B cell response six months after SARS-CoV-2 mRNA vaccination in people living with HIV under antiretroviral therapy

BackgroundSARS-CoV-2 mRNA vaccines have demonstrated high immunogenicity in healthy subjects and preliminary results for people living with HIV (PLWHIV) are promising too. We have previously reported the persistence of spike-specific circulating IgG and memory B cells in healthy adults up to six months after mRNA SARS-CoV-2 vaccination. Unfortunately, limited longitudinal data are available for PLWHIV and no evidence of persistent spike-specific B cells have been reported yet. MethodsWe investigated the humoral response and the persistence of spike-specific memory B cells up to six months after vaccination with two doses of mRNA vaccines in 84 PLWHIV under ART and compared them to healthy controls (HCs). Humoral response was analyzed with enzyme-linked immunosorbent assay and with an angiotensin-converting enzyme 2 (ACE2) and receptor binding domain (RBD) inhibition assay. PBMCs were analyzed with a cytofluorimetric approach for B cell phenotyping. FindingsSpike-specific IgG peaked 1 month after second dose and persisted up to six months after vaccination with no significant differences compared to HCs. The stratification of patients according to CD4+ T cell count showed a significantly lower IgG response in case of CD4<350/{micro}l, remarking the relevance of immune reconstitution. The ability of IgG of blocking the binding between ACE2 and RBD was detected in 58{middle dot}4% of PLWHIV, compared to 86{middle dot}2% in HCs. The amount of circulating spike-specific memory B cells detected in PLWHIV six months after vaccination was not significantly different from HCs, while there was prevalence of antigen-specific double negative (IgD-/CD27-) cells, compared to controls. InterpretationIn conclusion, the majority of PLWHIV developed spike-specific humoral and B cell responses that persist for at least six months after SARS-CoV-2 mRNA vaccination. However, hints of HIV-dependent immune impairment were revealed by altered spike-specific B cell phenotypes and by reduced spike-specific humoral response in patients with low CD4+ T cell count (<350/{micro}l).

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

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 immunity1,2. 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 doses3. 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 immunity3. 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.

The slower antibody response in myelofibrosis patients after two doses of mRNA SARS-CoV-2 vaccine calls for a third dose

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile categories with higher risk of mortality after COVID-19 disease compared to healthy people, including patients with myelofibrosis (MF). Available data on the vaccine immune response developed by MF patients, and the impact of the treatment with the inhibitor of JAK-STAT signaling ruxolitimib, are still fragmented to support an informed decision for a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second vaccine dose, but the response has a slower kinetic compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccination. A reduced ACE2/RBD inhibition binding activity of spike-specific antibodies was also observed, especially in ruxolitimib treated patients. Our results contribute to answer the open question on the induction of the antibody responses in MF patients following vaccination with COVID-19 mRNA vaccines, showing a slow kinetic that support the need for a third dose of SARS-CoV-2 mRNA vaccines.

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

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.

Evidence of SARS-Cov-2-specific memory B cells six months after vaccination with BNT162b2 mRNA vaccine

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cells generation and long-term persistence. Here, we investigated Spike-specific memory B cells and humoral responses in 145 subjects, up to six months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibody titers peaked 7 days after the second dose and significant titers and neutralizing activity were still observed after six months, despite a progressive decline over time. Concomitant to antibody reduction, Spike-specific memory B cells, mostly IgG class-switched, increased in blood of vaccinees and persisted six months after vaccination. Following in vitro restimulation, circulating memory B cells reactivated and produced Spike-specific antibodies. A high frequency of Spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at six months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with Spike-specific memory B cells that still persist six months after vaccination, playing a crucial role for rapid response to SARS-CoV-2 virus encounter. One Sentence SummarymRNA BNT162b2 vaccine elicits persistent spike-specific memory B cells crucial for rapid response to SARS-CoV-2 virus encounter

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired autophagy and reduced TNF production was demonstrated in HEK293 cells transfected with TLR3-L412F plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (P=0.038). An increased frequency of autoimmune disorders as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.

Extremely potent human monoclonal antibodies from convalescent Covid-19 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 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.

Clinical and molecular characterization of COVID-19 hospitalized patients

Clinical and molecular characterization by Whole Exome Sequencing (WES) is reported in 35 COVID-19 patients attending the University Hospital in Siena, Italy, from April 7 to May 7, 2020. Eighty percent of patients required respiratory assistance, half of them being on mechanical ventilation. Fiftyone percent had hepatic involvement and hyposmia was ascertained in 3 patients. Searching for common genes by collapsing methods against 150 WES of controls of the Italian population failed to give straightforward statistically significant results with the exception of two genes. This result is not unexpected since we are facing the most challenging common disorder triggered by environmental factors with a strong underlying heritability (50%). The lesson learned from Autism-Spectrum-Disorders prompted us to re-analyse the cohort treating each patient as an independent case, following a Mendelian-like model. We identified for each patient an average of 2.5 pathogenic mutations involved in virus infection susceptibility and pinpointing to one or more rare disorder(s). To our knowledge, this is the first report on WES and COVID-19. Our results suggest a combined model for COVID-19 susceptibility with a number of common susceptibility genes which represent the favorite background in which additional host private mutations may determine disease progression.

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

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.