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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.

Serological assays for SARS-CoV-2 infection are now widely available for use in diagnostic laboratories. Limited data are available on the performance characteristics in different settings, and at time periods remote from the initial infection. Validation of the Abbott (Architect SARS-CoV-2 IgG), DiaSorin (Liaison SARS-CoV-2 S1/S2 IgG) and Roche (Cobas Elecsys Anti-SARS-CoV-2) assays was undertaken utilising 217 serum samples from 131 participants up to 7 months following COVID-19 infection. The Abbott and DiaSorin assays were implemented into routine laboratory workflow, with outcomes reported for 2764 clinical specimens. Sensitivity and specificity were concordant with the range reported by the manufacturers for all assays. Sensitivity across the convalescent period was highest for the Roche at 95.2-100% (95% CI 81.0-100%), then the DiaSorin at 88.1-100% (95% CI 76.0-100%), followed by the Abbott 68.2-100% (95% CI 53.4-100%). Sensitivity of the Abbott assay fell from approximately 5 months; on this assay paired serum samples for 45 participants showed a significant drop in the signal-to-cut-off ratio and 10 sero-reversion events. When used in clinical practice, all samples testing positive by both DiaSorin and Abbott assays were confirmed as true positive results. In this low prevalence setting, despite high laboratory specificity, the positive predictive value of a single positive assay was low. Comprehensive validation of serological assays is necessary to determine the optimal assay for each diagnostic setting. In this low prevalence setting we found implementation of two assays with different antibody targets maximised sensitivity and specificity, with confirmatory testing necessary for any sample which was positive in only one assay.

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.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.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.These results suggest subtype-specific antibody-mediated protection persists for at least 6 months, which corresponds to the duration of a typical influenza season.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics., Antibody responses to SARS-CoV-2 are critical in the immune response to infection, but the potential cross-reactivity to other human corona viruses is poorly appreciated. Here the authors apply a systems based approach to characterise the antibody response in pre-pandemic cohorts and assess heterotypic reactivity to SARS-CoV-2.

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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts1-3, 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 interaction7-9, or indirectly prevent membrane fusion10, constitute an attractive modality for vaccine-elicited protection11. However, although prototypic S-based vaccines show promise in animal models12-14, 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.

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication.One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

The immune response in children with SARS-CoV-2 infection is not well understood.To compare seroconversion in nonhospitalized children and adults with mild SARS-CoV-2 infection and identify factors that are associated with seroconversion.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.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.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.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.

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.

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

ImportanceThe immune response in children with SARS-CoV-2 infection is not well understood.ObjectiveTo compare seroconversion in children and adults with non-hospitalized (mild) SARS-CoV-2 infection and to understand the factors that influence this.DesignParticipants were part of a household cohort study of SARS-CoV-2 infection. Weekly nasopharyngeal/throat swabs and blood samples were collected during the acute and convalescent period following PCR diagnosis for analysis.SettingParticipants were recruited at the Royal Children’s Hospital, Melbourne, Australia between May and October 2020.ParticipantsThose who had a SARS-CoV-2 PCR-positive nasal/throat swab.Main outcomes and measuresSARS-CoV-2 antibody and cellular responses in children and adults. Seroconversion was defined by seropositivity in all three serological assays.ResultsAmong 108 SARS-CoV-2 PCR-positive participants, 57 were children (median age: 4, IQR 2-10) and 51 were adults (median age: 37, IQR 34-45). Using three established serological assays, a lower proportion of children seroconverted compared with adults [20/54 (37.0%) vs 32/42 (76.2%); (pConclusion and RelevanceIn this non-hospitalized cohort with mild COVID-19, children were less likely to seroconvert than adults despite similar viral loads. This has implications for future protection following COVID-19 infection in children and for interpretation of serosurveys that involve children. Further research to understand why children are less likely to seroconvert and develop symptoms following SARS-CoV-2 infection, and comparison with vaccine responses may be of clinical and scientific importance.Key pointsQuestionWhat proportion of children with non-hospitalized (mild) SARS-CoV-2 infection seroconvert compared to adults?FindingsIn this cohort study conducted in 2020, we found the proportion of children who seroconverted to SARS-CoV-2 was half that in adults despite similar viral load.MeaningSerology is a less reliable marker of prior SARS-CoV-2 infection in children. SARS-CoV-2-infected children who do not seroconvert may be susceptible to reinfection. Our findings support strategies to protect children against COVID-19 including vaccination.

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.

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.

Significance Neutralizing antibodies are important for immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and as therapeutics for the prevention and treatment of COVID-19. We identified high-affinity nanobodies against SARS-CoV-2 receptor-binding domain and found that nanobody cocktails consisting of two noncompeting nanobodies were able to block ACE2 engagement with RBD variants present in human populations and potently neutralize both wild-type SARS-CoV-2 and the N501Y D614G variant at low concentrations. Prophylactic administration of nanobody cocktails reduced viral loads in mice infected with the N501Y D614G SARS-CoV-2 virus, showing that nanobody cocktails are useful as prophylactic agents against SARS-CoV-2., 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 104-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.

As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus; whether through infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure SARS-CoV-2 immunity, ideally with rapid turnaround and without the need for laboratory-based testing. Current rapid point-of-care (POC) tests measure antibodies (Ab) against the SARS-CoV-2 virus, however, these tests provide no information on whether the antibodies can neutralise virus infectivity and are potentially protective, especially against newly emerging variants of the virus. Neutralising Antibodies (NAb) are emerging as a strong correlate of protection, but most current NAb assays require many hours or days, samples of venous blood, and access to laboratory facilities, which is especially problematic in resource-limited settings. We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibodies from whole blood, with a result that can be determined by eye (semi-quantitative) or on a small instrument (quantitative), and results show high correlation with microneutralisation assays. This assay also provides a measure of total anti-RBD antibody, thereby providing evidence of exposure to SARS-CoV-2 or immunisation, regardless of whether NAb are present in the sample. By testing samples from immunised macaques, we demonstrate that this test is equally applicable for use with animal samples, and we show that this assay is readily adaptable to test for immunity to newly emerging SARS-CoV-2 variants. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (R2 =0.75, p

SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found 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. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/., Graphical Abstract, SARS-CoV-2 is the pathogen which is responsible for the global COVID-19 pandemic. Chang et al. demonstrate that the transcriptome of SARS-CoV-2 is dynamic and complex, with expression and relative proportions of viral mRNA changing to reflect the stage of infection in vitro. In contrast, the epi-transcriptome is stable throughout infection.

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 that immune escape can occur through two mechanisms, antibodies that fail to recognize mutations, along with antibodies that have reduced inhibitory capacity due to enhanced variant RBD-ACE2 affinity. A competitive approach where antibodies simultaneously compete with ACE2 for binding to the RBD may therefore more accurately reflect the physiological dynamics of infection. 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.

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., Current vaccine strategies for SARS-CoV-2 focus on eliciting neutralising antibodies to the spike protein (S), but differences in immunogenicity of full-length S versus receptor binding domain (RBD) only aren’t fully understood. Here, the authors show immunogenicity of different prime-boost strategies with S and/or RBD in mice and macaques.

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., Longitudinal analyses are needed to show how the immune response to Sars-Cov-2 infection changes over time. Here, the authors use multiple strategies to profile the change in immune cell responses from patients with convalescent COVID-19 over the course of ~5 months, showing that although neutralizing antibody responses drop off after ~4 months, B cell immune responses strengthen.

BackgroundSerological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. As we move into the era of vaccines, the detection of neutralising antibody will become increasingly important. Many serological tests have been developed under emergency use authorization, but their reliability remains unclear.MethodsWe evaluated the performance of six commercially-available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test, for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or neutralising antibodies and a subset of results were compared to microneutralisation.ResultsFor sera collected > 14 days post-symptom onset the Wantai total Ab performed best with highest sensitivity 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG,93.1% for GenScript Surrogate Virus Neutralization Test, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA and 83.3% for Wantai IgM. Specificity for the best performing assay was 99.5% and for the lowest 97.1%.ConclusionWantai ELISA, detecting total immunoglobulins against SARS-CoV-2 receptor binding domain, had the best performance. Antibody target, timing and longevity of the immune response, and the objectives of testing should be considered in test choice. ELISAs should be used within a confirmatory testing algorithm to ensure reliable results. ELISAs provide high quality results, with flexibility for test numbers without the need for manufacturer specific analyzers.

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.

Background Serological testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the coronavirus disease 2019 (COVID-19) pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. Methods We evaluated the performance of 6 commercially available enzyme-linked immunosorbent assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared with a cell culture–based microneutralization (MN) assay. We tested sera from patients with prior reverse transcription polymerase chain reaction–confirmed SARS-CoV-2 infection, prepandemic sera, and potential cross-reactive sera from patients with other non-COVID-19 acute infections. Results For sera collected >14 days post–symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% CI, 94.6%–100%), followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA, and 83.3% for Wantai IgM. Specificity for the best-performing assay was 99.5% for the Wantai total Ab, and for the lowest-performing assay it was 97.1% for sVNT (as per the Instructions for Use [IFU]). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG, and sVNT (as per IFU) with 97%, 97% and 95%, respectively; Wantai IgM had the poorest agreement at 93%. Conclusions Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture–based neutralization assay showed good result correlation between all assays. However, correlation between the cell-based neutralization test and some assays detecting Ab’s not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimize serological test algorithms for assessing antibody responses post–SARS-CoV-2 infection or vaccination.

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., We describe robust correlations across six SARS‐CoV‐2 serology assays detecting distinct antibody features. 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.

Much of our current understanding about novel coronavirus disease 2019 (COVID-19) comes from hospitalised patients. However, the spectrum of mild and subclinical disease has implications for population-level screening and control. Forty-nine participants were recruited from a group of 99 adults repatriated from a cruise ship with a high incidence of COVID-19. Respiratory and rectal swabs were tested by polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sera were tested for anti-SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and microneutralisation assay. Symptoms, viral shedding and antibody response were examined. Forty-five participants (92%) were considered cases based on either positive PCR or positive ELISA for immunoglobulin G. Forty-two percent of cases were asymptomatic. Only 15% of symptomatic cases reported fever. Serial respiratory and rectal swabs were positive for 10% and 5% of participants respectively about 3 weeks after median symptom onset. Cycle threshold values were high (range 31–45). Attempts to isolate live virus were unsuccessful. The presence of symptoms was not associated with demographics, comorbidities or antibody response. In closed settings, incidence of COVID-19 could be almost double that suggested by symptom-based screening. Serology may be useful in diagnosis of mild disease and in aiding public health investigations.

Background: We assessed the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a prefusion conformation by a novel molecular clamp (Sclamp). Methods: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020–ongoing; ClinicalTrials.gov NCT04495933). Healthy adults (18-55 years) received two doses of placebo, 5-μg, 15-μg, or 45-μg SARS-CoV-2 Sclamp, or one 45-μg dose of SARS-CoV-2 Sclamp followed by placebo, 28 days apart (n=120; 24 per group). Safety, humoral immunogenicity (ELISA, microneutralisation, pseudovirus neutralisation), and cellular immunogenicity (antigen-specific CD4+/CD8+ T-cells, antibody-secreting cells) were assessed up to 56 days after the first dose. Findings: The SARS-CoV-2 Sclamp vaccine was very well tolerated with few systemic reactions. All two-dose regimens elicited robust, broadly neutralising humoral responses. Geometric mean titres were higher than in sera from convalescent COVID-19 patients and strongly neutralised spike variants of concern, including N501Y. Moreover, humoral and cellular responses were highly correlated. However, antibodies elicited to a peptide sequence used in the molecular clamp derived from human immunodeficiency virus-1 (HIV-1) gp41 cross-reacted weakly with some HIV diagnostic screening tests. Interpretation: These first-in-human results demonstrate that a subunit vaccine comprising mammalian cell culture-derived, molecular clamp-stabilised recombinant spike protein formulated in a squalene-in-oil adjuvant elicits strong immune responses with an excellent safety profile. However, the gp41 peptide induced diagnostic interference, creates a likely barrier to widespread use and highlights the criticality of potential off-target immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. Clinical Trial Registration: ClinicalTrials.gov (NCT04495933). Funding: Coalition for Epidemic Preparedness Innovations; National Health and Medical Research Council, Queensland Government, and philanthropic sources. Declaration of Interests: KJC and DW report grants from the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government, during the conduct of the study; other from ViceBio Limited, outside the submitted work; and has patents pending (AU 2018241252; BR112019019813.0; CA 3057171; CH 201880022016.9; EP 18775234.0; IN 201917038666; ID P00201909145; IL 269534; JP 2019-553883; MX/a/2019/011599; NZ 757178; KR 0-2019-7031415; SG 11201908280S; US 16/498865). JB reports personal fees from CSL Limited, during the conduct of the study, and other from CSL Limited, outside the submitted work. WZ reports grants from the National Health and Medical Research Council of Australia, the Research Grants Council of the Hong Kong Special Administrative Region, China, and the Jack Ma Foundation, during the conduct of the study. SM-H reports grants from Canarian Foundation Doctor Manuel Morales, during the conduct of the study. KJS reports grants from the the Australian Medical Research Future Fund, during the conduct of the study. AWC reports grants from the Australian Medical Research Future Fund and a National Health and Medical Research Council of Australia Career Development Fellowship, during the conduct of the study. BDW reports grants from the National Health and Medical Research Council of Australia, the Australian Medical Research Future Fund, and the Victorian State Government, during the conduct of the study. PMH reports grants from the Australian Medical Research Future Fund, during the conduct of the study. DP reports grants from the National Health and Medical Research Council of Australia, the A2 Milk Foundation, and the Jack Ma Foundation, during the conduct of the study. CR reports grants from the Coalition for Epidemic Preparedness Innovations, during the conduct of the study. PRY reports grants from the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government, during the conduct of the study; grants from ViceBio Limited, outside the submitted work; and a patent issued (US 2020/0040042). FLM, Zl, DKW, PE, JAL, STMC, NM, SA, CLH, KH, PG, LH, THON, MHT, PT, JB, PCR, SN, SC, TH, KK, KS, and TPM have nothing to disclose. Ethics Approval Statement: The protocol was approved by the Alfred Health Human Research Ethics Committee (2020001376/334/20).

To better understand primary and recall T cell responses during COVID-19, it is important to examine unmanipulated SARS-CoV-2-specific T cells. Using peptide-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 towards subdominant epitopes – B7/N257, A2/S269 and A24/S1208 – CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequency in pre-pandemic samples, and at increased frequency 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 naïve 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/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells, and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses., Graphical Abstract, Examining unmanipulated SARS-CoV-2-specific T cells is important for understanding primary and recall responses during COVID-19. Nguyen et al. analyze ex vivo CD8+ T cells specific for SARS-CoV-2 epitopes and find that immunodominant B7/N105-specific CD8+ T cells are present at high frequencies in blood samples from unexposed, acute COVID-19, and convalescence individuals, which is underpinned by diverse TCR repertoires.

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., Children with SARS-CoV-2 infection are more likely to have mild symptoms and may be asymptomatic, but underlying reasons remain unclear. Here, the authors show cellular, cytokine and antibody response to SARS-CoV-2 infection in three children who repeatedly tested negative for the virus by PCR, despite high exposure in the household.

Efforts to develop and deploy effective vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continue at pace with more than 30 candidate vaccines now in clinical evaluation. Here we describe the preclinical development of an adjuvanted, prefusion-stabilised Spike (S) protein “Sclamp” subunit vaccine, from rational antigen design through to assessing manufacturability and vaccine efficacy. In mice, the vaccine candidate elicits high levels of neutralising antibodies to epitopes both within and outside the receptor binding domain (RBD) of S, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells. We also show protection in Syrian hamsters, which has emerged as a robust animal model for pulmonary SARS-CoV-2 infection. No evidence of vaccine enhanced disease was observed in animal challenge studies and pre-clinical safety was further demonstrated in a GLP toxicology study in rats. The Sclamp vaccine candidate is currently progressing rapidly through clinical evaluation in parallel with large-scale manufacture for pivotal efficacy trials and potential widespread distribution.

The durability of infection-induced SARS-CoV-2 immunity has major implications for public health mitigation and vaccine development. Animal studies1,2and the scarcity of confirmed re-infection3suggests immune protection is likely, although the durability of this protection is debated. Lasting immunity following acute viral infection requires maintenance of both serum antibody and antigen-specific memory B and T lymphocytes and is notoriously pathogen specific, ranging from life-long for smallpox or measles4, to highly transient for common cold coronaviruses (CCC)5. Neutralising antibody responses are a likely correlate of protective immunity and exclusively recognise the viral spike (S) protein, predominantly targeting the receptor binding domain (RBD) within the S1 sub-domain6. Multiple reports describe waning of S-specific antibodies in the first 2-3 months following infection7-12. However, extrapolation of early linear trends in decay might be overly pessimistic, with several groups reporting that serum neutralisation is stable over time in a proportion of convalescent subjects8,12-17. While SARS-CoV-2 specific B and T cell responses are readily induced by infection6,13,18-24, the longitudinal dynamics of these key memory populations remains poorly resolved. Here we comprehensively profiled antibody, B and T cell dynamics over time in a cohort recovered from mild-moderate COVID-19. We find that binding and neutralising antibody responses, together with individual serum clonotypes, decay over the first 4 months post-infection, as expected, with a similar decline in S-specific CD4+ and circulating T follicular helper (cTFH) frequencies. In contrast, S-specific IgG+ memory B cells (MBC) consistently accumulate over time, eventually comprising a significant fraction of circulating MBC. Modelling of the concomitant immune kinetics predicts maintenance of serological neutralising activity above a titre of 1:40 in 50% of convalescent subjects to 74 days, with probable additive protection from B and T cells. Overall, our study suggests SARS-CoV-2 immunity after infection is likely to be transiently protective at a population level. SARS-CoV-2 vaccines may require greater immunogenicity and durability than natural infection to drive long-term protection.

SummarySARS-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 assessed 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 was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were 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.

Background Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little premarket validation. Methods Performance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT). Results Sensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%–60.4%) to 67.9% (95% CI, 59.4%–75.6%), and specificities from 95.6% (95% CI, 89.2%–98.8%) to 100.0% (95% CI, 96.1%–100.0%). ELISA sensitivity for IgA or IgG detection was 67.9% (95% CI, 59.4%–75.6%), increasing to 93.8% (95% CI, 85.0%–98.3%) for samples >14 days post symptom onset. sVNT sensitivity was 60.9% (95% CI, 53.2%–68.4%), rising to 91.2% (95% CI, 81.8%–96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%–97.5%). Conclusions Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings., Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings where positive predictive values are low.

The rapid global spread of SARS-CoV-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. Multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. However, the immunogenic properties of the spike protein in humans are unresolved. To address this, we undertook an in-depth characterisation of humoral and cellular immunity against SARS-CoV-2 spike in humans following mild to moderate SARS-CoV-2 infection. We find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ACE2/RBD interaction. Expanded populations of spike-specific memory B cells and circulating T follicular helper cells (cTFH) were detected within convalescent donors, while responses to the receptor binding domain (RBD) constitute a minor fraction. Using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific cTFH subsets. Thus both qualitative and quantitative features of B and T cell immunity to spike constitute informative biomarkers of the protective potential of novel SARS-CoV-2 vaccines.

T-cell responses to SARS-CoV-2-derived peptide pools have been documented, however it remains largely unclear whether prominent SARS-CoV-2-specific T cell populations originate from naive or pre-existing memory sets. As HLA-B*07:02-restricted N105-113 epitope (B7/N105) is the most dominant SARS-CoV-2 CD8+ T-cell specificity to date, we dissected CD8+ T-cell responses directed at this epitope by direct ex vivo analyses in peripheral blood mononuclear cells (PBMCs) from acute and convalescent COVID-19 patients, and pre-pandemic PBMCs, tonsils, lungs and spleens. Using peptide-HLA tetramers, immunodominant B7/N105+CD8+ T-cells were detected at a high frequency (∼2.18x10-4) in COVID-19 patients, comparable to the well-established influenza-specific A2/M158+CD8+ T-cell population. Remarkably, frequencies of B7/N105+CD8+ T-cells were also readily detectable in pre-pandemic PBMCs and tonsils (at 6.55x10-5 and 2.76x10-4, respectively), although they mainly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. Ex vivo TCRαβ analyses revealed that a diverse TCRαβ repertoire and promiscuity in αβ TCR pairing underlie such high naive precursor frequencies of B7/N105+CD8+ T-cells. Overall, our study demonstrates that high precursor frequency and plasticity of TCRα-TCRβ pairing underpin immunodominance of SARS-CoV-2-specific B7/N105+CD8+ T-cell responses and advocates for vaccine strategies which include the nucleocapsid protein to elicit immunodominant CD8+ T-cell responses in HLA-B*07:02 individuals. Funding: This work was supported by theClifford Craig Foundation to KLF and KK, NHMRC Leadership Investigator Grant to KK (1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of theHong Kong Special Administrative Region, China (#T11-712/19-N) to KK, the Victorian Government (SJK, AKW), MRFF award (#2002073) to SJK and AKW, MRFFAward (#1202445) to KK, NHMRC program grant 1149990 (SJK) and NHMRC project grant 1162760 (AKW). AKW is supported by Emerging Leadership 1 Investigator Grant (#1173433), JAJ by an NHMRC Early Career Fellowship (ECF) (#1123673), KK by NHMRC SeniorResearch Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship(#1137285) and SJK by NHMRC Senior Principal Research Fellowship (#1136322). CES has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (#792532). JR is supported by an ARC Laureate fellowship. JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. LH is supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne. Ethical Approval: Experiments conformed to the Declaration of Helsinki Principles and theAustralian National Health and Medical Research Council Code of Practice. Written informed consents were obtained from all blood donors prior to the study. Lung and spleen tissues were obtained from deceased organ donors after written informed consents from the next of kin.Written informed consents were obtained from participants’ parental or guardians for underage tonsil tissue donors. The study was approved by the Alfred Hospital (#280/14), Austin Health (HREC/63201/Austin-2020); the University of Melbourne (#2057366.1, #2056901.1,#2056689, #2056761, #1442952, #1955465, and #1443389), the Australian Red CrossLifeblood (ID 2015#8), the Tasmanian Health and Medical (ID H0017479) and the James Cook University (H7886) Human Research Ethics Committees.

SARS-CoV-2 causes a spectrum of illness, ranging from asymptomatic to severe COVID-19. As the immunological basis for severity remains ill-defined, we analysed 78 SARS-CoV-2-infected individuals at acute and/or convalescent timepoints, up to 102 days post-symptom onset, quantifying 184 innate and adaptive immunological parameters. Acute COVID-19 in hospitalised patients was associated with high levels of IL-6, IL-18 and IL-10, elevated neutrophil-to-lymphocyte and neutrophil-to-T cell ratios, and high proportions of activated CD38+ neutrophils, CD38+ eosinophils, CD38+ HLA-DRlo monocytes, CD38+CD56dim NK cells, CD38+ γδ T cells, antibody-secreting cells, CD38+ICOS+ circulating T follicular helper (cTFH) cells, CD38+HLA-DR+CD4+ T cells, effector CD27-CD45RA+ and CD38+ CD8+ T cells. During convalescence, elevated seroconversion and neutralising antibodies were prominent and correlated with acute CXCR3+ cTFH cell activation. Strikingly, intensive care unit (ICU) patients with severe COVID-19 displayed higher levels of soluble IL-6R, IL-18, and hyperactivation of innate, adaptive and myeloid compartments than ward patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients during acute and convalescent phases of SARS-CoV-2 infection, and integrate key cellular pathways of complex perturbed immune networks that underpin severe COVID-19, providing important insights into potential biomarkers and immunotherapies. Funding: This work was supported by the Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grant to KK (#1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of the Hong Kong Special Administrative Region, China (#T11-712/19-N) to KK, the Jack Ma Foundation to KK, KS, DIG and AWC, the a2 milk foundation to KS, the Victorian Government MRFF award (#2002073) to SJK, DIG and AKW, MRFF Award (#1202445) to KK, NHMRC program grant 1149990 (SJK), NHMRC project grant 1162760 (AKW), NHMRC Program Grant (#1071916) to KK and DCJ, NHMRC Program Grant (1113293) to DIG, the Merridew Foundation to NJCK. KK is supported by NHMRC Senior Research Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship (#1137285), KS by an NHMRC Investigator grant (#1177174), SYCT by a NHMRC Career Development Fellowship (#1145033), DIG by an NHMRC Senior Principal Research Fellowship (#1117766) and SJK by NHMRC Senior Principal Research Fellowship (#1136322). SRL is supported by an NHMRC program grant (#1149990) and practitioner fellowship (#1135851). LH and WZ are supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne. XJ is supported by China Scholarship Council-University of Melbourne joint Scholarship. CES has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (#792532). JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. TMA and FM-W are International Society for Advancement of Cytometry Marylou Ingram Scholars. GHP is supported by the Australian Government Research Training Program (RTP) Scholarship. JAJ is supported by an NHMRC Early Career Fellowship (ECF) (#1123673). EBC. is supported by a NHMRC Peter Doherty Fellowship (#1091516). KK and AC were supported by the University of Melbourne Dame Kate Campbell Fellowship. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: The study was approved by the Alfred Hospital (#280/14), Melbourne Health (HREC/17/MH/53), Monash Health (HREC/15/MonH/64/2016.196), The University of Melbourne (#2056689, #2056761, #1442952, #1955465 and #1443389) and James Cook University (H7886) Human Research Ethics Committees.