A cell-based, spike protein binding assay highlights differences in antibody neutralising capacity for SARS-CoV-2 variants (preprint)

The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original "Wuhan" SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.

Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern (preprint)

CD4 + T-cells are essential for protection against viruses including SARS-CoV-2. Mutations in SARS-CoV-2 variants of concern (VOC) can enhance infectivity and reduce antibody recognition. CD4 + T-cell sensitivity to mutations is less well understood because few epitopes have been mapped. Characterising > 100 SARS-CoV-2-specific CD4 + T-cell clones from convalescent healthcare workers, we mapped and HLAII restricted 21 epitopes across three viral proteins. Responses to the same spike epitopes were also present after vaccination of uninfected individuals. Lack of CD4 + T-cell cross-reactivity with endemic beta-coronaviruses suggests these responses arose from naïve T-cells rather than pre-existing cross-reactive coronavirus-specific T-cell responses. 10/17 spike epitopes were mutated in VOCs and CD4 + T-cell recognition of 7 was impaired, including 3 of 4 epitopes mutated in Omicron. Broad CD4 + T-cell targeting of epitopes likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new emerging mutations able to evade CD4 + T-cell immunity.

mRNA COVID-19 vaccines induce enhanced antibody and cellular responses compared to ChAdOx1 or natural infection in children (preprint)

We present a comprehensive analysis of antibody and cellular responses in children aged 12-16 years who received COVID-19 vaccination with ChAdOx1 (n=6) or mRNA vaccine (mRNA-1273 or BNT162b2, n=9) using a 12-week extended-interval schedule. mRNA vaccination of seropositive children induces high antibody levels, with one dose, but a second dose is required in infection-naïve children. Following a second ChAdOx1 dose, antibody titres were higher than natural infection, but lower than mRNA vaccination. Vaccination induced live virus neutralising antibodies against Alpha, Beta and Delta variants, however, a second dose is required in infection-naïve children. We found higher T-cell responses following mRNA vaccination than ChAdOx1. Phenotyping of responses showed predominantly early effector-memory CD4 T cell populations, with a type-1 cytotoxic cytokine signature, with IL-10. These data demonstrate mRNA vaccination induces a co-ordinated superior antibody and robust cellular responses in children. Seronegative children require a prime-boost regime for optimal protection.

Robust SARS-CoV-2-specific and heterologous immune responses after natural infection in elderly residents of Long-Term Care Facilities (preprint)

Long term care facilities (LTCF) provide residential and/or nursing care support for frail and elderly people and many have suffered from a high prevalence of SARS-CoV-2 infection. Although mortality rates have been high in LTCF residents there is little information regarding the features of SARS-CoV-2-specific immunity after infection in this setting or how this may influence immunity to other infections. We studied humoral and cellular immunity against SARS-CoV-2 in 152 LTCF staff and 124 residents over a prospective 4-month period shortly after the first wave of infection and related viral serostatus to heterologous immunity to other respiratory viruses and systemic inflammatory markers. LTCF residents developed high levels of antibodies against spike protein and RBD domain which were stable over 4 months of follow up. Nucleocapsid-specific responses were also elevated in elderly donors but showed waning across all populations. Antibodies showed stable and equivalent levels of functional inhibition against spike-ACE2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or RSV. SARS-CoV-2-specific cellular responses were equivalent across the life course but virus-specific populations showed elevated levels of activation in older donors. LTCF residents who are survivors of SARS-CoV-2 infection thus show robust and stable immunity which does not impact responses to other seasonal viruses. These findings augur well for relative protection of LTCF residents to re-infection. Furthermore, they underlie the potent influence of previous infection on the immune response to Covid-19 vaccine which may prove to be an important determinant of future vaccine strategy. One sentence summeryCare home residents show waning of nucleocapsid specific antibodies and enhanced expression of activation markers on SARS-CoV-2 specific cells

Children develop strong and sustained cross-reactive immune responses against spike protein following SARS-CoV-2 infection (preprint)

SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis for this is unclear. We studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults. Antibody profiles in children were strong with high titres against spike protein and receptor binding domain (RBD). SARS-CoV-2 seroconversion in children strongly boosted antibody responses against seasonal beta-coronaviruses, partly through cross-recognition of the S2 domain, indicating a broad humoral response that was not seen in adults. T cell responses against spike were also >2-fold higher in children compared to adults and displayed a strong Th1 cytokine profile. SARS-CoV-2 spike-reactive cellular responses were present in more than half the seronegative children, indicating pre-existing cross-reactive responses or sensitization against SARS-CoV-2. Importantly, all children retained high antibody titres and cellular responses for more than 6 months after infection whilst relative antibody waning was seen in adults. Children thus distinctly generate robust, cross-reactive and sustained immune responses after SARS-CoV-2 infection with focussed specificity against spike protein. These observations demonstrate several novel features of SARS-CoV-2-specific immune responses in children and may provide insights into relative clinical protection in this group. Such information on the profile of natural infection will help to guide the introduction of vaccination regimens into the paediatric population.