B cell maturation restored ancestral germlines to control Omicron BA.2.86 (preprint)

The unceasing interplay between SARS-CoV-2 and the human immune system has led to a continuous maturation of the virus and B cell response providing an opportunity to track their evolution in real time. We longitudinally analyzed the functional activity of almost 1,000 neutralizing human monoclonal antibodies (nAbs) isolated from vaccinated people, and from individuals with hybrid and super hybrid immunity (SH), developed after three mRNA vaccine doses and two breakthrough infections. The most potent neutralization and Fc functions against highly mutated variants, including BA.2.86, were found in the SH cohort. Despite different priming, epitope mapping revealed a convergent maturation of the functional antibody response. Neutralization was mainly driven by Class 1/2 nAbs while Fc functions were induced by Class 3/4 antibodies. Remarkably, broad neutralization was mediated by restored IGHV3-53/3-66 B cell germlines which, after heterogenous exposure to SARS-CoV-2 S proteins, increased their level of somatic hypermutations. Our study shows the resilience of the human immune system which restored previously expanded germlines and activated naive B cells to broaden the antibody repertoire of antibodies to control future SARS-CoV-2 variants.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody (preprint)

SUMMARY/ABSTRACTThree 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 (preprint)

A growing body of evidence shows that 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 not-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.

mRNA vaccines and hybrid immunity use different B cell germlines to neutralize Omicron BA.4 and BA.5 (preprint)

SARS-CoV-2 omicron BA.4 and BA.5, characterized by high transmissibility and ability to escape natural and vaccine induced immunity, are rampaging worldwide. To understand the escape mechanisms, we tested the neutralizing activity against omicron BA.4 and BA.5 of a panel of 482 human monoclonal antibodies that had been isolated from people who received two or three mRNA vaccine doses or from people that had been vaccinated after infection. None of the antibodies isolated after two vaccine doses neutralized omicron BA.4 and BA.5, while these variants were neutralized by approximately 15% of antibodies obtained from people that received three doses or had been vaccinated after infection. Remarkably, the antibodies isolated after three vaccine doses targeted mainly the receptor binding domain (RBD) Class 1/2 epitope region and were encoded by the IGHV1-69 and IGHV3-66 B cell germlines, while the antibodies isolated after infection recognized mostly the RBD Class 3 epitope region and the NTD, and were encoded by the IGHV2-5;IGHJ4-1 and IGHV1-24;IGHJ4-1 germlines. 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 COVID-19.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly (preprint)

CTL-mediated killing of virally infected or malignant cells is orchestrated at a specialized intercellular junction, the immune synapse (IS). We hypothesized that SARS-CoV-2 may target IS assembly in CTLs to escape killing. We show that primary human CD8+ T cells strongly upregulate the expression of ACE2, the Spike protein receptor, during differentiation to CTLs. CTL pre-incubation 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 polarisation to the IS, resulting in impaired target cell killing. These defects were reversed by anti-Spike antibodies that interfere with ACE2 binding and were reproduced by ACE2 engagement with Angiotensin-II or an anti-ACE2 antibody, but not by the ACE2 product Ang (1-7). 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 the elimination of infected cells.

COVID-19 mRNA third dose induces a unique hybrid immunity-like antibody response (preprint)

The continuous evolution of SARS-CoV-2 generated highly mutated variants, like omicron BA.1 and BA.2, able to escape natural and vaccine-induced primary immunity. The administration of a third dose of mRNA vaccines induces a secondary response with increased protection. We investigated, at single-cell level, the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses. A total of 4,100 spike protein specific memory B cells were single cell sorted and 350 neutralizing antibodies were identified. The third dose increased the antibody neutralization potency and breadth against all SARS-CoV-2 variants of concern as previously observed with hybrid immunity. However, the B cell repertoire that stands behind the response is dramatically different. The increased neutralizing response was largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization such as IGHV3-53;IGHJ6-1 and IGHV3-66;IGHJ4-1. Divergently to hybrid immunity, cross-protection after a third dose was mainly guided by Class 1/2 antibodies encoded by IGHV1-58;IGHJ3-1 and IGHV1-69;IGHJ4-1 germlines. The IGHV2-5;IGHJ3-1 germline, which induced broadly cross-reactive Class 3 antibodies after infection or viral vector vaccination, was not induced by a third mRNA dose. Our data show that while neutralizing breadth and potency can be improved by different immunization regimens, each of them has a unique molecular signature which should be considered while designing novel vaccines and immunization strategies.

Anatomy of Omicron neutralizing antibodies in COVID-19 mRNA vaccinees (preprint)

SARS-CoV-2 vaccines, administered to billions of people worldwide, are mitigating 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 (B.1.1.529), and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine1, were tested for neutralization against the Omicron variant and SARS-CoV-1 virus. Cross-neutralizing antibodies were isolated from 100% of seropositive and 20% of seronegative vaccinees. Only 14.2% and 4.0% of tested antibodies neutralized the Omicron variant and SARS-CoV-1 respectively. These nAbs recognized mainly the SARS-CoV-2 receptor binding domain (RBD) and targeted class 3 and class 4 epitope regions on the SARS-CoV-2 spike protein. Antibodies targeting class 1/2 epitope regions only rarely showed cross-neutralization activity. Cross-protective antibodies derived from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1 and IGHV1-69;IGHV4-1. Only 15.6% and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralized Omicron and SARS-CoV-1 respectively. Our data provide evidence 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 (preprint)

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). To date there have been four major variants (Alpha, Beta, Gamma, Delta) that have tested the efficacy of the vaccines and have led to some breakthrough infections amongst vaccinated populations. Here, we evaluate the potency of a previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryo-EM. We show that mAb J08 is unique because it has low nanomolar affinity against the VoCs, binds high on the receptor binding domain (RBD) ridge and is therefore unaffected by most mutations, and can bind in the RBD-up and -down conformations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy. One sentence summaryMonoclonal antibody J08 can potently neutralize wild-type SARS-CoV-2 and variants of concern by binding to the ridge of the receptor binding domain in up and down conformations and thereby avoid the effects of current escape mutations.

Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern (preprint)

To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naive and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naive people. Of these, 944 from naive and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naive people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naive people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.

A single intramuscular injection of monoclonal antibody MAD0004J08 induces in healthy adults SARS-CoV-2 neutralising antibody titres exceeding those induced by infection and vaccination (preprint)

BackgroundThe emerging threat represented by SARS-CoV-2 variants, demands the development of therapies for better clinical management of COVID-19. MAD0004J08 is an extremely potent Fc-engineered monoclonal antibody (mAb) able to neutralise in vitro all current SARS-CoV-2 variants of concern (VoCs). This ongoing study, evaluates safety, pharmacokinetics and SARS-CoV-2 sera neutralization effect of MAD0004J08 when administered as single dose intramuscularly in healthy adults. MethodWe conducted a dose escalation study with sequential enrolment of three cohorts, each with an increasing dose level of MAD0004J08 (48mg, 100mg and 400mg). Within each cohort, 10 young healthy adults were randomized with 4:1 ratio to a single intramuscular (i.m.) injection of MAD0004J08 or placebo. The primary endpoint is the proportion of subjects with severe and/or serious treatment emergent adverse events (TEAEs) within 7 days post-treatment. Secondary endpoints reported in this paper are the proportion of subjects with solicited TEAEs up 7 days post dosing, MAD0004J08 serum concentrations and neutralising activity versus the original SARS-COV-2 Wuhan virus at different timepoints post-dosing. As post-hoc analyses, we compared the sera neutralising titres of subjects who received MAD0004J08 with those of people that had received the COVID-19 BNT162b2 mRNA vaccine in the previous sixty days (n=10) and COVID-19 convalescent patients (n=20), and assessed serum neutralisation activity against the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) SARS-CoV-2 variants of concern. FindingsA total of 30 subjects, 10 per cohort, were enrolled and randomized. Data up to 30 days were available and analysed in this report. No severe TEAEs were reported in any of the cohorts in the 7 days post-treatment. MAD0004J08 was detected in the sera of treated subjects within few hours post-administration and reached almost maximal levels on day 8. The geometric mean neutralising titres (GMT) assessed against the original Wuhan virus peaked on day 8 and ranged 226 - 905, 905 - 2,560, and 1,280 - 5,120 for cohort 1, 2 and 3 respectively. The sera neutralising GMT in MAD0004J08 treated subjects in all the three cohorts were found to be 1{middle dot}5-54{middle dot}5-fold higher compared to sera from convalescent patients and 1{middle dot}83- 76{middle dot}4-fold higher compared to sera from COVID-19 vaccinees. Finally, GMT in MAD0004J08 treated subjects showed high neutralising titres versus the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) SARS-CoV-2 VoCs. InterpretationA single dose administration of MAD0004J08 via i.m. route is safe and well tolerated and results in a rapid systemic distribution of the MAD0004J08 and sera neutralising titres higher than COVID-19 convalescent and vaccinated subjects. A single dose administration of MAD0004J08 is also sufficient to effectively neutralise major SARS-CoV-2 variants of concern. Based on these results, a Phase 2-3 trial is ongoing to further assess the safety, dosage, and efficacy of MAD0004J08 in asymptomatic or mild-moderate symptomatic COVID-19 patients. FundingEU Malaria Fund, Ministero dello Sviluppo Economico, Ministero della Salute, Regione Toscana, Toscana Life Sciences Sviluppo and European Research Council. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PUBMED, MEDLINE and MedRxiv for clinical trials, meta-analyses and randomized controlled trials evaluating the antibody neutralization titres vs. different SARS-CoV-2 variants of concern obtained from subjects who received monoclonal antibodies for the treatment of COVID-19 using the following search terms: ("COVID-19" OR "SARS-CoV-2") AND ("monoclonal antibody" OR "neutralising antibody") AND ("variants" OR "variants of concern"). No relevant studies were identified. Added value of this studyThis is the first human study assessing safety, PK and neutralising potential of MAD0004J08, a monoclonal antibody against SARS-CoV-2 wild type Wuhan virus and variants of concern, administered intramuscularly at low dosages (48, 100 and 400 mg). MAD0004J08 showed to be safe and well tolerated in the tested dose range. Anti-spike antibodies were detected in the sera of tested SARS-CoV-2 negative healthy adults few hours post-injection. In addition, the sera obtained from MAD0004J08treated subjects, showed to have high neutralisation titres against the Wuhan virus, the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) variants of concern. Implications of all the available evidenceA potent monoclonal antibody such as MAD0004J08, capable of neutralising multiple variants of concern of SARS-CoV-2 rapidly and long lastingly when given as a single intramuscular injection. The antibody, presently tested in a phase 2-3 efficacy trial, can be a major advancement in the prophylaxis and clinical management of COVID-19, because of its broad spectrum, ease of use in non-hospital settings and economic sustainability.

SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma (preprint)

To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, 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 and South Africa 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.

Extremely potent human monoclonal antibodies from convalescent Covid-19 patients (preprint)

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 Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. One Sentence SummaryExtremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.

Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients (preprint)

In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. One Sentence SummaryNeutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.