Immune imprinting revealed by SARS-CoV-2 Omicron infection prior to vaccination (preprint)

Immune imprinting or original antigenic sin (OAS) originally referred to a phenomenon of suboptimal immune response to a repeat exposure to a virus that was antigenically distinct from the original virus infection. OAS has been implicated in higher mortality in young people during the 2009-10 H1N1 pandemic where the elderly (H1N1 exposure in childhood) appeared relatively well protected compared to younger individuals whose first influenza infection was not H1N1. Immune imprinting is part of a rapid recall system and is highly effective against a slowly evolving virus (drifting) but not antigenically shifting viruses such as influenza and SARS CoV-2. As predicted by OAS, suboptimal neutralization responses to the highly divergent SARS-COV-2 lineage Omicron have been observed in animal models and individuals previously vaccinated with primary course of ancestral (Wu-1) vaccine. Due to the rapid scale up of vaccine before emergence of the antigenically distinct Omicron variant, it is unknown whether immunological imprinting for occurs in the context of SARS-COV-2 infection itself. We longitudinally assessed humoral responses to primary two dose Ad26.COV2.S Wu-hu-1 based vaccination in a Nigerian population following the global emergence of Omicron. At study entry in Jan 2023, we found 93% and 58% of pre-vaccination participants previously exposed to ancestral Wu-1 and Omicron virus respectively by anti-N IgG and anti-receptor binding domain (RBD) IgG Wu-1 and Omicron -specific antibodies. In participants with no evidence of prior exposure to Omicron, neutralisation against Wu-1 was significantly higher than Omicron variants as expected. However, serum neutralisation titres in participants who were anti-RBD Omicron IgG positive were paradoxically 2-fold lower for Omicron BA.1 as compared to Wu-1. This is clear evidence for imprinted immunity from the ancestral pre-omicron lineage viruses, and remarkably these old responses to Wu-1 were able to dominate over more recent, likely multiple, Omicron lineage infections. Furthermore, in these participants with prior exposure to Omicron and evidence of imprinting, we observed that further Omicron infection and Wu-1 based vaccine was associated with boosting of responses across variants with equalisation of neutralisation titres for Wu-1 and Omicron variants. However, omicron responses did not surpass ancestral responses, suggesting persistence of imprinting and only partial mitigation. Although neutralization responses at high titres were observed post dose 1 vaccination against ancestral and Omicron variants BA.1, BA.2, BA.4 in nearly all participants, neutralisation against the highly immune evasive XBB recombinant variant remained substantially lower, with a second vaccine dose providing very modest boosting. These data highlight immune imprinting against SARS-CoV-2 prior to vaccination and its persistence thereafter. In present day unvaccinated populations where serum neutralisation responses to pre-Omicron variants dominate, use of an omicron variant based vaccine should be used in preference to Wu-1 based vaccine to override imprinting and provide broader protection for vulnerable populations such as the elderly or those with compromised immunity.

Defining neutralization and allostery by antibodies against COVID-19 variants (preprint)

The changing landscape of mutations in the SARS-CoV-2 Spike protein is linked to the emergence of variants, immune-escape and reduced efficacy of the existing repertoire of anti-viral antibodies. A major factor that contributes to the functional activity of the neutralizing antibodies are the intrinsic quaternary changes that occur as a result of antibody-Spike trimer interactions. In this study, we reveal the conformational dynamics and allosteric perturbations linked to binding of human monoclonal antibodies and the viral Spike protein. We identify epitope hotspots of known and novel antibodies, and associated changes in Spike dynamics that define weak, moderate and strong neutralizing antibodies. We show the impact of mutations in Wuhan, Delta, and Omicron variants of concern (VoCs) and differences observed in the antibody-induced conformational changes and illustrate how these render certain antibodies ineffective. Our comparative analyses of the antibody-footprints on Spike variants reveal how antibodies with similar binding affinities may induce destabilizing and stabilizing allosteric effects. These differences have important implications for neutralization efficacy and for developing new antibodies targeting emerging variants. Our results provide mechanistic insights into the functional modes and synergistic behavior of human antibodies against COVID-19, and provide a rationale to design effective antiviral strategies.

SARS-CoV-2 Antibody Responses to AZD1222 Vaccination in West Africa (preprint)

There is a paucity of real-world data on vaccine elicited neutralising antibody responses for AZD1222, in African populations following vaccination scale up. Here, we first measured baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using both flow cytometric based analysis of binding antibodies coupled with pseudotyped virus neutralisation assays in two study cohorts from West Africa: Nigerian healthcare workers; (n = 140) and a Ghanaian general community cohort (n = 527). We found that 44% and 28% of pre-vaccination participants showed IgG anti-N positivity, increasing to 59% and 42% respectively with anti-receptor binding dominan (RBD) IgG specific antibodies. The increased prevalence of prior exposure using anti RBD antibodies was corroborated by Pseudotyped virus (PV) neutralizing antibody assays, indicating that overall, 50% of prior infections were missed by N antibody testing. PV titres (serum dilution required to inhibit 50% of infection, ID50) against wild type following 2-dose vaccination regimen were [145 (4.5) to 2579 (4.2) vs 57 (3.0) to 1049 (6.7)] (GMT ± s.d), delta [75 (3.0) to 549 (3.7) vs 37 (2.4) to 453 (7.4)] (GMT ± s.d) and omicron variants [37 (2.4) to 453 (7.4) vs 29 (1.8) to 95 (5.3)] (GMT ± s.d) in the Nigerian (1 month) vs Ghanaian participants (2 months) post vaccination (total n = 94). Previous IgG anti-N was associated with significantly higher neutralizing antibody levels with an observed 3.5-fold [1423 (3.9) vs 4674 (3.4)] (GMT ± s.d) and 2.7-fold [779 (7.1) vs 2128 (4.8) (GMT ± s.d) difference between N positive and negative participants in the Nigerian and Ghanaian cohorts respectively. We also observed serological evidence from N, S and RBD antibodies of breakthrough infection in 8/49 (16%) of Nigerian vaccinees over only 2 months, with neutralisation profiles suggesting delta variant infection consistent with the sampling period when this variant was known to dominate. Importantly, neutralising antibodies waned at 3 months after second dose vs 1 month post second-dose 1695 (4.3)] vs 2579 (4.2) in the Nigerian population who were N negative throughout. IgG anti-N was also observed to wane below cut-off in a total 19/94 (20%) of subjects highlighting the need for a combination of additional markers to characterise previous infection. We conclude that AZD1222 is immunogenic in two independent real world West African cohorts with high background seroprevalence and incidence of breakthrough infection in 2021. Waning titres at 6 months post second dose indicates the need for booster dosing after AZD1222 in the African setting despite hybrid immunity from previous infection.

Transmission of B.1.617.2 Delta Variant Between Vaccinated Healthcare Workers (preprint)

Breakthrough infections with SARS-CoV-2 Delta variant have been reported in vaccine recipients and in individuals infected with previous variants. Studies of viral spread within hospital settings have highlighted the potential for transmission between patients and health care workers, and highlighted the benefits of high-grade respiratory protection for health care workers. However the extent to which vaccination is preventative of viral spread in health care settings is less well studies. We here analyse data from health care workers in two hospitals in India, constructing probable transmission networks from epidemiological and virus genome sequence data using a suite of computational approaches. A maximum likelihood reconstruction of transmission involving known cases of infection suggests a high probability that doubly vaccinated health care workers transmitted SARS-CoV-2 between each other, and highlights potential cases of virus transmission between individuals who had received two doses of vaccine. Our findings support the need for ongoing infection control measures even in highly vaccinated populations.

SARS-CoV-2 Omicron spike mediated immune escape and tropism shift (preprint)

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

Role of Microalgae in Global CO2 Sequestration: Physiological Mechanism, Recent Development, Challenges, and Future Prospective

The rising concentration of global atmospheric carbon dioxide (CO2) has severely affected our planet’s homeostasis. Efforts are being made worldwide to curb carbon dioxide emissions, but there is still no strategy or technology available to date that is widely accepted. Two basic strategies are employed for reducing CO2 emissions, viz. (i) a decrease in fossil fuel use, and increased use of renewable energy sources;and (ii) carbon sequestration by various biological, chemical, or physical methods. This review has explored microalgae’s role in carbon sequestration, the physiological apparatus, with special emphasis on the carbon concentration mechanism (CCM). A CCM is a specialized mechanism of microalgae. In this process, a sub-cellular organelle known as pyrenoid, containing a high concentration of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), helps in the fixation of CO2. One type of carbon concentration mechanism in Chlamydomonas reinhardtii and the association of pyrenoid tubules with thylakoids membrane is represented through a typical graphical model. Various environmental factors influencing carbon sequestration in microalgae and associated techno-economic challenges are analyzed critically.

The impact of hypoxia on B cells in COVID-19 (preprint)

Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This brought to mind the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. We demonstrated the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes we also observed in B cell VHL-deficient mice. This was corroborated by hypoxia-related transcriptional changes in COVID-19 patients, and by similar B cell abnormalities in mice kept in hypoxic conditions, including reduced marginal zone and germinal center B cells. Thus hypoxia might contribute to B cell pathology in COVID-19, and in other hypoxic states. Through this mechanism it may impact on COVID-19 outcome, and be remediable through early oxygen therapy.

Age-related heterogeneity in immune responses to SARS-CoV-2 following BNT162b2 vaccination (preprint)

Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks and other countries are likely to follow suit given the demand for mRNA vaccines and ongoing uncontrolled transmission. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Median age was 81 years amongst 101 participants after the first dose of the BNT162b2 vaccine. Geometric mean neutralisation titres in participants over 80 years old after the first dose were lower than in younger individuals [83.4 (95% CI 52.0-133.7) vs 46.6 (95% CI 33.5-64.8) p 0.01]. A lower proportion of participants 80 years and older achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (21% vs 51%, p 0.003). Binding IgG responses correlated with neutralisation. Sera from participants in both age groups showed significantly lower neutralisation potency against B.1.1.7 Spike pseudotyped viruses as compared to wild type. The adjusted ORs for inadequate neutralisation in the 80 years and above age group were 3.7 (95% CI 1.2-11.2) and 4.4 (95% CI 1.5-12.6) against wild type and B.1.1.7 pseudotyped viruses. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T- cell IFN𝛾 and IL-2 responses were impaired in the older age group after 1 dose and although IFN𝛾 increased between vaccine doses, IL-2 responses did not significantly increase. There was a significantly higher risk of suboptimal neutralising antibody and T cell response following first dose vaccination with BNT162b2 in half of participants above the age of 80, persisting up to 12 weeks. These high risk populations warrant specific measures in order to mitigate against vaccine failure, particularly where SARS-CoV-2 variants of concern are circulating.

Age-Related Heterogeneity in Neutralising Antibody Responses to SARS-CoV-2 Following BNT162b2 Vaccination (preprint)

Background: Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2.Methods: We did a prospective cohort study of individuals presenting for first dose vaccination. Following the first and second doses of the BNT162b2 vaccine, we measured IFNγ T cell responses, as well as binding antibody (IgA, IgG and IgG1-4) responses to Spike and Spike RBD. We also measured neutralising antibody responses to Spike in sera using a lentiviral pseudotyping system. We correlated age with immune responses and compared responses after the first and second doses.Findings: Median age was 63.5 years amongst 42 participants. Three weeks after the first dose a lower proportion of participants over 80 years old achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (8/17 versus 19/24, p=0.03). Geometric mean neutralisation titres in this age group after the first dose were lower than in younger individuals (p<0.001). Binding IgA and IgG1 and 3 responses developed post vaccination, as observed in natural infection. T- cell responses were not different in those above or below 80 years. Following the second dose, 50% neutralising antibody titres were above 1:20 in all individuals and there was no longer a difference by age grouping.Interpretation: A high proportion of individuals above the age of 80 have suboptimal neutralising antibody responses following first dose vaccination with BNT162b2, cautioning against extending the dosing interval in this high risk population.Funding Statement: RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). DAC is supported by a Wellcome Trust Clinical PhD Research Fellowship. KGCS is the recipient of a Wellcome Investigator Award (200871/Z/16/Z). This research was supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, the Cambridge Clinical Trials Unit (CCTU), the NIHR BioResource and Addenbrooke’s Charitable Trust. JAGB is supported by the Medical Research Council (MC_UP_1201/16). IATM is funded by a SANTHE award.Declaration of Interests: None to declare. Ethics Approval Statement: The study was approved by the East of England – Cambridge Central Research Ethics Committee (17/EE/0025).

SARS-CoV-2 B.1.1.7 escape from mRNA vaccine-elicited neutralizing antibodies (preprint)

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Early Immune Pathology and Persistent Dysregulation Characterise Severe COVID-19 (preprint)

In a study of 207 SARS-CoV2-infected individuals with a range of severities followed over 12 weeks from symptom onset, we demonstrate that an early robust immune response, without systemic inflammation, is characteristic of asymptomatic or mild disease. Those presenting to hospital had delayed adaptive responses and systemic inflammation already evident at around symptom onset. Such early evidence of inflammation suggests immunopathology may be inevitable in some individuals, or that preventative intervention might be needed before symptom onset. Viral load does not correlate with the development of this pathological response, but does with its subsequent severity. Immune recovery is complex, with profound persistent cellular abnormalities correlating with a change in the nature of the inflammatory response, where signatures characteristic of increased oxidative phosphorylation and reactive-oxygen species-associated inflammation replace those driven by TNF and IL-6. These late immunometabolic inflammatory changes and unresolved immune cell defects, if persistent, may contribute to “long COVID”.Funding: We are grateful for the generous support of CVC Capital Partners, the Evelyn Trust (20/75), UKRI COVID Immunology Consortium, Addenbrooke’s Charitable Trust (12/20A) and the NIHR Cambridge Biomedical Research Centre for their financial support. K.G.C.S. is the recipient of a Wellcome Investigator Award (200871/Z/16/Z); M.P.W. is the recipient of Wellcome Senior Clinical Research Fellowship (108070/Z/15/Z); C.H. was funded by a Wellcome COVID-19 Rapid Response DCF and the Fondation Botnar; N.M. was funded by the MRC (CSF MR/P008801/1) and NHSBT (WPA15-02); I.G.G. is a Wellcome Senior Fellow and was supported by funding from the Wellcome (Ref: 207498/Z/17/Z).Conflict of Interest: The authors declare they have no competing interests.

Recurrent independent emergence and transmission of SARS-CoV-2 Spike amino acid H69/V70 deletions (preprint)

SARS-CoV-2 Spike amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide deletion in the S gene, which results in loss of two amino acids: H69 and V70. Of particular note this deletion, 𝚫H69/V70, often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F. One of the 𝚫H69/V70+ N501Y lineages, B.1.1.7, is comprised of over 4000 SARS-CoV-2 genome sequences from the UK and includes eight other S gene mutations: RBD (N501Y and A570D), S1 (𝚫H69/V70 and 𝚫144/145) and S2 (P681H, T716I, S982A and D1118H). Some of these mutations have presumably arisen as a result of the virus evolving from immune selection pressure in infected individuals and at least one, lineage B.1.1.7, potentially from a chronic infection. Given our recent evidence that 𝚫H69/V70 enhances viral infectivity (Kemp et al. 2020), its effect on virus fitness appears to be independent to the RBD changes. Enhanced surveillance for the 𝚫H69/V70 deletion with and without RBD mutations should be considered as a priority. Permissive mutations such as 𝚫H69/V70 have the potential to enhance the ability of SARS-CoV-2 to generate new variants, including vaccine escape variants, that would have otherwise significantly reduced viral fitness.