Response of lymphopenic apheresis platelet donors to COVID-19 vaccination

Background: Recent studies have shown that several frequent apheresis platelet donors (more than 20 donations/year for several years) have CD4+ T cell counts below the normal limit (<400 cells/μl) while the levels of other blood cell types (including B cells) remain normal. This lymphopenia does not appear to be associated with an increased susceptibility to infections and cancers, suggesting that it does not affect immune function of the platelet donors with low CD4+ T cell counts. Aims: To assess the immune capacity of apheresis platelet donors with lymphopenia by taking advantage of the recent SARS-CoV-2 vaccination campaign. Methods: Forty-three apheresis platelet donors who donate at least five times per year were recruited. At least half of these participants were frequent donors and were thus more likely to have low CD4+ T cell counts. Their baseline CD4+ T cell count was determined by flow cytometry. The levels of antibodies targeting the SARS-CoV-2 Spike receptor binding domain (RBD) were measured by ELISA before and after vaccination. The level of antibodies against native full-length Spike was determined by flow cytometry using transfected cells expressing different Spike variants of concern (D614G, Delta, Omicron). The avidity of RBD antibodies produced after the first and second dose of vaccine was determined using a modified ELISA in which a chaotropic agent (urea 8 M) was added. The functional properties of vaccine-elicited SARSCoV- 2 antibodies were measured using ADCC and neutralization assays. Results: Of the 43 participants, 27 had pre-vaccination CD4+ T cell counts below 400 cells/μl (low CD4 group) and 16 participants had CD4+ T cell counts in the normal range (400-1600 CD4/μl;normal CD4 group). The levels of RBD-binding antibody did not significantly differ between the low and normal CD4 groups for all three isotypes (IgG, IgA and IgM) after the first and second doses of vaccine. As expected given the maturation of immune response, the avidity of RBD-binding antibodies present in the plasma collected after the second dose of vaccine was significantly higher than that measured in plasma recovered after the first dose. However, the increase was of similar magnitude in both CD4 groups. Recognition of the three Spike variants, as measured by flow cytometry, did not differ significantly between the low and normal CD4 groups. Finally, the antibody(Fc)- mediated effector function (measured by ADCC using cells expressing the wild type Spike) and neutralization capacity of the three variants of concern were comparable for the two groups, after receiving two vaccine doses. Summary/Conclusions: Our data show that low CD4+ T cell counts in apheresis platelet donors do not impair their response to antigenic challenge such as COVID-19 vaccination. This finding is consistent with the previously lack of increased susceptibility of platelet donors with lymphopenia to infections and cancers. Work remains to be done to understand the physiological mechanism behind the low number of CD4+ T cells in the peripheral blood of several plateletpheresis donors.

A LONG-INTERVAL VACCINE REGIMEN LEADS to STRONG HUMORAL RESPONSES AGAINST SARS-CoV-2

Background: While the standard regimen of the BNT162b2 mRNA vaccine includes two doses administered three weeks apart, some public health authorities decided to space them in a context of vaccine scarcity. This decision raised concerns about vaccine efficacy, notably against the many circulating variants. In this study, we analyzed the longitudinal humoral responses from before the first dose to 4 months after the second dose in a cohort of SARS-CoV-2 naïve and previously infected (PI) individuals, with an interval of sixteen weeks between the two doses. We compared these responses to those elicited in individuals receiving the three weeks dose interval. Methods: We measured the level of antibodies recognizing SARS-CoV-2 Spike or its receptor-binding domain, and the capacity of these antibodies to neutralize several variants of concern (VOCs) and other human coronaviruses. We also measured B cell responses and Fc-mediated effector functions (ADCC) elicited by vaccination. Results: We observed that in PI individuals, the first dose led to strong humoral responses that could not be significantly improved further upon administration of a second dose. In the naïve individual's group, the first dose induced weak neutralizing activity but strong Fc-mediated functions and the administration of the second dose 16 weeks after led to a significant increase of humoral responses, achieving similar levels to those measured in PI individuals. In both groups, we observed that plasmas were able to recognize and neutralize the Spike of different VOCs but also SARS-CoV-1. Conclusion: Our results show that individuals that received the extended BNT162b2 vaccine interval developed strong humoral responses. For the naïve donors, these responses were superior to those elicited by the three-week dose interval and comparable to the PI responses after one or two doses.

SARS-CoV-2 SPIKE EXPRESSION at the SURFACE of INFECTED HUMAN AIRWAY EPITHELIAL CELLS

Background: Emerging evidence points out to potential benefits from Fc-mediated effector functions in SARS-CoV-2 infection. Some Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) require recognition of the antigen at the surface of infected cells. Methods: To evaluate the expression levels of SARS-CoV-2 Spike at the surface of infected airway epithelial cells, we developed an intracellular staining against SARS-CoV-2 nucleocapsid (N). This assay allows the distinction between infected versus uninfected cells. Human primary airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. Infected cells were identified with an anti-N antibody and cell surface expression of Spike measured with the conformational-independent anti-S2 CV3-25 antibody. Results: We found robust SARS-CoV-2 Spike expression at the cell surface of pAECs. Infected cells were readily recognized with plasma from convalescent and vaccinated individuals. Importantly, recognition of SARS-CoV-2 infected cells strongly correlated with Fc-mediated effector functions measured in a cohort of vaccinated naïve and previously-infected individuals. Conclusion: Altogether, our findings further support the importance of measuring Fc-mediated effector function in infection and vaccination settings for SARS-CoV-2.

ANTIGENICITY of the MU (B.1.621) and A.2.5 SARS-CoV-2 SPIKES

Background: The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Recently identified Mu (B.1.621) and A.2.5 variants carry some mutations shared by other variants of concerns (VOCs). For example, N501Y and E484K mutations in the receptor-binding domain (RBD) domain detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is now present in A.2.5. Here, we evaluated the capacity of Mu and A.2.5 Spikes to interact with angiotensin-converting enzyme 2 (ACE2) and performed binding and neutralization assays with plasma from vaccinated individuals. In addition, to better understand their antigenic properties, we compared both Mu and A.2.5 with Alpha, Beta, Gamma and Delta VOCs Spikes. Methods: Cells expressing the different Spikes were interrogated for their capacity to interact with the ACE2 receptor using a recombinant ACE2-Fc recombinant protein. We also evaluated their recognition by plasma from BNT162b2 vaccinated individuals. Biolayer interferometry (BLI) was used to measure the binding kinetics of selected RBD mutants to soluble ACE2 (sACE2). Finally, we evaluated the susceptibility of pseudoviral particles bearing the different Spikes to neutralization by plasma from vaccinated individuals. Results: All SARS-CoV-2 S-glycoprotein variants were recognized less efficiently by plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals compared to D614G Spike with the exception of B.1.1.7 S-glycoprotein. Enhanced ACE2 interaction by the Spikes tested was associated with a decrease in the off-rate of the ACE2-RBD interaction. Pseudoviral particles bearing the Spike of Mu variant were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta and Delta Spikes. Conclusion: Plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals efficiently recognized all the Spikes tested. The decreased neutralization susceptibility of pseudoviral particles expressing the Mu Spike was similar to Beta and Delta, thus underscoring the importance of functionally tracking emerging variants. In summary, our results highlight the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization from each emerging variant.

Multicenter Evaluation of the Clinical Performance and the Neutralizing Antibody Activity Prediction Properties of 10 High-Throughput Serological Assays Used in Clinical Laboratories.

As the coronavirus disease 2019 (COVID-19) pandemic second wave is emerging, it is of the upmost importance to screen the population immunity in order to keep track of infected individuals. Consequently, immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and positive predictive values are needed to obtain an accurate epidemiological picture. As more data accumulate about the immune responses and the kinetics of neutralizing-antibody (nAb) production in SARS-CoV-2-infected individuals, new applications are forecast for serological assays such as nAb activity prediction in convalescent-phase plasma from recovered patients. This multicenter study, involving six hospital centers, determined the baseline clinical performances, reproducibility, and nAb level correlations of 10 commercially available immunoassays. In addition, three lateral-flow chromatography assays were evaluated, as these devices can be used in logistically challenged areas. All assays were evaluated using the same patient panels in duplicate, thus enabling accurate comparison of the tests. Seven immunoassays examined in this study were shown to have excellent specificity (98 to 100%) and good to excellent positive predictive values (82 to 100%) when used in a low (5%)-seroprevalence setting. We observed sensitivities as low as 74% and as high as 95% at ≥15 days after symptom onset. The determination of optimized cutoff values through receiver operating characteristic (ROC) curve analyses had a significant impact on the diagnostic resolution of several enzyme immunoassays by increasing the sensitivity significantly without a large trade-off in specificity. We found that spike-based immunoassays seem to be better correlates of nAb activity. Finally, the results reported here will add to the general knowledge of the interlaboratory reproducibility of clinical performance parameters of immunoassays and provide new evidence about nAb activity prediction.