High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells.

Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)- specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, rather than the effector T- bet+, cytotoxic granzymes+ and perforin+ cells seen in high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, that were absent in individuals with low antibody levels. However, vaccination in low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD- region should be prioritized in booster vaccines. One Sentence SummaryIndividuals with low neutralising antibody titres may be at risk of SARS-CoV-2 re-infection due to a failure to generate a high quality CD4 T cell response specific for receptor binding domain (RBD), including memory CD4 T cells that proliferate in vitro in response to RBD, and which are also therefore an important target for vaccine design.

SARS-CoV-2 Omicron: reduction of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in the first world. In late 2021, the Omicron (B.1.1.529) virus variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant demonstrated higher numbers of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. We assessed the impact of Omicron infection on the ability of: serum from vaccinated and / or previously infected individuals; concentrated human IgG from plasma donors, and licensed monoclonal antibody therapies to neutralise virus in vitro. There was a 17 to 22-fold reduction in neutralisation titres across all donors who had a detectable neutralising antibody titre to the Omicron variant. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 16-fold. Of all therapeutic antibodies tested, significant neutralisation of the Omicron variant was only observed for Sotrovimab, with other monoclonal antibodies unable to neutralise Omicron in vitro. These results have implications for ongoing therapy of individuals infected with the Omicron variant.

COVID-19 convalescents exhibit deficient humoral and T cell responses to variant of concern Spike antigens at 12 month post-infection

BackgroundThe duration and magnitude of SARS-CoV-2 immunity after infection, especially with regard to the emergence of new variants of concern (VoC), remains unclear. Here, immune memory to primary infection and immunity to VoC was assessed in mild-COVID-19 convalescents one year after infection and in the absence of viral re-exposure or COVID-19 vaccination. MethodsSerum and PBMC were collected from mild-COVID-19 convalescents at [~]6 and 12 months after a COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15-40). Serum titers of RBD and Spike-specific Ig were quantified by ELISA. Virus neutralisation was assessed against homologous, pseudotyped virus and homologous and VoC live viruses. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped by activation-induced marker assay, while T cell functionality was assessed by intracellular cytokine staining using peptides specific to homologous Spike virus antigen and four VoC Spike antigens. FindingsAt 12 months after mild-COVID-19, >90% of convalescents remained seropositive for RBD-IgG and 88.9% had circulating RBD-specific memory B cells. Despite this, only 51.2% convalescents had serum neutralising activity against homologous live-SARS-CoV-2 virus, which decreased to 44.2% when tested against live B.1.1.7, 4.6% against B.1.351, 11.6% against P.1 and 16.2%, against B.1.617.2 VoC. Spike and non-Spike-specific T cells were detected in >50% of convalescents with frequency values higher for Spike antigen (95% CI, 0.29-0.68% in CD4+ and 0.11-0.35% in CD8+ T cells), compared to non-Spike antigens. Despite the high prevalence and maintenance of Spike-specific T cells in Spike high-responder convalescents at 12 months, T cell functionality, measured by cytokine expression after stimulation with Spike epitopes corresponding to VoC was severely affected. InterpretationsSARS-CoV-2 immunity is retained in a significant proportion of mild COVID-19 convalescents 12 months post-infection in the absence of re-exposure to the virus. Despite this, changes in the amino acid sequence of the Spike antigen that are present in current VoC result in virus evasion of neutralising antibodies, as well as evasion of functional T cell responses. FundingThis work was funded by project grants from The Hospital Research Foundation and Womens and Childrens Hospital Foundation, Adelaide, Australia. MGM is THRF Early Career Fellow. BGB is THRF Mid-Career Fellow. This project has been supported partly with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016 to A.S. and Contract No. 75N9301900065 to A.S, D.W. Evidence before this studyWe regularly searched on PubMed and Google Scholar in June-October 2021 using individual or combinations of the terms "long-term immunity", "SARS-CoV-2", "antigenic breadth", "variant of concern" and "COVID-19". We found studies that had assessed immune correlates at multipe time points after COVID-19 disease onset in convalescents, but not the antigenic breadth of T cells and antibodies and not in relation to VoC. Other immune studies in virus naive vaccinees, or vaccinated convalescents evaluated VoC-specific immunity, but not in convalescents that have not been vaccinated. In summary, we could not find long-term studies providing and in-depth evaluation of functionality of humoral and cell-mediated immunity, combined with addressing the adaptability of these immune players to VoC. Added value of this studyThe window of opportunity to conduct studies in COVID-19 convalescents (i.e. natural immunity to SARS-CoV-2) is closing due to mass vaccination programs. Here, in a cohort of unvaccinated mild-COVID-19 convalescents, we conducted a comprehensive, longitudinal, long-term immune study, which included functional assays to assess immune fitness against antigenically different VoC. Importantly, the cohort resided in a SARS-CoV-2-free community for the duration of the study with no subsequent re-exposure or infection. Our findings reveal a deeply weakened humoral response and functional vulnerability of T cell responses to VoC Spike antigens. Implications of all the available evidenceThis study provides a valuable snapshot of the quality of SARS-CoV-2 natural immunity and its durability in the context of a pandemic in which new variants continuously emerge and challenge pre-existing immune responses in convalescents and vacinees. Our results serve as a warning that delays in vaccination programs could lead to an increase in re-infection rates of COVID-19 convalescents, caused by virus variants that escape humoral and cell-mediated immune responses. Furthermore, they reinforce the potential benefit of booster vaccination that is tuned to the active variants.

SARS-CoV-2 neutralizing antibodies; longevity, breadth, and evasion by emerging viral variants

The SARS-CoV-2 antibody neutralization response and its evasion by emerging viral variants are unknown. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 RT-PCR-confirmed COVID-19 individuals with detailed demographics and followed up to seven months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization were associated with COVID-19 severity. A subgroup of high responders maintained high neutralizing responses over time, representing ideal convalescent plasma therapy donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal plasma donors and vaccine monitoring and design. One Sentence SummaryNeutralizing antibody responses to SARS-CoV-2 are sustained, associated with COVID19 severity, and evaded by emerging viral variants

SARS Coronavirus-2 microneutralisation and commercial serological assays correlated closely for some but not all enzyme immunoassays

BackgroundSerological testing for SARS-CoV-2 specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence, and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. MethodsSera from individuals recovered from patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n=200), and negative control sera collected prior to the COVID-19 pandemic (n=100) were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. ResultsNeutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. ConclusionsThese results suggest the marker used (total Ab vs IgG vs IgA), and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrate their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

SARS-CoV-2 in human milk is inactivated by Holder pasteurization but not cold storage

As the COVID-19 pandemic evolves, human milk banks worldwide continue to provide donor human milk to vulnerable infants who lack access to mothers own milk. Under these circumstances, ensuring the safety of donor human milk is paramount, as the risk of vertical transmission of SARS-CoV-2 is not well understood. Here, we investigate the inactivation of SARS-CoV-2 in human milk by pasteurisation, and the stability of SARS-CoV-2 in human milk under cold storage (freezing or refrigeration). Following heating to 63{degrees}C or 56{degrees}C for 30 minutes, SARS-CoV-2 replication competent (i.e. live) virus was undetected in both human milk and the control medium. Cold storage of SARS-CoV-2 in human milk (either at 4{degrees}C or - 30{degrees}C) did not significantly impact infectious viral load over a 48 hour period. Our findings demonstrate that SARS-CoV-2 can be effectively inactivated by Holder pasteurisation, and confirm that existing milk bank processes will effectively mitigate the risk of transmission of SARS-COV-2 to vulnerable infants through pasteurised donor human milk.