Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection.

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.

Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Patients with treated indolent lymphomas immunized with BNT162b2 have reduced anti-spike neutralizing IgG to SARS-CoV-2 variants, but preserved antigen-specific T cell responses.

Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID-19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti-spike immunoglobulin G (IgG) was determined by a high-sensitivity flow-cytometric assay, in addition to live-virus neutralization. Antigen-specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti-spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25-fold lower than TN (245 898) and HC (228 255, p = .0002 for both). Anti-spike IgG correlated with lymphocyte count (r = .63; p = .002), and time from treatment (r = .56; p = .007), on univariate analysis, but only with lymphocyte count on multivariate analysis (p = .03). In the WM cohort, median anti-spike IgG MFI in BTKi patients (39 039) was reduced compared to TN (220 645, p = .0008) and HC (p < .0001). Anti-spike IgG correlated with neutralization of the delta variant (r = .62, p < .0001). Median neutralization titer for WM BTKi (0) was lower than HC (40, p < .0001) for early-clade and delta. All cohorts had functional T cell responses. Median anti-spike IgG decreased 4-fold from second to third dose (p = .004). Only 5 of 29 poor initial responders assessed after third vaccination demonstrated seroconversion and improvement in neutralization activity, including to the omicron variant.

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

Background: 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. Methods: We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Findings: 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, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Interpretation: 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.

BACKGROUND: Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. 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, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway. METHODS: Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat. FINDINGS: Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat. INTERPRETATION: Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).

Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia.

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

SARS-CoV-2 Omicron variant escapes neutralizing antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

High-Titer Neutralizing Antibodies against the SARS-CoV-2 Delta Variant Induced by Alhydroxyquim-II-Adjuvanted Trimeric Spike Antigens.

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel). Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titer NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralizing against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titers against multiple SARS-CoV-2 variants; notably, high titers against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern. IMPORTANCE There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process. CoVac-II demonstrates strong and persistent immunity after vaccination of mice, and is highly immunogenic in multiple animal models, including rabbits and horses. We further show that prior immunity can be boosted using a recombinant spike antigen from the Beta variant; importantly, plasma from boosted mice effectively neutralize multiple SARS-CoV-2 variants in vitro, including Delta. The strong humoral and Th1-biased immunogenicity of CoVac-II is driven by use of Alhydroxiquim-II (AHQ-II), the first adjuvant in an authorized vaccine that acts through the dual Toll-like receptor (TLR)7 and TLR8 pathways, as part of the Covaxin vaccine. Our data suggest AHQ-II/spike protein combinations could constitute safe, affordable, and mass-manufacturable COVID-19 vaccines for global distribution.

A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilising immunity against SARS-CoV-2 infection.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Immunizations with diverse sarbecovirus receptor binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

SARS Coronavirus-2 Microneutralisation and Commercial Serological Assays Correlated Closely for Some but Not All Enzyme Immunoassays.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

Virus isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for diagnostic and research purposes.

Isolation of the new pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for diagnostic and research purposes including assessment of novel therapeutics. Several primary and continuous cell lines are currently used, and new organoid and engineered cell lines are being developed for improved investigation and understanding of the human immune response to this virus. Here we review the growth of SARS-CoV-2 in reference standard cell lines, engineered cell lines and new developments in this field.

Evaluation of Commercially Available Viral Transport Medium (VTM) for SARS-CoV-2 Inactivation and Use in Point-of-Care (POC) Testing.

Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore® Molecular Transport Medium (MTM) and COPAN eNAT™ completely inactivate high-titre SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent.

SARS-CoV-2 in human milk is inactivated by Holder pasteurisation but not cold storage.

AIM: As the COVID-19 pandemic evolves, human milk banks world-wide continue to provide donor human milk to vulnerable infants who lack access to mother's own milk. Under these circumstances, ensuring the safety of donor human milk is paramount, as the risk of vertical transmission of SARS-CoV-2 is not fully understood. Here, we investigate the inactivation of SARS-CoV-2 in human milk by pasteurisation and the stability of SARS-CoV-2 in human milk under cold storage. METHODS: SARS-CoV-2 was experimentally inoculated into human milk samples from healthy donors or into a control medium. Triplicates of each sample were layered onto uninfected cells after Holder pasteurisation (63°C for 30 min), heating to 56°C for 30 min, or after 48 h of storage at 4°C or -30°C. Infectious titres of virus were determined at 72 h post-infection by endpoint titration. RESULTS: Following heating to 63°C or 56°C for 30 min, replication competent (i.e. live) SARS-CoV-2 was undetected in both human milk and the control medium. Cold storage of SARS-CoV-2 in human milk (either at 4°C or -30°C) did not significantly impact infectious viral load over a 48 h period. CONCLUSION: SARS-CoV-2 is effectively inactivated by Holder pasteurisation, suggesting that existing milk bank processes will effectively mitigate the risk of transmission of SARS-COV-2 to vulnerable infants through pasteurised donor human milk. The demonstrated stability of SARS-CoV-2 in refrigerated or frozen human milk may assist in the development of guidelines around safe expressing and storing of milk from COVID-19 infected mothers.