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.

Human complement Factor H and Properdin act as soluble pattern recognition receptors and differentially modulate SARS-CoV-2 Infection (preprint)

Severe cases of SARS-CoV-2 infection are characterised by an imbalanced immune response, excessive inflammation, and the development of acute respiratory distress syndrome, which can lead to multiorgan failure and death. Several studies have demonstrated dysregulated complement activity as an indicator of immunopathogenesis in the SARS-CoV-2 infection. Notably, the complement alternative pathway has been implicated in driving the excessive inflammation during severe SARS-CoV-2 infection. Reduced levels of factor H (FH), a down-regulator of the alternative pathway, and increased levels of properdin (Factor P/FP), the only known up-regulator of the alternative pathway, have been observed in individuals with severe COVID-19 infection. The present study investigated the complement activation-independent, and a more direct role of FH and FP against SARS-CoV-2 infection. Using direct ELISA, the interactions of FH and FP with the SARS-CoV-2 spike (S) and receptor binding domain (RBD) were assessed. Using S protein expressing lentiviral pseudotypes, the cell binding and luciferase-based virus entry assays were employed to assess the potential modulatory effects of FH, FP, and recombinant thrombospondin repeats 4 and 5 (TSR4+5) on SARS-CoV-2 cell entry. We also evaluated the immunomodulatory functions of FH and FP in the cytokine response triggered by SARS-CoV-2 pseudotypes via RT-qPCR. SARS-CoV-2 S and RBD proteins were found to bind both FH and FP. Treatment of A549 cells expressing human ACE2 and TMPRSS2 with FP or TSR4+5 resulted in increased cell entry and binding of SARS-CoV-2 pseudotypes. In silico studies revealed that FP increases affinity between SARS-CoV-2 and host ACE2. The impact of FP on viral cell entry and binding was reversed by anti-FP antibody treatment in A549-hACE2+TMPRSS2 cells. However, FH treatment reduced the cell entry and binding of SARS-CoV-2 lentiviral pseudotypes. Furthermore, the A549-hACE2+TMPRSS2 cells challenged with SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes pre-treated with FP or TSR4+5, exhibited upregulation of the transcripts of pro-inflammatory cytokines, such as IL-1{beta}, IL-8, IL-6, TNF-, IFN- and RANTES (as well as NF-kB). Conversely, FH pre-treatment downregulated the expression of these pro-inflammatory cytokines. Treatment of A549-hACE2+TMPRSS2 cells with FP increased S protein-mediated NF-kB activation, while FH treatment reduced it. These findings suggest that FH may act as an inhibitor of SARS-CoV-2 cell entry and binding, thereby attenuating the infection-associated inflammatory response in a complement activation-independent manner. FP may contribute to viral cell entry, binding, and exacerbating the immune response. That may result in potentially influencing the severity of the infection.

Efficient Viral Capture and Inactivation from Bioaerosols Using Electrostatic Precipitation. (preprint)

The presence of infectious viral particles in bioaerosols generated during laparoscopic surgery places surgical staff at significant risk of infection and represents a major cause of nosocomial infection. These factors contributed to the postponement and cancellation of countless surgical procedures during the early stages of the ongoing COVID-19 pandemic, causing backlogs, increased waiting times for surgical procedures and excess deaths indirectly related to the pandemic. The development and implementation of devices that effectively inactivate viral particles from bioaerosols would be beneficial in limiting or preventing the spread of infections from such bioaerosols. Here, we sought to evaluate whether electrostatic precipitation (EP) is a viable means to capture and inactivate both non-enveloped (Adenovirus) and enveloped (SARS-CoV-2 Pseudotyped Lentivirus) viral particles present in bioaerosols. We developed a closed-system model to mimic the release of bioaerosols during laparoscopic surgery. Known concentrations of each virus were aerosolised into the model system, exposed to EP using a commercially available system (UltravisionTM, Alesi Surgical Limited, UK) and collected in a BioSampler for analysis. Using qPCR to quantify viral genomes and transduction assays to quantify biological activity, we show that both enveloped and non-enveloped viral particles were efficiently captured and inactivated by EP. Both capture and inactivation could be further enhanced when increasing the voltage to 10kV, or when using two UltravisionTM discharge electrodes together at 8kV. This study highlights EP as an efficient means for capturing and inactivating viral particles present in bioaerosols. The use of EP may limit the spread of diseases, reducing nosocomial infections and potentially enable the continuation of surgical procedures during periods of viral pandemics.

Novel intranasal vaccine targeting SARS-CoV-2 receptor binding domain to mucosal microfold cells and adjuvanted with TLR3 agonist Riboxxim™ elicits strong antibody and T-cell responses in mice (preprint)

SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin (cCPE), which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza.

Human Surfactant Protein D Facilitates SARS-CoV-2 Pseudotype Binding and Entry in DC-SIGN Expressing Cells, and Downregulates Spike protein Induced Inflammation (preprint)

Pattern recognition receptors are crucial for innate anti-viral immunity, including C-type lectin receptors. Two such examples are Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) which are soluble and membrane-bound C-type lectin receptors, respectively. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction as an antigen-presenting cell with naive T cells to mount an anti-viral immune response. Both SP-D and DC-SIGN have been shown to interact with various viruses, including HIV-1, Influenza A virus and SARS-CoV-2. SARS-CoV-2 is an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of -helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP- 1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. The RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-, IFN-, IL-1{beta}, IL- 6, IL-8, and RANTES (as well as NF-{kappa}B) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction of SARS- CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.

Endemic Seasonal Coronavirus Neutralisation and COVID-19 severity (preprint)

The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs) and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2.We use functional, neutralising assays to investigate cross reactive antibodies and their relationship with COVID-19 severity. We analysed neutralisation of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63.We found that although HCoV neutralisation was very common there was little evidence that these antibodies neutralised SARS-CoV-2. Despite no evidence in cross neutralisation, levels of NL63 neutralisating antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients (preprint)

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

Standardised, quantitative neutralisation responses to SARS-CoV-2 Variants of Concern by convalescent anti-sera from first wave infections of UK Health Care Workers and Patients (preprint)

One of the defining criteria of Variants of Concern (VOC) is their ability to evade pre-existing immunity, increased transmissibility, morbidity and/or mortality. Here we examine the capacity of convalescent plasma, from a well defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK, to neutralise B.1.1.298 variant and three VOCs; B.1.1.7, B.1.351 and P.1. Furthermore, to enable lab to lab, country to country comparisons we utilised the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation findings in International Units. These findings demonstrate a significant reduction in the ability of first wave convalescent plasma to neutralise the VOCs. In addition, Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Widespread use of the WHO International Standard by laboratories in different countries will allow for cross-laboratory comparisons, to benchmark and to establish thresholds of protection against SARS-CoV-2 and levels of immunity in different settings and countries.

Towards Internationally standardised humoral Immune Correlates of Protection from SARS CoV 2 infection and COVID-19 disease (preprint)

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.