Attenuated humoral responses in HIV infection after SARS-CoV-2 vaccination are linked to global B cell defects and cellular immune profiles

People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can have residual immune dysfunction and often display poorer responses to vaccination. We assessed in a cohort of PLWH (n=110) and HIV negative controls (n=64) the humoral and spike-specific B-cell responses following 1, 2 or 3 SARS-CoV-2 vaccine doses. PLWH had significantly lower neutralizing antibody (nAb) titers than HIV-negative controls at all studied timepoints. Moreover, their neutralization breadth was reduced with fewer individuals developing a neutralizing response against the Omicron variant (BA.1) relative to controls. We also observed a delayed development of neutralization in PLWH that was underpinned by a reduced frequency of spike-specific memory B cells (MBCs) and pronounced B cell dysfunction. Improved neutralization breadth was seen after the third vaccine dose in PLWH but lower nAb responses persisted and were associated with global, but not spike-specific, MBC dysfunction. In contrast to the inferior antibody responses, SARS-CoV-2 vaccination induced robust T cell responses that cross-recognized variants in PLWH. Strikingly, a subset of PLWH with low or absent neutralization had detectable functional T cell responses. These individuals had reduced numbers of circulating T follicular helper cells and an enriched population of CXCR3+CD127+CD8+ T cells after two doses of SARS-CoV-2 vaccination, which may compensate for sub-optimal serological responses in the event of infection. Therefore, normalisation of B cell homeostasis could improve serological responses to vaccines in PLWH and evaluating T cell immunity could provide a more comprehensive immune status profile in these individuals and others with B cell imbalances.

Atypical B cells and impaired SARS-CoV-2 neutralisation following booster vaccination in the elderly.

Age is a major risk factor for hospitalization and death after SARS-CoV-2 infection, even in vaccinees. Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here we show that individuals 70 or older who received a primary two dose schedule with AZD1222 and booster third dose with mRNA vaccine achieved significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared to those younger than 70. One month after the booster neither the concentration of serum binding anti spike IgG antibody, nor the frequency of spike-specific B cells showed differences by age grouping. However, the impaired neutralization potency and breadth post-third dose in the elderly was associated with enrichment of circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. Single cell RNA sequencing confirmed an expansion of TBX21-, ITGAX-expressing B cells in the elderly that enriched for B cell activation/receptor signalling pathway genes. Importantly we also observed impaired T cell responses to SARS-CoV-2 spike peptides in the elderly post-booster, both in terms of IFNgamma and IL2 secretion, as well as a decrease in T cell receptor signalling pathway genes. This expansion of atypical B cells and impaired T cell responses may contribute to the generation of less affinity-matured antibodies, with lower neutralizing capacity post-third dose in the elderly. Altogether, our data reveal the extent and potential mechanistic underpinning of the impaired vaccine responses present in the elderly after a booster dose, contributing to their increased susceptibility to COVID-19 infection.

Commercial immunoglobulin products now contain neutralising antibodies against SARS-CoV-2 spike antibody which are detectable in patient serum

Antibody-deficient patients respond poorly to COVID-19 vaccination and are at risk of severe or prolonged infection. Prophylaxis with anti-SARS-CoV-2 monoclonal antibodies has been considered. We here demonstrate that many immunoglobulin preparations now contain neutralising anti-SARS-CoV-2 antibodies which are transmitted to patients in good concentrations, albeit with significant differences between products.

Low seropositivity and sub-optimal neutralisation rates in patients fully vaccinated against COVID-19 with B cell malignancies.

Patients with haematological malignancies are at increased risk of severe disease and death from COVID-19 and are less likely to mount humoral immune responses to COVID-19 vaccination, with the B cell malignancies a particularly high-risk group. Our COV-VACC study is evaluating the immune response to COVID-19 vaccination in patients with B cell malignancies. Eligible patients were either receiving active treatment or had received treatment within the last 24 months. Patients were vaccinated with either the BNT162b2 (Pfizer-BioNTech) (n=41) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca) (n=14) vaccines. The median age of participants was 60 years (range: 27-82) and 50% were receiving systemic anti-cancer therapy (SACT) at the time of vaccination. This interim analysis from the first 55 participants describes anti-S seropositivity rates, neutralising antibody activity and association with peripheral lymphocyte subsets. After the first vaccine dose, 36% overall had detectable anti-S antibodies rising to 42% after the second dose. Sera from seropositive patients was assessed for neutralisation activity in vitro. Of the seropositive patients after first dose (n=17), only 41% were able to neutralise SARS-CoV-2 pseudotyped virus with a 50% inhibitory dilution factor (ID50) of >1:50. After two doses (n=21) 57% of the seropositive patients had detectable neutralisation activity (median ID50 of 1:469, range 1:70 - 1:3056). Total blood lymphocyte, CD19, CD4 and CD56 counts were significantly associated with seropositivity. Patients vaccinated more than 6 months after completing therapy were significantly more likely to develop antibodies than those within 6 months of treatment or on active treatment; OR: 5.93 (1.29 - 27.28). Our data has important implications for patients with B cell malignancies as we demonstrate a disconnect between anti-S seropositivity and virus neutralisation in vitro following vaccination against COVID-19. Urgent consideration should be given to revaccinating patients with B-cell malignancies after completion of anti-cancer treatment as large numbers currently remain at high risk of infection with the increasing transmission of SARS-CoV-2 in many countries.

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2 infection

Individuals with likely exposure to the highly infectious SARS-CoV-2 do not necessarily develop PCR or antibody positivity, suggesting some may clear sub-clinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-5. We hypothesised that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-26-12, would expand in vivo to mediate rapid viral control, potentially aborting infection. We studied T cells against the replication transcription complex (RTC) of SARS-CoV-2 since this is transcribed first in the viral life cycle13-15and should be highly conserved. We measured SARS-CoV-2-reactive T cells in a cohort of intensively monitored healthcare workers (HCW) who remained repeatedly negative by PCR, antibody binding, and neutralisation for SARS-CoV-2 (exposed seronegative, ES). 16-weeks post-recruitment, ES had memory T cells that were stronger and more multispecific than an unexposed pre-pandemic cohort, and more frequently directed against the RTC than the structural protein-dominated responses seen post-detectable infection (matched concurrent cohort). The postulate that HCW with the strongest RTC-specific T cells had an abortive infection was supported by a low-level increase in IFI27 transcript, a robust early innate signature of SARS-CoV-2 infection16. We showed that the RNA-polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and was preferentially targeted by T cells from UK and Singapore pre-pandemic cohorts and from ES. RTC epitope-specific T cells capable of cross-recognising HCoV variants were identified in ES. Longitudinal samples from ES and an additional validation cohort, showed pre-existing RNA-polymerase-specific T cells expanded in vivo following SARS-CoV-2 exposure, becoming enriched in the memory response of those with abortive compared to overt infection. In summary, we provide evidence of abortive seronegative SARS-CoV-2 infection with expansion of cross-reactive RTC-specific T cells, highlighting these highly conserved proteins as targets for future vaccines against endemic and emerging Coronaviridae.

SARS-CoV-2-specific memory B cells can persist in the elderly despite loss of neutralising antibodies

Memory B cells (MBC) can provide a recall response able to supplement waning antibodies with an affinity-matured response better able to neutralise variant viruses. We studied a cohort of vulnerable elderly care home residents and younger staff, a high proportion of whom had lost neutralising antibodies (nAb), to investigate their reserve immunity from SARS-CoV-2-specific MBC. Class-switched spike and RBD-tetramer-binding MBC with a classical phenotype persisted five months post-mild/asymptomatic SARS-CoV-2 infection, irrespective of age. Spike/RBD-specific MBC remained detectable in the majority who had lost nAb, although at lower frequencies and with a reduced IgG/IgA isotype ratio. Functional spike/S1/RBD-specific recall was also detectable by ELISpot in some who had lost nAb, but was significantly impaired in the elderly, particularly to RBD. Our findings demonstrate persistence of SARS-CoV-2-specific MBC beyond loss of nAb, but highlight the need for careful monitoring of functional defects in RBD-specific B cell immunity in the elderly. One sentence summaryCirculating class-switched spike and RBD-specific memory B cells can outlast detectable neutralising antibodies but are functionally constrained in the elderly.

Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARS-CoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV

There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH. One Sentence SummaryAdaptive immune responses to SARS-CoV-2 in the setting of HIV infection

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike

The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ~45% showed neutralizing activity of which ~20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

The impact of Spike mutations on SARS-CoV-2 neutralization

Multiple SARS-CoV-2 vaccines have shown protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, Spike. Antibodies from SARS-CoV-2 infection neutralize the virus by focused targeting of Spike and there is limited serum cross-neutralization of the closely-related SARS-CoV. As new SARS-CoV-2 variants are rapidly emerging, exemplified by the B.1.1.7, 501Y.V2 and P.1 lineages, it is critical to understand if antibody responses induced by infection with the original SARS-CoV-2 virus or the current vaccines will remain effective against virus variants. In this study we evaluate neutralization of a series of mutated Spike pseudotypes including a B.1.1.7 Spike pseudotype. The analyses of a panel of Spike-specific monoclonal antibodies revealed that the neutralizing activity of some antibodies was dramatically reduced by Spike mutations. In contrast, polyclonal antibodies in the serum of patients infected in early 2020 remained active against most mutated Spike pseudotypes. The majority of serum samples were equally able to neutralize the B.1.1.7 Spike pseudotype, however potency was reduced in a small number of samples (3 of 36) by 5-10-fold. This work highlights that changes in the SARS-CoV-2 Spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their impact on vaccine efficacy.

SARS-CoV-2 recruits a haem metabolite to evade antibody immunity

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

Neutralising antibodies drive Spike mediated SARS-CoV-2 evasion

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and{Delta} H69/{Delta}V70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing{Delta} H69/{Delta}V70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The{Delta} H69/{Delta}V70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against{Delta} H69/{Delta}V70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

SARS-CoV-2 antibody responses in patients with aggressive haematological malignancies

The development of antibody responses to SARS-CoV-2 is an indicator of seroprevalence and may afford protection from infection. It has been presumed that antibody responses to SARS-CoV-2 will be impaired in patients with aggressive haematological malignancy (PHM) due to underlying immunological dysfunction caused by malignancy or systemic anti-cancer treatment (SACT), placing them at increased risk. Here we analysed longitudinal serum samples from ten hospitalised PHM with aggressive disease and on SACT, collected up to 103 days post-onset of COVID-19 symptoms. We found that the majority (8/9) of PHM with confirmed SARS-CoV-2 infection seroconverted and developed antibodies to the major SARS-CoV-2 antigens (S1 and N) with most (6/8) produced neutralising antibody responses. Furthermore, the dynamics of antibody responses were broadly similar to that reported for the general population, except for a possible delay to seroconversion. Our finding that PHM on SACT can make functional antibody responses to SARS-CoV-2 has important implications for patient management and serological monitoring of SARS-CoV-2 in high-risk groups.

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.