potential, toward improved control of Mtb. Albeit antibodies may not be essential in natural protection, functional antibodies induced by vaccination may be of added value and contribute to host protection.

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.

Melanoma cells secrete melanosomes, large extracellular vesicles that reinforce tumor growth and survival signaling. To determine whether these vesicles elicit functional humoral immunity, we immunized mice with melanoma-derived melanosomes and analyzed the resulting antibody responses. This approach induced B cell expansion and generated antibodies directed against heat shock protein 70 (HSP70) present on the surface of melanosomes. Functionally, anti-HSP70 monoclonal antibodies inhibited growth in murine B16 and human MNT-1 melanoma cells independently of immune effector cells while suppressing key transcriptional programs involved in proliferation. In vivo, passive administration of anti-HSP70 antibodies delayed tumor onset and improved survival in a prophylactic B16 model. Moreover, in patients with metastatic melanoma treated with immune checkpoint blockade, higher serum anti-HSP70 immunoglobulin G (IgG) levels were associated with complete response compared with progressive disease. Together, these findings identify an extracellular vesicle-antibody axis that constrains melanoma survival signaling and has biomarker and therapeutic implications.

Abstract The generation of a diverse and self-tolerant B cell repertoire is essential for adaptive immunity and is achieved through V(D)J recombination. In mice, Igκ is the dominant light chain, whereas Igλ rearrangement typically occurs in response to nonproductive or autoreactive Igκ recombination, a process termed receptor editing. Recombination at the RS element deletes the Igκ constant exon, silencing the locus and enabling Igλ expression. However, the epigenetic regulatory framework that orchestrates and governs receptor editing remains poorly defined. Here, we identify a CTCF-binding insulator element (CBE) within the 3′ Igκ super-enhancer (3′-SEκ) that regulates receptor editing and directs the κ-to-λ switch required for Igλ⁺ B-cell development. Mechanistically, loss of this CBE activates an insulated enhancer within the 3′-SEκ, causing aberrant Vκ rearrangements and altered chromatin interactions through disrupted loop extrusion dynamics. Notably, loss of this CBE in mice leads to increased autoantibody production by ten weeks of age, demonstrating that CBE-mediated chromatin architecture shapes B cell fate by constraining autoreactive potential. Collectively, our findings define a novel CTCF-dependent cis-regulatory insulation checkpoint that connects chromatin loop extrusion to antigen-driven receptor editing, thereby enforcing B-cell tolerance.

Over the last 5 years, the world has been facing the Corona Virus Disease 2019 (COVID-19) pandemic caused by the SARS-CoV2 virus. While vaccination is a leading strategy to control virus spread, it demonstrated an ability to push the virus to evolve and generate vaccine-escape variants. Nevertheless, the official policy in Israel and around the world is vaccination, even in cases of previous SARS-CoV2 infection. Many studies were published regarding vaccination protocols and antibody responses. However, data concerning the differences in humoral and cellular immune response between convalescent individuals who received a single vaccine dose, and SARS-CoV2 naïve vaccinated individuals, are still sparse. In this study we evaluated the humoral and cellular immune response of healthy convalescent individuals who received a single vaccine dose, and compared it with uninfected individuals who received three BNT162b2 mRNA vaccine doses. Humoral immune response was evaluated by testing the ability of donor samples to bind or inhibit the binding of different SARS-CoV2 variants Spike glycoprotein receptor binding domain (RBD) to ACE2. Cellular immunity was tested using flow cytometry to evaluate cytokine production in response to different SARS-CoV2 peptide mixes, including the Wuhan wild-type (WT) Spike glycoprotein (S)-peptide mix, Omicron-specific S-peptide mix and the Membrane (M) protein peptide mix. We show that the ability of convalescent, single-dose vaccinated, donors to bind RBD or inhibit ACE2:RBD interaction was comparable to that of 3-dose vaccinated-only donors, independent of the variant tested. In contrast, when testing cellular response, convalescent individuals showed increased IFNγ staining following WT-S peptide mix stimulation (average of 988.4 ± 687.1 vs 590.2 ± 397.1, p = 0.022 responding IFNγ+ cells per 106 CD4+ cells), and even more enhanced response to M-protein peptide mix (average of 2291 ± 4074.2 vs 239 ± 485.85, p = 0.023 responding IFNγ+ cells per 106 CD4+ cells). This research may provide information for future development of more effective vaccines and vaccination strategies.

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.

While antibodies have emerged as potential mediators of protective immunity against Mycobacterium tuberculosis (Mtb), their mechanisms of action remain incompletely understood. Here, we demonstrate that immune complexes of Mtb and monoclonal antibodies targeting the Mtb phosphate transporter subunit PstS1 robustly activate the NLRP3 inflammasome in human and murine macrophages, leading to enhanced interleukin-1β secretion. Surprisingly, antibody-mediated inflammasome activation occurred independently of cell-surface Fcγ receptors, as confirmed using Fc-domain glycosylation mutant mAbs and macrophages from Fcγ receptor-deficient mice. Crucially, NLRP3 is indispensable for early antibody-mediated protection in vivo, as both pharmacological inhibition, and genetic deletion of NLRP3 completely abolished protective effects of PstS1-specific antibodies in Mtb-infected mice. This mechanism extends beyond monoclonal antibodies, as polyclonal sera from intravenously BCG-immunized rhesus macaques also required NLRP3 for protective efficacy. Our findings reveal a previously unrecognized mechanism by which Mtb-specific antibodies enhance host defense through inflammasome activation, potentially informing novel approaches for tuberculosis vaccine development.

Monkeypox virus (MPXV) is the most pathogenic Poxvirus in circulation, yet key viral antigens remain immunologically unexplored. We isolate and characterize a panel of monoclonal antibodies (mAbs) targeting MPXV A28 (OPG153), an important membranal protein present on mature MPXV virions. From male convalescent individuals, we isolate anti-A28 mAbs alongside additional mAbs targeting the A35 and H3 proteins. Anti-A28 mAbs potently neutralize MPXV and Vaccinia virus (VACV) through complement-dependent mechanisms involving C1q and C3 deposition. High-resolution crystal structures of two anti-A28 mAbs, 10M2146 and 8M2110, in complex with VACV A26 reveal two distinct and highly conserved proximal epitopes within the N-terminal domain. Passive transfer of 8M2110 modestly attenuates disease in infected female mice. Moreover, immunization with A28 elicits antigen-specific B cells and robust neutralizing antibody responses and provides protection against lethal VACV challenge. These findings identify MPXV A28 as a promising central target for the induction of neutralizing antibodies and antiviral interventions.

Mpox is the most pathogenic Poxvirus in circulation. While several antigens have been identified as targets for neutralizing antibodies, many proteins remain unexplored. We isolated and characterized four monoclonal antibodies (mAbs) targeting the Mpox A28 (OPG153), a virulence factor present on mature Mpox virions. The antibodies were isolated from convalescent individuals, alongside 14 additional mAbs targeting the A35 and H3 proteins. Anti-A28 mAbs potently neutralized Mpox and Vaccinia virus (VACV) through complement-dependent mechanisms involving C1q and C3 deposition. High resolution crystal structures of Anti-A28 mAbs 10M2146 and 8M2110 in complex with VACV A26 revealed two proximal epitopes within the N-terminal domain. Passive transfer of 8M2110 attenuated disease in infected mice. Moreover, immunization with A28 elicited antigen-specific B cells and robust neutralizing antibody responses and provided complete protection against lethal VACV challenge. These findings support Mpox A28 as a promising target for the induction of neutralizing antibodies and antiviral interventions. ### Competing Interest Statement The authors have declared no competing interest. Israel Science Foundation, 3136/22, 638/23 Binational Science Foundation, 01031771 BMGF, INV-058519 Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS-MIE, the Vaccine Research Institute (VRI), ANR-10-LABX-77 Labex IBEID, ANR-10-LABX-62-IBEID the HERA projects DURABLE, grant 101102733

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.

Being able to accurately predict the three-dimensional structure of an Ab can facilitate Ab characterization and epitope prediction, with important diagnostic and clinical implications. In this study, we evaluated the ability of AlphaFold to predict the structures of 222 recently published, high-resolution Fab H and L chain structures of Abs from different species directed against different Ags. We show that although the overall Ab prediction quality is in line with the results of CASP14, regions such as the complementarity-determining regions (CDRs) of the H chain, which are prone to higher variation, are predicted less accurately. Moreover, we discovered that AlphaFold mispredicts the bending angles between the variable and constant domains. To evaluate the ability of AlphaFold to model Ab-Ag interactions based only on sequence, we used AlphaFold-Multimer in combination with ZDOCK to predict the structures of 26 known Ab-Ag complexes. ZDOCK, which was applied on bound components of both the Ab and the Ag, succeeded in assembling 11 complexes, whereas AlphaFold succeeded in predicting only 2 of 26 models, with significant deviations in the docking contacts predicted in the rest of the molecules. Within the 11 complexes that were successfully predicted by ZDOCK, 9 involved short-peptide Ags (18-mer or less), whereas only 2 were complexes of Ab with a full-length protein. Docking of modeled unbound Ab and Ag was unsuccessful. In summary, our study provides important information about the abilities and limitations of using AlphaFold to predict Ab-Ag interactions and suggests areas for possible improvement.

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.