Generation of SARS-CoV-2 escape mutations by monoclonal antibody therapy.

COVID-19 patients at risk of severe disease may be treated with neutralising monoclonal antibodies (mAbs). To minimise virus escape from neutralisation these are administered as combinations e.g. casirivimab+imdevimab or, for antibodies targeting relatively conserved regions, individually e.g. sotrovimab. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to detect emerging drug resistance in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the antibody epitopes and for casirivimab+imdevimab multiple mutations are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination

Most existing studies characterising SARS-CoV-2-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of ACE-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations. Graphical Yin et al. utilize two informative systems for evaluating overall T cell responses to SARS-CoV-2 and variants, enabling greater understanding of T cell responses to the virus, cross-reactivity to viral variants and the differences between vaccine- and infection-induced immunity to SARS-CoV-2, and other emerging viruses in the future.

Evolution of long-term vaccine induced and hybrid immunity in healthcare workers after different COVID19 vaccine regimens: a longitudinal cohort study

Background Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARS-CoV-2. However, the maintenance of such responses – and hence protection from disease – requires careful characterisation. In a large prospective study of UK healthcare workers (Protective immunity from T cells in Healthcare workers (PITCH), within the larger SARS-CoV-2 immunity & reinfection evaluation (SIREN) study) we previously observed that prior infection impacted strongly on subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. Methods Here, we report longer follow up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination. Findings We make three observations: Firstly, the dynamics of humoral and cellular responses differ;binding and neutralising antibodies declined whereas T and memory B cell responses were maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels, broadened neutralising activity against variants of concern including omicron BA.1, BA.2 and BA.5, and boosted T cell responses above the 6-month level post dose 2. Thirdly, prior infection maintained its impact driving larger and broader T cell responses compared with never-infected people – a feature maintained until 6 months after the third dose. Conclusions Broadly cross-reactive T cell responses are well maintained over time – especially in those with combined vaccine and infection-induced immunity ("hybrid” immunity) – and may contribute to continued protection against severe disease. Funding Department for Health and Social Care, Medical Research Council Graphical abstract Moore et al. studied antibody and cellular responses to COVID-19 vaccines before and after dose 3. Antibody responses waned, but T cell responses were well maintained. T cells recognised Omicron variants better and for longer than antibodies. Differences due to vaccine regimen and previous infection evened out over time.

Rapid escape of new SARS-CoV-2 Omicron variants from BA.2 directed antibody responses

In November 2021 Omicron BA.1, containing a raft of new spike mutations emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or SARS-CoV-2 infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional RBD amino-acid substitutions compared to BA.2. We describe a panel of 25 potent mAbs generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titre of vaccine or BA.1, BA.2 or BA.4/5 immune serum. Graphical Dijokaite-Guraliuc et al. analyse potently neutralizing antibodies from vaccinated individuals with BA.2 breakthrough infections. The antibodies bind 3 sites on the receptor binding domain, 2 in common with early pandemic antibodies. Mutations in more recent variants map closely to these sites leading to reduced neutralization in all but one mAb.

Rapid escape of new SARS-CoV-2 Omicron variants from BA.2-directed antibody responses.

In November 2021, Omicron BA.1, containing a raft of new spike mutations, emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 and then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional receptor-binding domain (RBD) amino acid substitutions compared with BA.2. We describe a panel of 25 potent monoclonal antibodies (mAbs) generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titer of vaccine or BA.1, BA.2, or BA.4/5 immune serum.

Evolution of long-term vaccine-induced and hybrid immunity in healthcare workers after different COVID-19 vaccine regimens.

BACKGROUND: Both infection and vaccination, alone or in combination, generate antibody and T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the maintenance of such responses-and hence protection from disease-requires careful characterization. In a large prospective study of UK healthcare workers (HCWs) (Protective Immunity from T Cells in Healthcare Workers [PITCH], within the larger SARS-CoV-2 Immunity and Reinfection Evaluation [SIREN] study), we previously observed that prior infection strongly affected subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. METHODS: Here, we report longer follow-up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination. FINDINGS: We make three observations: first, the dynamics of humoral and cellular responses differ; binding and neutralizing antibodies declined, whereas T and memory B cell responses were maintained after the second vaccine dose. Second, vaccine boosting restored immunoglobulin (Ig) G levels; broadened neutralizing activity against variants of concern, including Omicron BA.1, BA.2, and BA.5; and boosted T cell responses above the 6-month level after dose 2. Third, prior infection maintained its impact driving larger and broader T cell responses compared with never-infected people, a feature maintained until 6 months after the third dose. CONCLUSIONS: Broadly cross-reactive T cell responses are well maintained over time-especially in those with combined vaccine and infection-induced immunity ("hybrid" immunity)-and may contribute to continued protection against severe disease. FUNDING: Department for Health and Social Care, Medical Research Council.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused successive global waves of infection. These variants, with multiple mutations in the spike protein, are thought to facilitate escape from natural and vaccine-induced immunity and often increase in affinity for ACE2. The latest variant to cause concern is BA.2.75, identified in India where it is now the dominant strain, with evidence of wider dissemination. BA.2.75 is derived from BA.2 and contains four additional mutations in the receptor-binding domain (RBD). Here, we perform an antigenic and biophysical characterization of BA.2.75, revealing an interesting balance between humoral evasion and ACE2 receptor affinity. ACE2 affinity for BA.2.75 is increased 9-fold compared with BA.2; there is also evidence of escape of BA.2.75 from immune serum, particularly that induced by Delta infection, which may explain the rapid spread in India, where where there is a high background of Delta infection. ACE2 affinity appears to be prioritized over greater escape.

Persistence of immunity against Omicron BA.1 and BA.2 variants following homologous and heterologous COVID-19 booster vaccines in healthy adults after a two-dose AZD1222 vaccination.

OBJECTIVES: The SARS-CoV-2 Omicron variant presents numerous mutations potentially able to evade neutralizing antibodies (NAbs) elicited by COVID-19 vaccines. Therefore, this study aimed to provide evidence on a heterologous booster strategy to overcome the waning immunity against Omicron variants. METHODS: Participants who completed the Oxford/AstraZeneca (hereafter AZD1222) vaccine dose for 5-7 months were enrolled. The reactogenicity and persistence of immunogenicity in both humoral and cellular response after a homologous or heterologous booster with the AZD1222 and messenger RNA (mRNA) vaccines (BNT162b2, full, or half-dose mRNA-1273) administered 6 months after primary vaccination were determined. RESULTS: A total of 229 individuals enrolled, and waning of immunity was observed 5-7 months after the AZD1222-primed vaccinations. Total receptor-binding domain (RBD) immunoglobulin (Ig) levels, anti-RBD IgG, and focus reduction neutralization test against Omicron BA.1 and BA.2 variants and T cell response peaked at 14-28 days after booster vaccination. Both the full and half dose of mRNA-1273 induced the highest response, followed by BNT162b2 and AZD1222. At 90 days, the persistence of immunogenicity was observed among all mRNA-boosted individuals. Adverse events were acceptable for all vaccines. CONCLUSION: A heterologous mRNA booster provided a significantly superior boost of binding and NAbs levels against the Omicron variant compared with a homologous booster in individuals with AZD1222-primed vaccinations.

Comparison of the reactogenicity and immunogenicity of a reduced and standard booster dose of the mRNA COVID-19 vaccine in healthy adults after two doses of inactivated vaccine.

The coronavirus disease 2019 (COVID-19) pandemic has been a serious healthcare problem worldwide since December 2019. The third dose of heterologous vaccine was recently approved by World Health Organization. The present study compared the reactogenicity and immunogenicity of the reduced and standard third booster dose of the BNT162b2 and mRNA-1273 vaccine in adults who previously received the two-dose CoronaVac vaccine. Results showed that headache, joint pain, and diarrhea were more frequent in the 15 µg- than the 30 µg-BNT162b2 groups, whereas joint pain and chilling were more frequent in the 100 µg- than the 50 µg-mRNA-1273 groups. No significant differences in immunogenicity were detected. These findings demonstrate that the reduced dose of the mRNA vaccines elicited antibody responses against the SARS-CoV-2 delta and omicron variants that were comparable to the standard dose. The reduced dose could be used to increase vaccine coverage in situations of limited global vaccine supply.

Effects of boosted mRNA and adenoviral-vectored vaccines on immune responses to omicron BA.1 and BA.2 following the heterologous CoronaVac/AZD1222 vaccination.

The coronavirus 2019 omicron variant has surged rapidly and raises concerns about immune evasion even in individuals with complete vaccination, because it harbors mutations. Here we examine the capability of booster vaccination following CoronaVac/AZD1222 prime to induce neutralizing antibodies (NAbs) against omicron (BA.1 and BA.2) and T-cell responses. A total of 167 participants primed with heterologous CoronaVac/AZD1222 for 4-5 months were enrolled, to receive AZD1222, BNT162b2, or mRNA-1273 as a third dose. Reactogenicity was recorded. Immunogenicity analyses of severe acute respiratory syndrome coronavirus 2-binding antibodies were measured using enzyme-linked immunosorbent assay. The NAb titers against omicron BA.1 and BA.2 were determined using the focus reduction neutralization test (FRNT50) and total interferon-γ responses were measured to observe the T-cell activation. A substantial loss in neutralizing potency to omicron variant was found at 4-5 months after receiving the heterologous CoronaVac/AZD1222. Following booster vaccination, a significant increase in binding antibodies and neutralizing activities toward delta and omicron variants was observed. Neutralization to omicron BA.1 and BA.2 were comparable, showing the highest titers after boosted mRNA-1273 followed by BNT162b2 and AZD1222. In addition, individuals boosted with messenger RNA (mRNA) vaccines develop a T-cell response to spike protein, whereas those boosted with AZD1222 did not. Reactogenicity was mild to moderate without serious adverse events. Our findings demonstrated that mRNA booster vaccination is able to overcome waning immunity to provide antibodies that neutralize omicron BA.1 and BA.2, as well as a T-cell response.

Strong Correlations between the Binding Antibodies against Wild-Type and Neutralizing Antibodies against Omicron BA.1 and BA.2 Variants of SARS-CoV-2 in Individuals Following Booster (Third-Dose) Vaccination.

This study examined the neutralizing activity and receptor-binding domain (RBD) antibody levels against wild-type and omicron BA.1 and BA.2 variants in individuals who received three doses of COVID-19 vaccination. The relationship between the anti-RBD IgG against wild-type and live virus neutralizing antibody titers against omicron BA.1 and BA.2 variants was examined. In total, 310 sera samples from individuals after booster vaccination (third-dose) were tested for specific IgG wild-type SARS-CoV-2 RBD and the omicron BA.1 surrogate virus neutralization test (sVNT). The live virus neutralization assay against omicron BA.1 and BA.2 was performed using the foci-reduction neutralization test (FRNT50). The anti-RBD IgG strongly correlated with FRNT50 titers against BA.1 and BA.2. Non-linear regression showed that anti-RBD IgG at the cut-off value ≥148 BAU/mL and ≥138 BAU/mL were related to the threshold for FRNT50 titers ≥20 against BA.1 and BA.2, respectively. A moderate correlation was observed between the sVNT and FRNT50 titers. At FRNT50 titers ≥20, the predicted sVNT for BA.1 and BA.2 was ≥10.57% and ≥11.52%, respectively. The study identified anti-RBD IgG and sVNT levels that predict detectable neutralizing antibodies against omicron variants. Assessment and monitoring of protective immunity support vaccine policies and will help identify optimal timing for booster vaccination.

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.

The Omicron lineage of SARS-CoV-2, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa's Gauteng region uncovered two new sublineages, BA.4 and BA.5, which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences, and although closely related to BA.2, they contain further mutations in the receptor-binding domain of their spikes. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the serum from BA.1 vaccine breakthrough infections, there are, likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal outcomes with coronavirus disease 2019 (COVID-19) is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to an intensive care unit (ICU) with fatal COVID-19 outcomes, but not in individuals with nonfatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to an ICU with fatal and nonfatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an "original antigenic sin" type response.

Potent cross-reactive antibodies following Omicron breakthrough in vaccinees.

Highly transmissible Omicron variants of SARS-CoV-2 currently dominate globally. Here, we compare neutralization of Omicron BA.1, BA.1.1, and BA.2. BA.2 RBD has slightly higher ACE2 affinity than BA.1 and slightly reduced neutralization by vaccine serum, possibly associated with its increased transmissibility. Neutralization differences between sub-lineages for mAbs (including therapeutics) mostly arise from variation in residues bordering the ACE2 binding site; however, more distant mutations S371F (BA.2) and R346K (BA.1.1) markedly reduce neutralization by therapeutic antibody Vir-S309. In-depth structure-and-function analyses of 27 potent RBD-binding mAbs isolated from vaccinated volunteers following breakthrough Omicron-BA.1 infection reveals that they are focused in two main clusters within the RBD, with potent right-shoulder antibodies showing increased prevalence. Selection and somatic maturation have optimized antibody potency in less-mutated epitopes and recovered potency in highly mutated epitopes. All 27 mAbs potently neutralize early pandemic strains, and many show broad reactivity with variants of concern.

Heterologous versus homologous COVID-19 booster vaccination in previous recipients of two doses of CoronaVac COVID-19 vaccine in Brazil (RHH-001): a phase 4, non-inferiority, single blind, randomised study.

INTRODUCTION: The inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac, Sinovac) has been widely used in a two-dose schedule. We assessed whether a third dose of the homologous or a different vaccine could boost immune responses. METHODS: RHH-001 is a phase 4, participant masked, two centre, safety and immunogenicity study of Brazilian adults (18 years and older) in São Paulo or Salvador who had received two doses of CoronaVac 6 months previously. The third heterologous dose was of either a recombinant adenoviral vectored vaccine (Ad26.COV2-S, Janssen), an mRNA vaccine (BNT162b2, Pfizer-BioNTech), or a recombinant adenoviral-vectored ChAdOx1 nCoV-19 vaccine (AZD1222, AstraZeneca), compared with a third homologous dose of CoronaVac. Participants were randomly assigned (5:6:5:5) by a RedCAP computer randomisation system stratified by site, age group (18-60 years or 61 years and over), and day of randomisation, with a block size of 42. The primary outcome was non-inferiority of anti-spike IgG antibodies 28 days after the booster dose in the heterologous boost groups compared with homologous regimen, using a non-inferiority margin for the geometric mean ratio (heterologous vs homologous) of 0·67. Secondary outcomes included neutralising antibody titres at day 28, local and systemic reactogenicity profiles, adverse events, and serious adverse events. This study was registered with Registro Brasileiro de Ensaios Clínicos, number RBR-9nn3scw. FINDINGS: Between Aug 16, and Sept 1, 2021, 1240 participants were randomly assigned to one of the four groups, of whom 1239 were vaccinated and 1205 were eligible for inclusion in the primary analysis. Antibody concentrations were low before administration of a booster dose with detectable neutralising antibodies of 20·4% (95% CI 12·8-30·1) in adults aged 18-60 years and 8·9% (4·2-16·2) in adults 61 years or older. From baseline to day 28 after the booster vaccine, all groups had a substantial rise in IgG antibody concentrations: the geometric fold-rise was 77 (95% CI 67-88) for Ad26.COV2-S, 152 (134-173) for BNT162b2, 90 (77-104) for ChAdOx1 nCoV-19, and 12 (11-14) for CoronaVac. All heterologous regimens had anti-spike IgG responses at day 28 that were superior to homologous booster responses: geometric mean ratios (heterologous vs homologous) were 6·7 (95% CI 5·8-7·7) for Ad26.COV2-S, 13·4 (11·6-15·3) for BNT162b2, and 7·0 (6·1-8·1) for ChAdOx1 nCoV-19. All heterologous boost regimens induced high concentrations of pseudovirus neutralising antibodies. At day 28, all groups except for the homologous boost in the older adults reached 100% seropositivity: geometric mean ratios (heterologous vs homologous) were 8·7 (95% CI 5·9-12·9) for Ad26.COV2-S vaccine, 21·5 (14·5-31·9) for BNT162b2, and 10·6 (7·2-15·6) for ChAdOx1 nCoV-19. Live virus neutralising antibodies were also boosted against delta (B.1.617.2) and omicron variants (B.1.1.529). There were five serious adverse events. Three of which were considered possibly related to the vaccine received: one in the BNT162b2 group and two in the Ad26.COV2-S group. All participants recovered and were discharged home. INTERPRETATION: Antibody concentrations were low at 6 months after previous immunisation with two doses of CoronaVac. However, all four vaccines administered as a third dose induced a significant increase in binding and neutralising antibodies, which could improve protection against infection. Heterologous boosting resulted in more robust immune responses than homologous boosting and might enhance protection. FUNDING: Ministry of Health, Brazil.

Neutralizing Activities Against the Omicron Variant After a Heterologous Booster in Healthy Adults Receiving Two Doses of CoronaVac Vaccination.

BACKGROUND: The use of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (CoronaVac) against SARS-CoV-2 is implemented worldwide. However, waning immunity and breakthrough infections have been observed. Therefore, we hypothesized that the heterologous booster might improve the protection against the delta and omicron variants. METHODS: A total of 224 individuals who completed the 2-dose CoronaVac for 6 months were included. We studied reactogenicity and immunogenicity after a heterologous booster with the inactivated vaccine (BBIBP), the viral vector vaccine (AZD1222), and the messenger ribonucleic acid (mRNA) vaccine (both BNT162B2 and mRNA-1273). We also determined immunogenicity at 3- and 6-month boosting intervals. RESULTS: The solicited adverse events were mild to moderate and well tolerated. Total receptor binding domain (RBD) immunoglobulin (Ig), anti-RBD IgG, focus reduction neutralization test (FRNT50) against delta and omicron variants, and T-cell response were highest in the mRNA-1273 group followed by the BNT162b2, AZD1222, and BBIBP groups, respectively. We also witnessed a higher total Ig anti-RBD in the long-interval than in the short-interval group. CONCLUSIONS: All 4 booster vaccines significantly increased binding and neutralizing antibodies in individuals immunized with 2 doses of CoronaVac. The present evidence may benefit vaccine strategies to thwart variants of concern, including the omicron variant.