Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

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.

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.

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.

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma.

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

Role of a fluid-phase PRR in fighting an intracellular pathogen: PTX3 in Shigella infection.

Shigella spp. are pathogenic bacteria that cause bacillary dysentery in humans by invading the colonic and rectal mucosa where they induce dramatic inflammation. Here, we have analyzed the role of the soluble PRR Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity. Mice that had been intranasally infected with S. flexneri were rescued from death by treatment with recombinant PTX3. In vitro PTX3 exerts the antibacterial activity against Shigella, impairing epithelial cell invasion and contributing to the bactericidal activity of serum. PTX3 is produced upon LPS-TLR4 stimulation in accordance with the lipid A structure of Shigella. In the plasma of infected patients, the level of PTX3 amount only correlates strongly with symptom severity. These results signal PTX3 as a novel player in Shigella pathogenesis and its potential role in fighting shigellosis. Finally, we suggest that the plasma level of PTX3 in shigellosis patients could act as a biomarker for infection severity.

The Orchestra and Its Maestro: Shigella's Fine-Tuning of the Inflammasome Platforms.

Shigella spp. are the causative agents of bacillary dysentery, leading to extensive mortality and morbidity worldwide. These facultative intracellular bacteria invade the epithelium of the colon and the rectum, inducing a severe inflammatory response from which the symptoms of the disease originate. Shigella are human pathogens able to manipulate and subvert the innate immune system surveillance. Shigella dampens inflammasome activation in epithelial cells. In infected macrophages, inflammasome activation and IL-1ß and IL-18 release lead to massive neutrophil recruitment and greatly contribute to inflammation. Here, we describe how Shigella hijacks and finely tunes inflammasome activation in the different cell populations involved in pathogenesis: epithelial cells, macrophages, neutrophils, DCs, and B and T lymphocytes. Shigella emerges as a "sly" pathogen that switches on/off the inflammasome mechanisms in order to optimize the interaction with the host and establish a successful infection.

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS.

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroideslacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation.

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1ß in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Thermophiles as potential source of novel endotoxin antagonists: the full structure and bioactivity of the lipo-oligosaccharide from Thermomonas hydrothermalis.

Thermomonas hydrothermalis is a Gram-negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell-wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure-dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system-an antagonistic effect, as shown in studies of LPS structure-activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo-oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.

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.

Chemistry and biology of the potent endotoxin from a Burkholderia dolosa clinical isolate from a cystic fibrosis patient.

This is the first report of the chemical and biological properties of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia dolosa IST4208, an isolate recovered from a cystic fibrosis (CF) patient in a Portuguese CF center. B. dolosa is a member of the Burkholderia cepacia complex, a group of closely related species that are highly problematic and opportunistic pathogens in CF. B. dolosa infection leads to accelerated loss of lung function and decreased survival. The structural determination of its endotoxin was achieved using a combination of chemistry and spectroscopy, and has revealed a novel endotoxin structure. The purified LOS was tested for its immunostimulatory activity on human HEK 293 cells expressing TLR-4, MD-2, and CD-14. In these assays, the LOS showed strong proinflammatory activity.

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.

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.

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.

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.

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.

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.

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.