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Objectives: PICOBOO is a randomised, adaptive trial evaluating the immunogenicity, reactogenicity, and safety of COVID-19 booster strategies. We report data for second boosters among individuals 50-<70 years old primed with AZD1222 (50-<70y-AZD1222) until Day 84., Methods: Contributed equally as first authors.Immunocompetent adults who received any first booster >three months prior were eligible. Participants were randomly allocated to BNT162b2, mRNA-1273 or NVX-CoV2373 1:1:1. The concentrations of ancestral anti-spike immunoglobulin was summarised as the geometric mean concentrations (GMC). Reactogenicity and safety outcomes were captured. Additional analyses including neutralising antibodies were performed on a subset. ACTRN12622000238774., Results: Between Mar 2022-Aug 2023, 743 participants were recruited and had D28 samples; 155 belonged to the 50-<70y-AZD1222 stratum. The mean adjusted GMCs (95% credible intervals) were 20,690 (17,555-23,883), 23,867 (20,144-27,604) and 8,654 (7,267-9,962) U/mL at D28 following boosting with BNT162b2, mRNA-1273 and NVX-CoV2372, respectively, and 10,976 (8,826-13,196), 15,779 (12,512-19,070) and 6,559 (5 220-7 937) U/mL by D84. IgG against Omicron BA.5 was 2.7-2.9 times lower than the ancestral strain. Limited neutralisation against Omicron subvariants was found following all vaccines. Severe reactogenicity events were <4%., Conclusions: All vaccines were immunogenic with more rapid waning after mRNA vaccines. These data support boosting with vaccines with greater specificity for circulating Omicron subvariants., Competing Interests: Declaration of Competing Interest KF and TS are members of the Australian Technical Advisory Group on Immunisation (ATAGI) which advises the government on vaccine policy; their involvement as investigators on this trial has been declared to ATAGI. MP is involved in an ovarian cancer clinical trial that received funding from AstraZeneca. MP was involved in performing immunological assays on biological specimens obtained from participants in this trial, but was not involved in participant recruitment, data collection or the analysis of results. SNF leads the UK National Institute for Health and Care Research funded trial of third and fourth dose COVID-19 boosters. SNF acts on behalf of University Hospital Southampton NHS Foundation Trust, UK as an Investigator and/or providing consultative advice on clinical trials and studies of vaccines funded or sponsored by vaccine manufacturers including Moderna, Sanofi, Janssen, Pfizer, AstraZeneca, GlaxoSmithKline, Novavax, Seqirus, Medimmune, Merck and Valneva vaccines and antimicrobials. UW is an investigator on clinical trials of vaccines funded or sponsored by vaccine manufactures including Moderna, Sanofi, Pfizer, Merck and GlaxoSmithKline. PR also reports acting on behalf of University of Western Australia, as an Investigator and/or providing consultative advice on clinical trials and studies of vaccines funded or sponsored by vaccine manufacturers including Moderna, Sanofi, Janssen, Pfizer, AstraZeneca, GlaxoSmithKline, Novavax, Seqirus, Merck and Clover Biopharmaceutical vaccines. They receive no personal financial payment for this work. The other authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and became the globally dominant variant by January 2022. Authentic virus and pseudovirus systems have shown Omicron spike has an increased dependence on the endosomal pathway for entry., Methods: We investigated the entry mechanisms of Omicron, Delta, and ancestral viruses in cell models that represent different parts of the human respiratory tract, including nasal epithelial cells (hNECs), large-airway epithelial cells (LAECs), small-airway epithelial cells, and embryonic stem cell-derived type II alveolar cells., Findings: Omicron had an early replication advantage in LAECs, while Delta grew to higher titers in all cells. Omicron maintained dependence on serine proteases for entry in all culture systems. While serine protease inhibition with camostat was less robust for Omicron in hNECs, endosomal entry was not enhanced., Conclusions: Our findings demonstrate that entry of Omicron BA.1 SARS-CoV-2 is dependent on serine proteases for entry throughout the respiratory tract., Funding: This work was supported by The Medical Research Future Fund (MRF9200007; K.S., J.M.P.) and the DHHS Victorian State Government grant (Victorian State Government; DJPR/COVID-19; K.S, J.M.P.). K.S. is supported by a National Health and Medical Research Council of Australia Investigator grant (APP1177174)., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Background: SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG 1 F c -fusion protein, and an mRNA encoding a membrane-anchored RBD., Methods: 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 μg, N = 32), mRNA vaccine (10, 20, or 50 μg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29., Clinicaltrials: govNCT05272605., Findings: No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4 + and CD8 + T cell activation., Interpretation: There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains., Funding: Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors., Competing Interests: Declaration of interests The vaccines evaluated in this Phase I study were the result of independent University of Melbourne and Monash University research and development, with funding provided by the Australian Government's Medical Research Future Fund (MRFF), the National Health and Medical Research Council (NHMRC), the Victorian Government (mRNA Victoria) and philanthropic funders. The MF59 for the protein-RBD candidate was donated by CSL Seqirus. One author (S.R.) is an employee of CSL Seqirus and he also has an adjunct (honorary) appointment to the University of Melbourne. G.D., N.G., D.P., D.I.G. are named inventors on 2 provisional patents for the RBD-Fc dimer vaccine in this study. T.M.N. has been a DSMB member for vaccine studies conducted by Moderna, Clover, Novavax, CSL Seqirus, and SK Bioscience Korea, and has received payment for advisory roles on vaccines from AstraZeneca, Moderna, MSD, Sanofi, CSL, and Pfizer. G.D. received salary support from philanthropic funds from IFM Investors Pty Ltd. S.L. received consulting fees from several companies related to HIV research, and honoraria from MSD and Gilead. H.McQ. received consulting fees from Ena Respiratory Pty Ltd. K.S. was a member of a DSMB for a vaccine study in Thailand. H.alW. received consulting and research funding from CSL Seqirus. D.I.G. received research support from CSL., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years., Methods: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 μg, 15 μg, or 45 μg vaccine, or one 45 μg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined., Findings: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4 + T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants., Interpretation: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development., Funding: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government., Competing Interests: Declaration of interests KJC, DW, and PRY report grants from the Medical Research Future Fund of the Australian Government, the Coalition for Epidemic Preparedness Innovations, the Queensland Government, the Paul Ramsay Foundation, the Lott, and the a2 Milk Company; contract research funding and/or consulting fees from ViceBio Pty Ltd; and a patent (PAT-02207-WO-01); KJC and DW report two additional patents (PAT-02387-WO-01, PAT-02432-EP-01). FLM reports owning shares in CSL. JKB reports consultancy fees from The University of Queensland. PT reports grants from The University of Queensland paid to his employer. KS reports grants from the Jack Ma Foundation paid to his institution; travel support from the World Health Organisation; and membership of the WHO Technical Advisory Group on COVID-19 Vaccines (chair) and the Data Safety Monitoring Board for a COVID-19 booster vaccine study in Thailand. TPM reports grants from the Medical Research Future Fund of the Australian Government and the Coalition for Epidemic Preparedness Innovations; honoraria from The University of Melbourne; and travel support from Moderna. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines., Methods: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine., Findings: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5., Interpretation: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial., Funding: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company., Competing Interests: Declaration of interests Two provisional patents (PCT/AU2021/051553 and PCT/AU2023/050093) covering the RBD-Fc vaccines described in this study, and underlying technology, have been submitted through The University of Melbourne, with D.I.G., G.D., N.A.G., D.C.J. and D.F.J.P. as co-inventors. C.W.T. and L.-f.W. are co-inventors of a patent on the surrogate virus neutralization test (sVNT) platform. S.R.L. receives consulting fees from ViiV, Vaxxinity, Esfam, Abbvie, and Gilead. S.R.L. has received honoraria from Merck, Sharpe and Dohme, and from Gilead. K.S. is on a DSMB for a vaccine study in Thailand, and is Chair of the WHO Technical Advisory Group on COVID-19 vaccines (TAG-CO-VAC). T.N. receives research contracts to conduct clinical trials, with funding to institution from Moderna, SanofiPasteur, GSK, Iliad Biotechnologies, Dynavax, Seqirus, Janssen, MSD. T.N. receives consulting fees from GSK, Seqirus, MSD, SanofiPasteur, AstraZeneca, Moderna, BioNet, Pfizer. T.N. serves on DSMBs for Seqirus, Clover, Moderna, Emergent, Serum Institute of India, SK Bioscience Korea, Emergent Biosolutions, Novavax. S.R. is an employee of CSL Seqirus that is a maker of influenza vaccines. D.C.J. is a founder and shareholder of Ena Respiratory. C.Y.W. and W.Z. are shareholders of Ena Respiratory. D.I.G. has received research funding from CSL for an unrelated project. All other authors declare no conflict of interests., (Copyright © 2023. Published by Elsevier B.V.)

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4 + /CD8 + T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. H.A.M. and B.Y.C. are currently consulting for Ena Respiratory. B.W.T. has received research funding from MSD, Seqirus, and Sanofi and is on the advisory board for Moderna, CSL-Behring, and Takeda., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In Australia, seasonal inactivated influenza vaccine is typically offered in April. However, the onset, peak and end of a typical influenza season vary, and optimal timing for vaccination remains unclear. Here, we investigated vaccine-induced antibody response kinetics over 6 months in different age groups., Methods: We conducted a prospective serosurvey among 71 adults aged 18-50 years, 15 community-dwelling ('healthy') and 16 aged-care facility resident ('frail') older adults aged ≥65 years who received the 2018 southern hemisphere vaccines. Sera were collected at baseline, and 1, 2, 4, and 6 months post-vaccination. Antibody titres were measured by haemagglutination inhibition or microneutralisation assays. Geometric mean titres were estimated using random effects regression modelling and superimposed on 2014-2018 influenza season epidemic curves., Results: Antibody titres peaked 1.2-1.3 months post-vaccination for all viruses, declined by 3 months post-vaccination but, notably, persisted above baseline after 6 months in all age groups by 1.3- to 1.5-fold against A(H1N1)pdm09, 1.7- to 2-fold against A(H3N2), 1.7- to 2.1-fold against B/Yamagata and 1.8-fold against B/Victoria. Antibody kinetics were similar among different age groups. Antibody responses were poor against cell-culture grown compared to egg-grown viruses., Conclusions: These results suggest subtype-specific antibody-mediated protection persists for at least 6 months, which corresponds to the duration of a typical influenza season., (© 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens., Competing Interests: Authors PMH and BW are inventors on a provisional patent filing by the Burnet Institute. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wines, Kurtovic, Trist, Esparon, Lopez, Chappin, Chan, Mordant, Lee, Gherardin, Patel, Hartley, Pymm, Cooney, Beeson, Godfrey, Burrell, van Zelm, Wheatley, Chung, Tham, Subbarao, Kent and Hogarth.)

Background: Household studies are crucial for understanding the transmission of SARS-CoV-2 infection, which may be underestimated from PCR testing of respiratory samples alone. We aim to combine the assessment of household mitigation measures; nasopharyngeal, saliva, and stool PCR testing; along with mucosal and systemic SARS-CoV-2-specific antibodies, to comprehensively characterize SARS-CoV-2 infection and transmission in households., Methods: Between March and September 2020, we obtained samples from 92 participants in 26 households in Melbourne, Australia, in a 4-week period following the onset of infection with ancestral SARS-CoV-2 variants., Results: The secondary attack rate was 36% (24/66) when using nasopharyngeal swab (NPS) PCR positivity alone. However, when respiratory and nonrespiratory samples were combined with antibody responses in blood and saliva, the secondary attack rate was 76% (50/66). SARS-CoV-2 viral load of the index case and household isolation measures were key factors that determine secondary transmission. In 27% (7/26) of households, all family members tested positive by NPS for SARS-CoV-2 and were characterized by lower respiratory Ct values than low transmission families (Median 22.62 vs. 32.91; IQR 17.06-28.67 vs. 30.37-34.24). High transmission families were associated with enhanced plasma antibody responses to multiple SARS-CoV-2 antigens and the presence of neutralizing antibodies. Three distinguishing saliva SARS-CoV-2 antibody features were identified according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP)), suggesting that adults and children generate distinct mucosal antibody responses during the acute phase of infection., Conclusion: Utilizing respiratory and nonrespiratory PCR testing, along with the measurement of SARS-CoV-2-specific local and systemic antibodies, provides a more accurate assessment of infection within households and highlights some of the immunological differences in response between children and adults., (© 2022 The Authors. Pediatric Allergy and Immunology published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2 , which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chang, Gleeson, Rawlinson, De Paoli-Iseppi, Zhou, Mordant, Londrigan, Clark, Subbarao, Stinear, Coin and Pitt.)

Importance: The immune response in children with SARS-CoV-2 infection is not well understood., Objective: To compare seroconversion in nonhospitalized children and adults with mild SARS-CoV-2 infection and identify factors that are associated with seroconversion., Design, Setting, and Participants: This household cohort study of SARS-CoV-2 infection collected weekly nasopharyngeal and throat swabs and blood samples during the acute (median, 7 days for children and 12 days for adults [IQR, 4-13] days) and convalescent (median, 41 [IQR, 31-49] days) periods after polymerase chain reaction (PCR) diagnosis for analysis. Participants were recruited at The Royal Children's Hospital, Melbourne, Australia, from May 10 to October 28, 2020. Participants included patients who had a SARS-CoV-2-positive nasopharyngeal or oropharyngeal swab specimen using PCR analysis., Main Outcomes and Measures: SARS-CoV-2 immunoglobulin G (IgG) and cellular (T cell and B cell) responses in children and adults. Seroconversion was defined by seropositivity in all 3 (an in-house enzyme-linked immunosorbent assay [ELISA] and 2 commercial assays: a SARS-CoV-2 S1/S2 IgG assay and a SARS-CoV-2 antibody ELISA) serological assays., Results: Among 108 participants with SARS-CoV-2-positive PCR findings, 57 were children (35 boys [61.4%]; median age, 4 [IQR, 2-10] years) and 51 were adults (28 women [54.9%]; median age, 37 [IQR, 34-45] years). Using the 3 established serological assays, a lower proportion of children had seroconversion to IgG compared with adults (20 of 54 [37.0%] vs 32 of 42 [76.2%]; P < .001). This result was not associated with viral load, which was similar in children and adults (mean [SD] cycle threshold [Ct] value, 28.58 [6.83] vs 24.14 [8.47]; P = .09). In addition, age and sex were not associated with seroconversion within children (median age, 4 [IQR, 2-14] years for both seropositive and seronegative groups; seroconversion by sex, 10 of 21 girls [47.6%] vs 10 of 33 boys [30.3%]) or adults (median ages, 37 years for seropositive and 40 years for seronegative adults [IQR, 34-39 years]; seroconversion by sex, 18 of 24 women [75.0%] vs 14 of 18 men [77.8%]) (P > .05 for all comparisons between seronegative and seropositive groups). Symptomatic adults had 3-fold higher SARS-CoV-2 IgG levels than asymptomatic adults (median, 227.5 [IQR, 133.7-521.6] vs 75.3 [IQR, 36.9-113.6] IU/mL), whereas no differences were observed in children regardless of symptoms. Moreover, differences in cellular immune responses were observed in adults compared with children with seroconversion., Conclusions and Relevance: The findings of this cohort study suggest that among patients with mild COVID-19, children may be less likely to have seroconversion than adults despite similar viral loads. This finding has implications for future protection after SARS-CoV-2 infection in children and for interpretation of serosurveys that involve children. Further research to understand why seroconversion and development of symptoms are potentially less likely in children after SARS-CoV-2 infection and to compare vaccine responses may be of clinical and scientific importance.

Background: Seroprotection and seroconversion rates are not well understood for 2-dose inactivated influenza vaccination (IIV) schedules in autologous hematopoietic stem cell transplantation (autoHCT) patients., Methods: A randomized, single-blind, controlled trial of IIV in autoHCT patients in their first year post-transplant was conducted. Patients were randomized 1:1 to high-dose (HD) IIV followed by standard dose (SD) vaccine (HD-SD arm) or 2 SD vaccines (SD-SD arm) 4 weeks apart. Hemagglutination inhibition (HI) assay for IIV strains was performed at baseline, 1, 2, and 6 months post-first dose. Evaluable primary outcomes were seroprotection (HI titer ≥40) and seroconversion (4-fold titer increase) rates and secondary outcomes were geometric mean titers (GMTs), GMT ratios (GMRs), adverse events, influenza-like illness (ILI), and laboratory-confirmed influenza (LCI) rates and factors associated with seroconversion., Results: Sixty-eight patients were enrolled (34/arm) with median age of 61.5 years, majority male (68%) with myeloma (68%). Median time from autoHCT to vaccination was 2.3 months. For HD-SD and SD-SD arms, percentages of patients achieving seroprotection were 75.8% and 79.4% for H1N1, 84.9% and 88.2% for H3N2 (all P > .05), and 78.8% and 97.1% for influenza-B/Yamagata (P = .03), respectively. Seroconversion rates, GMTs and GMRs, and number of ILI or LCIs were not significantly different between arms. Adverse event rates were similar. Receipt of concurrent cancer therapy was independently associated with higher odds of seroconversion (OR, 4.3; 95% CI, 1.2-14.9; P = .02)., Conclusions: High seroprotection and seroconversion rates against all influenza strains can be achieved with vaccination as early as 2 months post-autoHCT with either 2-dose vaccine schedules., Clinical Trials Registration: Australian New Zealand Clinical Trials Registry: ACTRN12619000617167., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing., Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques., Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test., Interpretation: Taken together, the COVID-19 NAb-test TM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care., Funding: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation., Competing Interests: Declaration of Competing Interest A provisional patent covering the COVID-19 NAb-test(TM) test and underlying technology has been submitted through The University of Melbourne. Dr. Fulford reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Van reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Mr. Van has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Dr. Gherardin reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Ms. Zheng reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Ms. Zheng has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Ciula has nothing to disclose. Prof. Drummer has nothing to disclose. Mr. Redmond has nothing to disclose. Dr. Tan has nothing to disclose. Ms. Boo has nothing to disclose. Dr. Center has nothing to disclose. Dr. Li has nothing to disclose. Dr. Grimley has nothing to disclose. Dr. Wines reports grants from Australian Govt, Medical Research Future Fund, grants from NHMRC, National Health and Medical Research Council GNT1145303, Australia, during the conduct of the study; In addition, Dr. Wines has a patent drafted for use of ACE2-Fc pending. Dr. Nguyen has nothing to disclose. Ms. Mordant has nothing to disclose. Dr. Ellenberg has nothing to disclose. Dr. Rowntree has nothing to disclose. Lukasz Kedzierski has nothing to disclose. Prof. Cheng reports that he is a member of government advisory committees advising on COVID policy. Prof. Doolan has nothing to disclose. Prof. Matthews reports grants from Curran Foundation, during the conduct of the study; grants from Gilead sciences, grants from Abbvie Inc outside the submitted work. Dr. Bond has nothing to disclose. Prof. Hogarth reports grants from NHMRC, grants from MRFF, during the conduct of the study; In addition, Prof. Hogarth has a patent Antiviral pending. A/Prof. McQuilten reports grants from Medical Research Future Fund, during the conduct of the study. Prof. Subbarao has nothing to disclose. Prof. Kedzierska has nothing to disclose. Dr. Juno has nothing to disclose. Dr. Wheatley has nothing to disclose. Prof. Kent has nothing to disclose. Prof. Williamson has nothing to disclose. Prof. Purcell reports grants from Victorian Government DHHS, grants from Medical Research Future Fund, other from Jack Ma Foundation, during the conduct of the study; In addition, Prof. Purcell has a patent PCT/AU2021/050839 pending. Prof. Anderson reports grants from Government of the State of Victoria, during the conduct of the study; other from Nanjing BioPoint Diagnostic Technology, outside the submitted work; In addition, Prof. Anderson has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Prof. Godfrey reports grants from Victorian Government DHHS, and from Australian Research Council, during the conduct of the study; grants from Medical Research Future Fund, other from Jack Ma Foundation, grants from National Health and Medical Research Council, outside the submitted work; In addition, Prof. Godfrey has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending., (Copyright © 2021. Published by Elsevier B.V.)

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests. A provisional patent covering human mAbs isolated from convalescent donors has been submitted through the University of Melbourne. A provisional patent covering human mAbs isolated from phage display has been submitted through the Walter and Eliza Hall Institute., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp])., Methods: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 μg, 15 μg, or 45 μg, or one dose of sclamp vaccine at 45 μg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933., Findings: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 μg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 μg dose (7492, 4959-11 319), and the two 45 μg dose cohorts (8770, 5526-13 920 in the two-dose 45 μg cohort; 8793, 5570-13 881 in the single-dose 45 μg cohort); 4 weeks after the second dose (day 57) with two 5 μg doses (102 400, 64 857-161 676), with two 15 μg doses (74 725, 51 300-108 847), with two 45 μg doses (79 586, 55 430-114 268), only a single 45 μg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 μg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 μg doses (GMT 228, 95% CI 146-356), two 15 μg doses (230, 170-312), and two 45 μg doses (239, 187-307)., Interpretation: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response., Funding: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2-RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8 + T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 + T cells directed toward subdominant epitopes (B7/N 257 , A2/S 269 , and A24/S 1,208 ) CD8 + T cells specific for the immunodominant B7/N 105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 + T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N 105 + CD8 + T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N 105 -specific CD8 + T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle., Competing Interests: Declaration of interests L.J.M.C., M.E.P., J.G., R.D.P.-I., and M.B.C. have received support from ONT to present their findings at scientific conferences. ONT played no role in study design, execution, analysis, or publication. L.J.M.C. has received research funding from ONT unrelated to this project., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 10 4 -fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2., Competing Interests: Competing interest statement: P.P., A.A., and W.-H.T. are inventors on a provisional patent covering the nanobodies described in this manuscript., (Copyright © 2021 the Author(s). Published by PNAS.)

Objectives: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia)., Methods: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat., Results: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4 + and cytotoxic CD8 + T cells in vivo . In the Syrian hamster challenge model ( n  = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level., Conclusion: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models., Competing Interests: KJC, DW and PRY are inventors of the ‘Molecular Clamp’ patent, US 2020/0040042., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3 + cT FH 1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assess prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD is associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augments neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines are comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

Objectives: As the world transitions into a new era of the COVID-19 pandemic in which vaccines become available, there is an increasing demand for rapid reliable serological testing to identify individuals with levels of immunity considered protective by infection or vaccination., Methods: We used 34 SARS-CoV-2 samples to perform a rapid surrogate virus neutralisation test (sVNT), applicable to many laboratories as it circumvents the need for biosafety level-3 containment. We correlated results from the sVNT with five additional commonly used SARS-CoV-2 serology techniques: the microneutralisation test (MNT), in-house ELISAs, commercial Euroimmun- and Wantai-based ELISAs (RBD, spike and nucleoprotein; IgG, IgA and IgM), antigen-binding avidity, and high-throughput multiplex analyses to profile isotype, subclass and Fc effector binding potential. We correlated antibody levels with antibody-secreting cell (ASC) and circulatory T follicular helper (cTfh) cell numbers., Results: Antibody data obtained with commercial ELISAs closely reflected results using in-house ELISAs against RBD and spike. A correlation matrix across ten measured ELISA parameters revealed positive correlations for all factors. The frequency of inhibition by rapid sVNT strongly correlated with spike-specific IgG and IgA titres detected by both commercial and in-house ELISAs, and MNT titres. Multiplex analyses revealed strongest correlations between IgG, IgG1, FcR and C1q specific to spike and RBD. Acute cTfh-type 1 cell numbers correlated with spike and RBD-specific IgG antibodies measured by ELISAs and sVNT., Conclusion: Our comprehensive analyses provide important insights into SARS-CoV-2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS-CoV-2-specific humoral responses., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

The durability of infection-induced SARS-CoV-2 immunity has major implications for reinfection and vaccine development. Here, we show a comprehensive profile of antibody, B cell and T cell dynamics over time in a cohort of patients who have recovered from mild-moderate COVID-19. Binding and neutralising antibody responses, together with individual serum clonotypes, decay over the first 4 months post-infection. A similar decline in Spike-specific CD4 + and circulating T follicular helper frequencies occurs. By contrast, S-specific IgG + memory B cells consistently accumulate over time, eventually comprising a substantial fraction of circulating the memory B cell pool. Modelling of the concomitant immune kinetics predicts maintenance of serological neutralising activity above a titre of 1:40 in 50% of convalescent participants to 74 days, although there is probably additive protection from B cell and T cell immunity. This study indicates that SARS-CoV-2 immunity after infection might be transiently protective at a population level. Therefore, SARS-CoV-2 vaccines might require greater immunogenicity and durability than natural infection to drive long-term protection.

The duration of the humoral immune response in children infected with severe acute respiratory syndrome coronavirus 2 is unknown. We detected specific IgG 6 months after infection in children who were asymptomatic or had mild symptoms of coronavirus disease. These findings will inform vaccination strategies and other prevention measures.

Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly mild or asymptomatic infections, but the underlying immunological differences remain unclear. Here, we describe clinical features, virology, longitudinal cellular, and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who tested repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children are similar to their parents at all timepoints. All family members have salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincide with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child have IgG antibody against the S1 protein and virus-neutralizing activity detected. Using a systems serology approach, we demonstrate higher levels of SARS-CoV-2-specific antibody features of these family members compared to healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological confirmation of infection, raising the possibility that immunity in children can prevent the establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may not identify exposed children, with implications for epidemiological and clinical studies across the life-span.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts 1-3 , most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2) 4-6 . Eliciting neutralizing antibodies that block S-ACE2 interaction 7-9 , or indirectly prevent membrane fusion 10 , constitute an attractive modality for vaccine-elicited protection 11 . However, although prototypic S-based vaccines show promise in animal models 12-14 , the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.