Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Deep-learning image analysis for high-throughput screening of opsono-phagocytosis-promoting monoclonal antibodies against Neisseria gonorrhoeae.

Antimicrobial resistance (AMR) is nowadays a global health concern as bacterial pathogens are increasingly developing resistance to antibiotics. Monoclonal antibodies (mAbs) represent a powerful tool for addressing AMR thanks to their high specificity for pathogenic bacteria which allows sparing the microbiota, kill bacteria through complement deposition, enhance phagocytosis or inhibit bacterial adhesion to epithelial cells. Here we describe a visual opsono-phagocytosis assay which relies on confocal microscopy to measure the impact of mAbs on phagocytosis of the bacterium Neisseria gonorrhoeae by macrophages. With respect to traditional CFU-based assays, generated images can be automatically analysed by convolutional neural networks. Our results demonstrate that confocal microscopy and deep learning-based analysis allow screening for phagocytosis-promoting mAbs against N. gonorrhoeae, even when mAbs are not purified and are expressed at low concentration. Ultimately, the flexibility of the staining protocol and of the deep-learning approach make the assay suitable for other bacterial species and cell lines where mAb activity needs to be investigated.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

High-resolution map of the Fc functions mediated by COVID-19-neutralizing antibodies.

A growing body of evidence shows that fragment crystallizable (Fc)-dependent antibody effector functions play an important role in protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To unravel the mechanisms that drive these responses, we analyzed the phagocytosis and complement deposition mediated by a panel of 482 human monoclonal antibodies (nAbs) neutralizing the original Wuhan virus, expressed as recombinant IgG1. Our study confirmed that nAbs no longer neutralizing SARS-CoV-2 Omicron variants can retain their Fc functions. Surprisingly, we found that nAbs with the most potent Fc function recognize the N-terminal domain, followed by those targeting class 3 epitopes in the receptor binding domain. Interestingly, nAbs direct against the class 1/2 epitopes in the receptor binding motif, which are the most potent in neutralizing the virus, were the weakest in Fc functions. The divergent properties of the neutralizing and Fc function-mediating antibodies were confirmed by the use of different B cell germlines and by the observation that Fc functions of polyclonal sera differ from the profile observed with nAbs, suggesting that non-neutralizing antibodies also contribute to Fc functions. These data provide a high-resolution picture of the Fc-antibody response to SARS-CoV-2 and suggest that the Fc contribution should be considered for the design of improved vaccines, the selection of therapeutic antibodies, and the evaluation of correlates of protection.

Prior SARS-CoV-2 Infection Enhances Initial mRNA Vaccine Response with a Lower Impact on Long-Term Immunity.

Spike-encoding mRNA vaccines in early 2021 effectively reduced SARS-CoV-2-associated morbidity and mortality. New booster regimens were introduced due to successive waves of distinct viral variants. Therefore, people now have a diverse immune memory resulting from multiple SARS-CoV-2 Ag exposures, from infection to following vaccination. This level of community-wide immunity can induce immunological protection from SARS-CoV-2; however, questions about the trajectory of the adaptive immune responses and long-term immunity with respect to priming and repeated Ag exposure remain poorly explored. In this study, we examined the trajectory of adaptive immune responses following three doses of monovalent Pfizer BNT162b2 mRNA vaccination in immunologically naive and SARS-CoV-2 preimmune individuals without the occurrence of breakthrough infection. The IgG, B cell, and T cell Spike-specific responses were assessed in human blood samples collected at six time points between a moment before vaccination and up to 6 mo after the third immunization. Overall, the impact of repeated Spike exposures had a lower improvement on T cell frequency and longevity compared with IgG responses. Natural infection shaped the responses following the initial vaccination by significantly increasing neutralizing Abs and specific CD4+ T cell subsets (circulating T follicular helper, effector memory, and Th1-producing cells), but it had a small benefit at long-term immunity. At the end of the three-dose vaccination regimen, both SARS-CoV-2-naive and preimmune individuals had similar immune memory quality and quantity. This study provides insights into the durability of mRNA vaccine-induced immunological memory and the effects of preimmunity on long-term responses.

High-throughput bactericidal assays for monoclonal antibody screening against antimicrobial resistant Neisseria gonorrhoeae.

Neisseria gonorrhoeae (gonococcus) is an obligate human pathogen and the etiological agent of the sexually transmitted disease gonorrhea. The rapid rise in gonococcal resistance to all currently available antimicrobials has become a significant public health burden and the need to develop novel therapeutic and prophylactic tools is now a global priority. While high-throughput screening methods allowed rapid discovery of extremely potent monoclonal antibodies (mAbs) against viral pathogens, the field of bacteriology suffers from the lack of assays that allow efficient screening of large panels of samples. To address this point, we developed luminescence-based (L-ABA) and resazurin-based (R-ABA) antibody bactericidal assays that measure N. gonorrhoeae metabolic activity as a proxy of bacterial viability. Both L-ABA and R-ABA are applicable on the large scale for the rapid identification of bactericidal antibodies and were validated by conventional methods. Implementation of these approaches will be instrumental to the development of new medications and vaccines against N. gonorrhoeae and other bacterial pathogens to support the fight against antimicrobial resistance.

Emerging roles of SARS-CoV-2 Spike-ACE2 in immune evasion and pathogenesis.

The COVID-19 pandemic, caused by SARS-CoV-2, has caused an estimated 5 billion infections and 20 million deaths by respiratory failure. In addition to the respiratory disease, SARS-CoV-2 infection has been associated with many extrapulmonary complications not easily explainable by the respiratory infection. A recent study showed that the SARS-CoV-2 spike protein, which mediates cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, signals through ACE2 to change host cell behavior. In CD8+ T cells, spike-dependent ACE2-mediated signaling suppresses immunological synapse (IS) formation and impairs their killing ability, leading to immune escape of virus-infected cells. In this opinion article, we discuss the consequences of ACE2 signaling on the immune response and propose that it contributes to the extrapulmonary manifestations of COVID-19.

mRNA vaccines and hybrid immunity use different B cell germlines against Omicron BA.4 and BA.5.

Severe acute respiratory syndrome 2 Omicron BA.4 and BA.5 are characterized by high transmissibility and ability to escape natural and vaccine induced immunity. Here we test the neutralizing activity of 482 human monoclonal antibodies isolated from people who received two or three mRNA vaccine doses or from people vaccinated after infection. The BA.4 and BA.5 variants are neutralized only by approximately 15% of antibodies. Remarkably, the antibodies isolated after three vaccine doses target mainly the receptor binding domain Class 1/2, while antibodies isolated after infection recognize mostly the receptor binding domain Class 3 epitope region and the N-terminal domain. Different B cell germlines are used by the analyzed cohorts. The observation that mRNA vaccination and hybrid immunity elicit a different immunity against the same antigen is intriguing and its understanding may help to design the next generation of therapeutics and vaccines against coronavirus disease 2019.

B cell analyses after SARS-CoV-2 mRNA third vaccination reveals a hybrid immunity like antibody response.

The continuous evolution of SARS-CoV-2 generated highly mutated variants able to escape natural and vaccine-induced primary immunity. The administration of a third mRNA vaccine dose induces a secondary response with increased protection. Here we investigate the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses at single-cell level. We sorted 4100 spike protein specific memory B cells identifying 350 neutralizing antibodies. The third dose increases the antibody neutralization potency and breadth against all SARS-CoV-2 variants as observed with hybrid immunity. However, the B cell repertoire generating this response is different. The increases of neutralizing antibody responses is largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization. Our data show that different immunization regimens induce specific molecular signatures which should be considered while designing new vaccines and immunization strategies.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.

2D NMR Analysis as a Sensitive Tool for Evaluating the Higher-Order Structural Integrity of Monoclonal Antibody against COVID-19.

The higher-order structure (HOS) of protein therapeutics has been confirmed as a critical quality parameter. In this study, we compared 2D 1H-13C ALSOFAST-HMQC NMR spectra with immunochemical ELISA-based analysis to evaluate their sensitivity in assessing the HOS of a potent human monoclonal antibody (mAb) for the treatment of coronavirus disease 2019 (COVID-19). The study confirmed that the methyl region of the 2D 1H-13C NMR spectrum is sensitive to changes in the secondary and tertiary structure of the mAb, more than ELISA immunoassay. Because of its highly detailed level of characterization (i.e., many 1H-13C cross-peaks are used for statistical comparability), the NMR technique also provided a more informative outcome for the product characterization of biopharmaceuticals. This NMR approach represents a powerful tool in assessing the overall higher-order structural integrity of mAb as an alternative to conventional immunoassays.

The neutralizing response to SARS-CoV-2 Omicron variants BA.1 and BA.2 in COVID-19 patients and homologous and heterologous vaccinees.

The rapid replacement of Omicron BA.1 by BA.2 sublineage is very alarming, raising the question of whether BA.2 can escape the immunity acquired after BA.1 infection. We compared the neutralizing activity toward the Omicron BA.1 and BA.2 sub-lineages in five groups: COVID-19 patients; subjects who had received two doses of mRNA vaccine; subjects naturally infected with SARS-CoV-2 who had received two doses of mRNA; and subjects who had received three doses of homologous or heterologous vaccine. The results obtained highlight the importance of vaccine boosters in eliciting neutralizing antibody responses against Omicron sub-lineages, and suggest that the adenovirus vectored vaccine elicits a lower response against BA.1 than against BA.2 sub-lineage.

Production of two SARS-CoV-2 neutralizing antibodies with different potencies in Nicotiana benthamiana.

Monoclonal antibodies are considered to be highly effective therapeutic tools for the treatment of mild to moderate COVID-19 patients. In the present work, we describe the production of two SARS-CoV-2 human IgG1 monoclonal antibodies recognizing the spike protein receptor-binding domain (RBD) and endowed with neutralizing activity (nAbs) in plants. The first one, mAbJ08-MUT, was previously isolated from a COVID-19 convalescent patient and Fc-engineered to prolong the half-life and reduce the risk of antibody-dependent enhancement. This nAb produced in mammalian cells, delivered in a single intramuscular administration during a Phase I clinical study, was shown to (i) be safe and effectively protect against major variants of concern, and (ii) have some neutralizing activity against the recently emerged omicron variant in a cytopathic-effect-based microneutralization assay (100% inhibitory concentration, IC100 of 15 µg/mL). The second antibody, mAb675, previously isolated from a vaccinated individual, showed an intermediate neutralization activity against SARS-CoV-2 variants. Different accumulation levels of mAbJ08-MUT and mAb675 were observed after transient agroinfiltration in Nicotiana benthamiana plants knocked-out for xylosil and fucosil transferases, leading to yields of ~35 and 150 mg/kg of fresh leaf mass, respectively. After purification, as a result of the proteolytic events affecting the hinge-CH2 region, a higher degradation of mAb675 was observed, compared to mAbJ08-MUT (~18% vs. ~1%, respectively). Both nAbs showed a human-like glycosylation profile, and were able to specifically bind to RBD and compete with angiotensin-converting enzyme 2 binding in vitro. SARS-CoV-2 neutralization assay against the original virus isolated in Wuhan demonstrated the high neutralization potency of the plant-produced mAbJ08-MUT, with levels (IC100 < 17 ng/mL) comparable to those of the cognate antibody produced in a Chinese hamster ovary cell line; conversely, mAb675 exhibited a medium neutralization potency (IC100 ~ 200 ng/mL). All these data confirm that plant expression platforms may represent a convenient and rapid production system of potent nAbs to be used both in therapy and diagnostics in pandemic emergencies.

Immune response to SARS-CoV-2 Omicron variant in patients and vaccinees following homologous and heterologous vaccinations.

The SARS-CoV-2 Omicron variant has rapidly replaced the Delta variant of concern. This new variant harbors worrisome mutations on the spike protein, which are able to escape the immunity elicited by vaccination and/or natural infection. To evaluate the impact and susceptibility of different serum samples to the Omicron variant BA.1, samples from COVID-19 patients and vaccinated individuals were tested for their ability to bind and neutralize the original SARS-CoV-2 virus and the Omicron variant BA.1. COVID-19 patients show the most drastic reduction in Omicron-specific antibody response in comparison with the response to the wild-type virus. Antibodies elicited by a triple homologous/heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1. Overall, these findings confirm that vaccination of COVID-19 survivors and booster dose to vaccinees with mRNA vaccines is the correct strategy to enhance the antibody cross-protection against Omicron variant BA.1.

Anatomy of Omicron BA.1 and BA.2 neutralizing antibodies in COVID-19 mRNA vaccinees.

SARS-CoV-2 vaccines, administered to billions of people worldwide, mitigate the effects of the COVID-19 pandemic, however little is known about the molecular basis of antibody cross-protection to emerging variants, such as Omicron BA.1, its sublineage BA.2, and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine, were tested for neutralization against the Omicron BA.1 and BA.2 variants, and SARS-CoV-1 virus. Only 14.2, 19.9 and 4.0% of tested antibodies neutralize BA.1, BA.2, and SARS-CoV-1 respectively. These nAbs recognize mainly the SARS-CoV-2 receptor binding domain (RBD) and target Class 3 and Class 4 epitope regions on the SARS-CoV-2 spike protein. Interestingly, around 50% of BA.2 nAbs did not neutralize BA.1 and among these, several targeted the NTD. Cross-protective antibodies derive from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1, IGHV2-5;IGHJ4-1 and IGHV1-69;IGHV4-1. Only 15.6, 20.3 and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralize Omicron BA.1, BA.2 and SARS-CoV-1 respectively. Our data provide evidence, at molecular level, of the presence of cross-neutralizing antibodies induced by vaccination and map conserved epitopes on the S protein that can inform vaccine design.

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody.

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryoelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

Immune response in COVID-19: what is next?

The coronavirus disease 2019 (COVID-19) has been a global pandemic for more than 2 years and it still impacts our daily lifestyle and quality in unprecedented ways. A better understanding of immunity and its regulation in response to SARS-CoV-2 infection is urgently needed. Based on the current literature, we review here the various virus mutations and the evolving disease manifestations along with the alterations of immune responses with specific focuses on the innate immune response, neutrophil extracellular traps, humoral immunity, and cellular immunity. Different types of vaccines were compared and analyzed based on their unique properties to elicit specific immunity. Various therapeutic strategies such as antibody, anti-viral medications and inflammation control were discussed. We predict that with the available and continuously emerging new technologies, more powerful vaccines and administration schedules, more effective medications and better public health measures, the COVID-19 pandemic will be under control in the near future.

Safety and serum distribution of anti-SARS-CoV-2 monoclonal antibody MAD0004J08 after intramuscular injection.

The emerging threat represented by SARS-CoV-2 variants, demands the development of therapies for better clinical management of COVID-19. MAD0004J08 is a potent Fc-engineered monoclonal antibody (mAb) able to neutralize in vitro all current SARS-CoV-2 variants of concern (VoCs) including the omicron variant even if with significantly reduced potency. Here we evaluated data obtained from the first 30 days of a phase 1 clinical study (EudraCT N.: 2020-005469-15 and ClinicalTrials.gov Identifier: NCT04932850). The primary endpoint evaluated the percentage of severe adverse events. Secondary endpoints evaluated pharmacokinetic and serum neutralization titers. A single dose administration of MAD0004J08 via intramuscular (i.m.) route is safe and well tolerated, resulting in rapid serum distribution and sera neutralizing titers higher than COVID-19 convalescent and vaccinated subjects. A single dose administration of MAD0004J08 is also sufficient to effectively neutralize major SARS-CoV-2 variants of concern (alpha, beta, gamma and delta). MAD0004J08 can be a major advancement in the prophylaxis and clinical management of COVID-19.

A new dawn for monoclonal antibodies against antimicrobial resistant bacteria.

Antimicrobial resistance (AMR) is a quickly advancing threat for human health worldwide and almost 5 million deaths are already attributable to this phenomenon every year. Since antibiotics are failing to treat AMR-bacteria, new tools are needed, and human monoclonal antibodies (mAbs) can fill this role. In almost 50 years since the introduction of the first technology that led to mAb discovery, enormous leaps forward have been made to identify and develop extremely potent human mAbs. While their usefulness has been extensively proved against viral pathogens, human mAbs have yet to find their space in treating and preventing infections from AMR-bacteria and fully conquer the field of infectious diseases. The novel and most innovative technologies herein reviewed can support this goal and add powerful tools in the arsenal of weapons against AMR.

Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.