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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.

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions.In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

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.

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.

Although ACE2 is the primary receptor for 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 aTLR-related cell surface receptor calledleucine-rich repeat-containing protein 15(LRRC15).LRRC15expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex.LRRC15mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe 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 intrans. 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.

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 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., Graphical Abstract, 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.

Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 exist, with evidence of antibody titres 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 six months after infection. While antibody titres are maintained, about 13% of the cohort’s neutralising 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 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire., Graphical Abstract, Abayasingam et al. report that despite the declining anti-RBD antibody titres and neutralising capacity of antibodies in the serum at six months, the memory B cells still contain RBD-specific reactivity that have the capacity to generate antibodies that can neutralise SARS-CoV-2 in vitro.

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.

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.

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 in50% 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.

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 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre 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 neutralising 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 titres against multiple SARS-CoV-2 variants; notably high titres 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.

Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for prediction of protection against reinfection. In a cohort of 24 participants, the association of disease severity and early immunological measurements on the maintenance of humoral immune responses 12 months post-infection were examined. All severely affected participants maintained a stable subset of SARS-CoV-2 receptor-binding domain (RBD) specific memory B cells (MBCs) and good neutralising antibody breadth against the majority of the variants of concern, including the Delta variant. Modelling these immune responses on vaccine efficacy data indicated a level equivalent to a vaccine efficacy of approximately 45-76% against symptomatic reinfection (variant dependent). Overall, these findings indicate durable humoral responses in most participants, provide an estimate of the level of protection and identifies the magnitude and phenotype of baseline antigen-specific CD4+ T cell response as a predictor of maintenance of both antibody neutralisation breadth and RBD-specific MBC levels at 12 months post-infection. Funding: The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication do not necessarily represent the position of the Australian Government. Research reported in this publication was supported by Snow Medical Foundation as an investigator-initiated study. The content is solely the responsibility of the authors. RAB, MM, CR and ARL are fellows funded by National Health and Medical Research Council (NHMRC). MWAC is in part funded by the Research Infrastructure Programme of UNSW. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District and the University of New South Wales, NSW Australia (ETH00520) and was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines and local regulatory requirements. Written informed consent was obtained from all participants before study procedures.

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. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of 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 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50μg doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

The SARS-CoV-2 antibody neutralization response and its evasion by emerging viral variants are unknown. 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 RT-PCR-confirmed COVID-19 individuals with detailed demographics and followed up to seven months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization were associated with COVID-19 severity. A subgroup of ‘high responders’ maintained high neutralizing responses over time, representing ideal convalescent plasma therapy donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal plasma donors and vaccine monitoring and design.One Sentence SummaryNeutralizing antibody responses to SARS-CoV-2 are sustained, associated with COVID19 severity, and evaded by emerging viral variants

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 stabilized, 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 neutralizing 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 neutralized 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.

BackgroundSerological 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.MethodsSera from individuals recovered from 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.ResultsNeutralising 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.ConclusionsThese 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 demonstrate 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.

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.

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.

As the COVID-19 pandemic evolves, human milk banks worldwide 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 well 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 (freezing or refrigeration). Following heating to 63°C or 56°C for 30 minutes, SARS-CoV-2 replication competent (i.e. live) virus 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 hour period. Our findings demonstrate that SARS-CoV-2 can be effectively inactivated by Holder pasteurisation, and confirm that existing milk bank processes will effectively mitigate the risk of transmission of SARS-COV-2 to vulnerable infants through pasteurised donor human milk.

Introduction Lymphoid malignancies are a risk factor for severe COVID-19. Vaccination with BNT162b2 protects the general population from severe disease, but recent studies have shown limited seroconversion after vaccination in patients with lymphoid malignancy. This reduced response is likely related to disease and treatment factors altering both humoral and cellular immunity. Assessing response in patients with the indolent lymphomas, Waldenström's Macroglobulinaemia (WM) and Follicular Lymphoma (FL), including cohorts on differing treatment regimens, may help elucidate some of these factors. Australia has had low prevalence of SARS-CoV-2 infection to date, affording a unique opportunity to assess efficacy to vaccination without the confounding impact of endemic infection. Methods Patients with WM and FL and controls were enrolled in a prospective study of immune response after two doses of BNT162b2 administered 21 days apart. The study had Human Research Ethics Committee approval and all patients gave informed consent prior to participation. Recruitment was targeted to obtain comparable proportions of controls to treatment cohorts. PBMC and sera were collected from participants immediately prior to the first dose (T1), at day 21 immediately prior to the second dose (T2), and day 49 (+/- 7d) (T3). Immune response was measured by: flow cytometric detection of anti-SARS-CoV-2 spike antibodies (ASAb), performed using our recently validated flow cytometric live cell assay (Tea et.al. PLoS Medicine 2021) with increased sensitivity compared to currently available commercial ELISAs; live virus neutralisation to a panel of SARS-CoV-2 variants of concern; and CD4+and CD8+ antigen-specific T cell responses. Statistical analysis of medians between cohorts were compared by the Mann-Whitney non-parametric test using Graphpad Prism. Initial ASAb IgG data for T1 and T2 is presented here. Complete immune response data at all time points will be available for the ASH meeting. Results Eighty-five participants received their first dose of BNT162b2 from 18 May 2021 to 7 June 2021 with a second dose 21 days later: 72 lymphoma patients (WM and FL) and 13 age-matched healthy volunteers (controls). Of 37 with WM [19 (51.3%) female, median 71 years (IQR 63-74)] 9 were treatment naïve (WMN), 15 had received rituximab-chemotherapy (WMT), and 13 were currently treated with a Bruton Tyrosine Kinase inhibitor (BTKi): 5 ibrutinib, 8 zanubrutinib (WMB). Of 35 patients with FL [16 (45.7%) female, median 65 years (IQR 54-71)], 11 were treatment naïve (FLN), and 24 had received rituximab-chemotherapy (FLT). Of the 13 controls 8 (61.5%) were female, median age 72 years (IQR 57-74)]. No participants had detectable ASAb at T1, confirming no prior SARS-CoV-2 exposure. Figure 1 shows ASAb results at T2. The median mean fluorescence intensity (MFI) of healthy controls: 60802 (IQR 17565 -78443), is higher than all WM: 0 (IQR 0 - 15010) p Conclusion These early serological data show measurable ASAb in all healthy controls 21 days post first dose of BNT162b2 vaccination. Treatment naïve patients had a better response than treated patients, and this did not differ significantly to healthy controls in the FL cohort. In WM, patients on BTKi had a significantly reduced response compared to treatment-naïve patients. This same reduction was not observed in the chemotherapy-rituximab cohort, but the characteristics of early responders versus non-responders, including time since last therapy, is being analysed. FL patients treated with chemotherapy-rituximab had a significantly reduced response compared to the treatment-naïve cohort. Time constraints before the abstract submission deadline prevented reporting of all mature vaccination response data. Measurement of ASAb one month after second vaccination, live virus neutralisation to a panel of SARS-CoV-2 variants of concern, and CD4+ and CD8+ antigen-specific T cell responses at T1, T2 and T3, to fully characterise the immune response to BNT162b2, will be reported at the ASH meeting. Figure 1 Figure 1. Disclosures Rankin: Novo Nordisk Pharmaceuticals: Other: Travel reimbursement to attend haemophilia academy. Kelleher: CSL/Sequiris: Honoraria; Aegros and CSL: Consultancy; Merck: Speakers Bureau; Aegros, CSL, Merek: Membership on an entity's Board of Directors or advisory committees. Brilot: Novartis: Research Funding; Biogen, Merck: Honoraria. Trotman: beigene: Research Funding; roche: Research Funding; PCYC: Research Funding; BMS: Research Funding; TAKEDA: Research Funding; JANSSEN: Research Funding.

F-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, provided Cdc42 and IQGAP1 were present. From these observations we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.