Immune profiling of COVID-19 vaccine responses in people with multiple sclerosis on B cell-depleting therapy (preprint)

Background and ObjectivePeople with multiple sclerosis (pwMS) receiving B cell-depleting therapies have impaired antibody responses to vaccination. In a proportion of individuals, repeat vaccination against COVID-19 leads to seroconversion. We sought to describe the immune phenotype of pwMS on ocrelizumab, and identify clinical and immunological determinants of an effective vaccine response. MethodsThis was a single-centre, prospective cohort study. Peripheral blood samples were collected from pwMS receiving ocrelizumab (n = 38) pre and post administration of a third dose of mRNA COVID-19 vaccine. Immunogenicity was measured by T cell IFN{gamma} ELISpot, antibody titres, and live virus neutralisation. Humoral immunity was benchmarked against pwMS receiving natalizumab (n = 15), and against a correlate of real-world protection (50% reduction in incidence of infection) from SARS-CoV-2 ancestral and omicron BA.5 variants. The peripheral immune phenotype was comprehensively assessed by flow cytometry, and potential clinical and phenotypic determinants of response to vaccination identified. ResultsImmune cell populations relevant to disease and vaccine response were altered in pwMS receiving ocrelizumab versus natalizumab treatment, including depleted CD20-expressing B cell, T cell and NK cell populations, and elevated CD27+CD38+ T cell and NK8 cell frequencies. Following a third vaccine dose, 51% of pwMS on ocrelizumab were seropositive for SARS-CoV-2 receptor-binding-domain IgG, and 25% and 14% met the threshold for effective neutralisation of live SARS-CoV-2 ancestral and omicron BA.5 virus, respectively. B cell frequency at the time of vaccination, but not time since ocrelizumab infusion, was positively correlated with antibody response, while a strong negative correlation was observed between CD56bright NK cell frequency and antibody response in the ocrelizumab group. In this exploratory cohort, CD3-CD20+ B cells (% of lymphocytes; OR=3.92) and CD56bright NK cells (% of NK cells; OR=0.94) were predictive of an effective neutralising antibody response in second dose non-responders (AUC: 0.98). DiscussionOcrelizumab treatment was associated with an altered immune phenotype, including recently described T cell and NK populations with potential roles in disease pathogenesis. However, seroconversion was severely impaired by ocrelizumab, and less than half of those who seroconverted following a third vaccine dose demonstrated effective immunity against SARS-CoV-2 ancestral or omicron BA.5. B cell frequency was associated with an effective antibody response, while immunomodulatory CD56bright NK cells were identified as a potential negative determinant of response in those with inadequate B cell numbers. Immune phenotype rather than time since ocrelizumab infusion may help to stratify individuals for prophylaxis.

TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2 (preprint)

Continued high levels spread of SARS-CoV-2 globally enabled accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in ACE2 and TMPRSS2 use by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on cleavage or non-cleavage of ACE2 by TMPRSS2 activity. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 was cleaved by TMPRSS2 and this led to either augmentation or progressive attenuation of pre-Omicron and Omicron lineages, respectfully. In contrast, TMPRSS2 resistant ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 spike activation. Therefore, our data support the tropism shift of Omicron lineages to be a function of evolution towards the use of uncleaved pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins.

Improvement of immune dysregulation and health-related quality of life in individuals with long COVID at 24-months following SARS-CoV-2 infection (preprint)

This study investigated the humoral and cellular immune responses in individuals with long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24-months. LC participants showed elevated spike and nucleocapsid IgG levels, higher neutralizing capacity, and increased spike- and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells at 3- and 8-months, but these differences did not persist at 24-months. Some LC participants had detectable IFN-{beta} and IFN-{gamma} that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at 24-month timepoint revealed similar immune cell proportions and reconstitution of naive T and B cell subsets in LC. No significant differences in exhaustion scores or antigen-specific T cell clones were observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24-months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count were associated with improvements in health-related quality of life.

mTOR inhibition improves the formation of functional T cell memory following COVID-19 vaccination of kidney transplant recipients (preprint)

Inadequate immune response to vaccination is a long-standing problem faced by immunosuppressed kidney transplant recipients (KTRs), requiring novel strategies to improve vaccine efficacy. In this study, the potential of mechanistic target of rapamycin inhibitors (mTORi) to improve T cell responses to COVID-19 vaccination was investigated. Following primary vaccination with adenoviral (ChAdOx1) or mRNA (BNT162b2) COVID-19 vaccines, KTRs receiving rapamycin demonstrated T cell responses greater than those of healthy individuals, characterized by increased frequencies of vaccine-specific central memory, effector memory and TEMRA T cells, in both the CD4+ and CD8+ compartments. Relative to standard-of-care triple therapy, mTORi-based therapy was associated with a 12-fold greater functional T cell response to primary vaccination of KTRs. The use of rapamycin to augment T cell responses to COVID-19 booster (third dose) vaccination was next investigated in a randomized, controlled trial. Immunosuppression modification with rapamycin was feasible and well-tolerated, but did not improve vaccine-specific T cell responses in this cohort. To understand the parameters for effective use of rapamycin as a vaccine adjuvant, mice were treated with rapamycin before primary or booster vaccination with ancestral and/or Omicron COVID-19 vaccines. Supporting the findings from KTRs, significant enhancement of functional and stem-like memory T cell responses was observed when rapamycin was administered from the time of primary, rather than booster, vaccination. Collectively, a positive effect of mTOR inhibitors on vaccine-induced T cell immunity against COVID-19 in humans was demonstrated.

Emergence and antibody evasion of BQ and BA.2.75 SARS-CoV-2 sublineages in the face of maturing antibody breadth at the population level (preprint)

The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two levels: (i) we tracked over 400,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using antibody pools. (ii) we mapped the antibody response at the individual level using blood from strigently curated vaccine and convalescent cohorts. In pooled antibody samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases we observed increased antibody breadth to variants that were yet to be in circulation. Resolution of viral neutralisation at the cohort level supported equivalent coverage across prior and emerging variants with emerging isolates BQ.1.1, XBB.1 and BR.2.1 the most evasive. Further, these emerging variants were resistant to Evusheld, whilst neutralization resistance to Sotrovimab was restricted to BQ.1.1 and further supported by lack of Spike glycoprotein binding to this variant. An outgrowth advantage through better utilization of TMPRSS2 was observed across BQ lineages and not those derived from BA.2.75. We conclude at this current point in time that variants derived from BQ lineages can evade antibodies at levels equivalent to their most evasive BA.2.75 counterparts but sustain an entry phenotype that would promote an additional outgrowth advantage.

Enhanced stability of the SARS CoV-2 spike glycoprotein trimer following modification of an alanine cavity in the protein core. (preprint)

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity formed by Ala1016 and Ala1020 that form part of the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. The trimer stabilizing effects of filling the Ala1016/Ala1020 cavity was linked to improved S glycoprotein membrane fusion function. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited very high titers of neutralizing antibodies to ancestral and Delta-derived viruses (1/2,700-1/5,110), while neutralization titer was somewhat reduced with Omicron BA.1 (1/210-1,1744). The antigens elicited antibody specificities that could compete with ACE2-Fc for binding to the receptor-binding motif (RBM) and NAbs directed to key neutralization epitopes within the receptor-binding domain (RBD), N-terminal domain (NTD) and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S ectodomain trimers in the absence of an external trimerization motif (T4 foldon). The VI mutation represents a method for producing an intrinsically stable trimeric S ectodomain glycoprotein vaccine in the absence of a foreign trimerization tag.

High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells. (preprint)

Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, rather than the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, that were absent in individuals with low antibody levels. However, vaccination in low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern. (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, the Omicron BA.1 variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant demonstrated higher numbers of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5 has now started to dominate globally, with the potential to supplant BA.2. To address the relative threat of BA.5, we determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a well characterised, genetically engineered ACE2/TMPRSS2 cell line. We then assessed the impact of BA.5 infection on humoral neutralisation in vitro, in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. The infectivity of virus in primary swabs and expanded isolates revealed whilst BA.1 and BA.2 are attenuated through ACE2/TMPRSS2, BA.5 infectivity is equivalent to that of an early 2020 circulating clade and has greater sensitivity to the TMPRSS2 inhibitor Nafamostat. As with BA.1, we observed BA.5 to significantly reduce neutralisation titres across all donors. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 7-fold with BA.5. Of all therapeutic antibodies tested, we observed a 14.3-fold reduction using Evusheld and 16.8 reduction using Sotrovimab when neutralising a Clade A versus BA.5 isolate. These results have implications for ongoing tracking and management of Omicron waves globally.

Rapid isolation and resolution immune evasion and viral fitness across contemporary SARS-CoV-2 variants (preprint)

From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern and 6 variants of interest) globally with a 4-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by allowing determining end point viral titers from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated and tested the first cases of this variant within Australia. In this setting we observed Omicron to diverge from other variants at two levels: Firstly, it ranks at the mots evasive to neutralisation antibodies compared to all VOCs and major VUIs. Secondly, it no longer engages TMPRSS2 during the late stages of fusion.

SARS-CoV-2 Omicron: evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in the first world. In late 2021, the Omicron (B.1.1.529) virus variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant a demonstrated higher number of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. We assessed the impact of Omicron infection on the ability of: serum from vaccinated and/or previously infected individuals; concentrated human IgG from plasma donors, and licensed monoclonal antibody therapies to neutralise the virus in vitro . There was a 17 to 27-fold reduction in neutralisation titres across all donors who had a detectable neutralising antibody titre to the Omicron variant. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 16-fold. Of all therapeutic antibodies tested, significant neutralisation of the Omicron variant was only observed for Sotrovimab, with other monoclonal antibodies unable to neutralise Omicron in vitro . These results have implications for ongoing therapy of individuals infected with the Omicron variant.

COVID-19 Vaccine Failure in Chronic Lymphocytic Leukemia and Monoclonal B-Lymphocytosis; Humoral and Cellular Immunity (preprint)

Chronic lymphocytic leukemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-Lymphocytosis (MBL) patients also have immune impairment. We evaluated humoral and cellular immune responses in 181 patients with CLL (160) and MBL (21) to correlate failed seroconversion (<50AU/mL SARS-CoV-2 II IgG assay, antibody to spike protein, Abbott Diagnostics) following each of 2 vaccine doses with clinical and laboratory parameters. Following first and second doses, 79.2% then 45% of CLL, and 50% then 9.5% of MBL respectively remained seronegative, indicating 2 vaccine doses are crucial. There was significant association between post-dose 2 antibody level with pre-vaccination reduced IgM (p<0.0001), IgG2 (p<0.035), IgG3 (p<0.046), and CLL therapy within 12 months (p<0.001) in univariate analysis. By multivariate analysis, reduced IgM (p<0.0002) and active therapy (p<0.0002) retained significance. There was no significant correlation with age, gender, CLL duration, IgG, IgA or lymphocyte subsets. Anti-spike protein levels varied widely and were lower in CLL, than MBL, and both lower than normal donors. Neutralization activity showed anti-spike levels <1000AU/mL were usually negative for both an early viral clade and the contemporary Delta variant. There were 72.9% of CLL and 53.3% of MBL who failed to reach anti-spike levels >1000AU/mL. In a representative subset of 32 CLL patients, 80% had normal T-cell responses by IFN{gamma} and IL-2 FluoroSpot assay. Failed seroconversion occurred in 36.6%% of treatment-naive patients, 52.9% treatment-naive with reduced IgM, 78.1% on therapy, and 85.7% on ibrutinib. Vaccination failure is very common in CLL, including early-stage disease.