Specific and off-target immune responses following COVID-19 vaccination with ChAdOx1-S and BNT162b2 vaccines-an exploratory sub-study of the BRACE trial.

BACKGROUND: The COVID-19 pandemic led to the rapid development and deployment of several highly effective vaccines against SARS-CoV-2. Recent studies suggest that these vaccines may also have off-target effects on the immune system. We sought to determine and compare the off-target effects of the adenovirus vector ChAdOx1-S (Oxford-AstraZeneca) and modified mRNA BNT162b2 (Pfizer-BioNTech) vaccines on immune responses to unrelated pathogens. METHODS: Prospective sub-study within the BRACE trial. Blood samples were collected from 284 healthcare workers before and 28 days after ChAdOx1-S or BNT162b2 vaccination. SARS-CoV-2-specific antibodies were measured using ELISA, and whole blood cytokine responses to specific (SARS-CoV-2) and unrelated pathogen stimulation were measured by multiplex bead array. FINDINGS: Both vaccines induced robust SARS-CoV-2 specific antibody and cytokine responses. ChAdOx1-S vaccination increased cytokine responses to heat-killed (HK) Candida albicans and HK Staphylococcus aureus and decreased cytokine responses to HK Escherichia coli and BCG. BNT162b2 vaccination decreased cytokine response to HK E. coli and had variable effects on cytokine responses to BCG and resiquimod (R848). After the second vaccine dose, BNT162b2 recipients had greater specific and off-target cytokine responses than ChAdOx1-S recipients. INTERPRETATION: ChAdOx1-S and BNT162b2 vaccines alter cytokine responses to unrelated pathogens, indicative of potential off-target effects. The specific and off-target effects of these vaccines differ in their magnitude and breadth. The clinical relevance of these findings is uncertain and needs further study. FUNDING: Bill & Melinda Gates Foundation, National Health and Medical Research Council, Swiss National Science Foundation and the Melbourne Children's. BRACE trial funding is detailed in acknowledgements.

Evolution of Humoral and Cellular Immunity Post-Breakthrough Coronavirus Disease 2019 in Vaccinated Patients With Hematologic Malignancy Receiving Tixagevimab-Cilgavimab.

Background: In-depth immunogenicity studies of tixagevimab-cilgavimab (T-C) are lacking, including following breakthrough coronavirus disease 2019 (COVID-19) in vaccinated patients with hematologic malignancy (HM) receiving T-C as pre-exposure prophylaxis. Methods: We performed a prospective, observational cohort study and detailed immunological analyses of 93 patients with HM who received T-C from May 2022, with and without breakthrough infection, during a follow-up period of 6 months and dominant Omicron BA.5 variant. Results: In 93 patients who received T-C, there was an increase in Omicron BA.4/5 receptor-binding domain (RBD) immunoglobulin G (IgG) antibody titers that persisted for 6 months and was equivalent to 3-dose-vaccinated uninfected healthy controls at 1 month postinjection. Omicron BA.4/5 neutralizing antibody was lower in patients receiving B-cell-depleting therapy within 12 months despite receipt of T-C. COVID-19 vaccination during T-C treatment did not incrementally improve RBD or neutralizing antibody levels. In 16 patients with predominantly mild breakthrough infection, no change in serum neutralization of Omicron BA.4/5 postinfection was detected. Activation-induced marker assay revealed an increase in CD4+ (but not CD8+) T cells post infection, comparable to previously infected healthy controls. Conclusions: Our study provides proof-of-principle for a pre-exposure prophylaxis strategy and highlights the importance of humoral and cellular immunity post-breakthrough COVID-19 in vaccinated patients with HM.

Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.

Laboratory evaluation of ELISA and indirect immunofluorescence assay in response to emergence of Japanese encephalitis virus genotype IV in Australia.

The unexpected recent emergence of Japanese encephalitis virus (JEV) genotype IV in multiple southern states of Australia necessitated an evaluation of JEV serological tests suitable for diagnosing acute infection and for seroprevalence studies. This study examined the analytical and clinical performance of two high-throughput JEV assays, Euroimmun immunofluorescence assay (IFA) and Euroimmun enzyme-linked immunosorbent assay (ELISA), across four cohorts; (1) surveillance of piggery workers in outbreak areas, (2) surveillance of residents in outbreak areas, (3) acute JEV infection and (4) post-JEV vaccination. ELISA and IFA IgM demonstrated minimal cross-reactivity (0-1.8%) with other endemic flaviviruses, with high sensitivity (100%) for acute JEV infection in this low endemicity setting. Differences in IgG serodynamics between the two assays suggest convalescent and paired testing with IgM are critical in diagnosing acute infection. High assay concordance was observed between ELISA and IFA when used in serosurveillance (97.4% agreement, Cohen' κ 0.74 [95% CI 0.614-0.860]) and vaccination cohorts (91.1% agreement, Cohen's κ 0.806 [95% CI 0.672-0.941]). In conclusion, this study highlights the clinical & epidemiological applications and limitations of these two commercial JEV assays.

Comparative Longitudinal Serological Study of Anti-SARS-CoV-2 Antibody Profiles in People with COVID-19.

Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.

Laboratory diagnosis of HIV: a contemporary overview in the Australian context.

Over the past decade there have been technical advances in human immunodeficiency virus (HIV) assays and updates to testing regulations that have substantially changed the landscape of laboratory testing for HIV. In addition, there have been significant changes in the epidemiology of HIV in Australia in the context of highly effective contemporary biomedical treatment and prevention strategies. Here, we provide an update on contemporary issues for the laboratory detection and confirmation of HIV in Australia. These include (1) the impact of early treatment and biological prevention strategies on the serological and virological detection of HIV; (2) the updated national HIV laboratory case definition and its interaction with testing regulations, public health and clinical guidelines; and (3) novel strategies for the laboratory detection of HIV, including the incorporation of HIV nucleic acid amplification tests (NAATs) into testing algorithms. These developments present an opportunity to develop a nationally consistent contemporary HIV testing algorithm that would result in optimisation and standardisation of HIV testing in Australia.

Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and γδ T cell perturbations.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Randomized Trial of BCG Vaccine to Protect against Covid-19 in Health Care Workers.

BACKGROUND: The bacille Calmette-Guérin (BCG) vaccine has immunomodulatory "off-target" effects that have been hypothesized to protect against coronavirus disease 2019 (Covid-19). METHODS: In this international, double-blind, placebo-controlled trial, we randomly assigned health care workers to receive the BCG-Denmark vaccine or saline placebo and followed them for 12 months. Symptomatic Covid-19 and severe Covid-19, the primary outcomes, were assessed at 6 months; the primary analyses involved the modified intention-to-treat population, which was restricted to participants with a negative test for severe acute respiratory syndrome coronavirus 2 at baseline. RESULTS: A total of 3988 participants underwent randomization; recruitment ceased before the planned sample size was reached owing to the availability of Covid-19 vaccines. The modified intention-to-treat population included 84.9% of the participants who underwent randomization: 1703 in the BCG group and 1683 in the placebo group. The estimated risk of symptomatic Covid-19 by 6 months was 14.7% in the BCG group and 12.3% in the placebo group (risk difference, 2.4 percentage points; 95% confidence interval [CI], -0.7 to 5.5; P = 0.13). The risk of severe Covid-19 by 6 months was 7.6% in the BCG group and 6.5% in the placebo group (risk difference, 1.1 percentage points; 95% CI, -1.2 to 3.5; P = 0.34); the majority of participants who met the trial definition of severe Covid-19 were not hospitalized but were unable to work for at least 3 consecutive days. In supplementary and sensitivity analyses that used less conservative censoring rules, the risk differences were similar but the confidence intervals were narrower. There were five hospitalizations due to Covid-19 in each group (including one death in the placebo group). The hazard ratio for any Covid-19 episode in the BCG group as compared with the placebo group was 1.23 (95% CI, 0.96 to 1.59). No safety concerns were identified. CONCLUSIONS: Vaccination with BCG-Denmark did not result in a lower risk of Covid-19 among health care workers than placebo. (Funded by the Bill and Melinda Gates Foundation and others; BRACE ClinicalTrials.gov number, NCT04327206.).

Robust SARS-CoV-2 T cell responses with common TCRαß motifs toward COVID-19 vaccines in patients with hematological malignancy impacting B cells.

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.

Prospective comprehensive profiling of immune responses to COVID-19 vaccination in patients on zanubrutinib therapy.

Zanubrutinib-treated and treatment-naïve patients with chronic lymphocytic leukaemia (CLL) or Waldenstrom's macroglobulinaemia were recruited in this prospective study to comprehensively profile humoral and cellular immune responses to COVID-19 vaccination. Overall, 45 patients (median 72 years old) were recruited; the majority were male (71%), had CLL (76%) and were on zanubrutinib (78%). Seroconversion rates were 65% and 77% following two and three doses, respectively. CD4+ and CD8+ T-cell response rates increased with third dose. In zanubrutinib-treated patients, 86% developed either a humoral or cellular response. Patients on zanubrutinib developed substantial immune responses following two COVID-19 vaccine doses, which further improved following a third dose.

Serological testing of blood donors to characterise the impact of COVID-19 in Melbourne, Australia, 2020.

Rapidly identifying and isolating people with acute SARS-CoV-2 infection has been a core strategy to contain COVID-19 in Australia, but a proportion of infections go undetected. We estimated SARS-CoV-2 specific antibody prevalence (seroprevalence) among blood donors in metropolitan Melbourne following a COVID-19 outbreak in the city between June and September 2020. The aim was to determine the extent of infection spread and whether seroprevalence varied demographically in proportion to reported cases of infection. The design involved stratified sampling of residual specimens from blood donors (aged 20-69 years) in three postcode groups defined by low (<3 cases/1,000 population), medium (3-7 cases/1,000 population) and high (>7 cases/1,000 population) COVID-19 incidence based on case notification data. All specimens were tested using the Wantai SARS-CoV-2 total antibody assay. Seroprevalence was estimated with adjustment for test sensitivity and specificity for the Melbourne metropolitan blood donor and residential populations, using multilevel regression and poststratification. Overall, 4,799 specimens were collected between 23 November and 17 December 2020. Seroprevalence for blood donors was 0.87% (90% credible interval: 0.25-1.49%). The highest estimates, of 1.13% (0.25-2.15%) and 1.11% (0.28-1.95%), respectively, were observed among donors living in the lowest socioeconomic areas (Quintiles 1 and 2) and lowest at 0.69% (0.14-1.39%) among donors living in the highest socioeconomic areas (Quintile 5). When extrapolated to the Melbourne residential population, overall seroprevalence was 0.90% (0.26-1.51%), with estimates by demography groups similar to those for the blood donors. The results suggest a lack of extensive community transmission and good COVID-19 case ascertainment based on routine testing during Victoria's second epidemic wave. Residual blood donor samples provide a practical epidemiological tool for estimating seroprevalence and information on population patterns of infection, against which the effectiveness of ongoing responses to the pandemic can be assessed.

Off-target effects of bacillus Calmette-Guérin vaccination on immune responses to SARS-CoV-2: implications for protection against severe COVID-19.

Background and objectives: Because of its beneficial off-target effects against non-mycobacterial infectious diseases, bacillus Calmette-Guérin (BCG) vaccination might be an accessible early intervention to boost protection against novel pathogens. Multiple epidemiological studies and randomised controlled trials (RCTs) are investigating the protective effect of BCG against coronavirus disease 2019 (COVID-19). Using samples from participants in a placebo-controlled RCT aiming to determine whether BCG vaccination reduces the incidence and severity of COVID-19, we investigated the immunomodulatory effects of BCG on in vitro immune responses to SARS-CoV-2. Methods: This study used peripheral blood taken from participants in the multicentre RCT and BCG vaccination to reduce the impact of COVID-19 on healthcare workers (BRACE trial). The whole blood taken from BRACE trial participants was stimulated with γ-irradiated SARS-CoV-2-infected or mock-infected Vero cell supernatant. Cytokine responses were measured by multiplex cytokine analysis, and single-cell immunophenotyping was made by flow cytometry. Results: BCG vaccination, but not placebo vaccination, reduced SARS-CoV-2-induced secretion of cytokines known to be associated with severe COVID-19, including IL-6, TNF-α and IL-10. In addition, BCG vaccination promoted an effector memory phenotype in both CD4+ and CD8+ T cells, and an activation of eosinophils in response to SARS-CoV-2. Conclusions: The immunomodulatory signature of BCG's off-target effects on SARS-CoV-2 is consistent with a protective immune response against severe COVID-19.

SARS-CoV-2 infection results in immune responses in the respiratory tract and peripheral blood that suggest mechanisms of disease severity.

Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples correlates with RBD-specific IgM and IgG levels. Cytokines/chemokines vary between respiratory samples and plasma, indicating that inflammation should be assessed in respiratory specimens to understand immunopathology. IFN-α2 and IL-12p70 in endotracheal aspirate and neutralization in sputum negatively correlate with duration of hospital stay. Diverse immune subsets are detected in respiratory samples, dominated by neutrophils. Importantly, dexamethasone treatment does not affect humoral responses in blood of COVID-19 patients. Our study unveils differential immune responses between respiratory samples and blood, and shows how drug therapy affects immune responses during COVID-19.

Comparison of Seroconversion in Children and Adults With Mild COVID-19.

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.

Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibodies in Australia After the First Epidemic Wave in 2020: A National Survey.

BACKGROUND: As of mid-2021, Australia's only nationwide coronavirus disease 2019 (COVID-19) epidemic occurred in the first 6 months of the pandemic. Subsequently, there has been limited transmission in most states and territories. Understanding community spread during the first wave was hampered by initial limitations on testing and surveillance. To characterize the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody seroprevalence generated during this time, we undertook Australia's largest national SARS-CoV-2 serosurvey. METHODS: Between June 19 and August 6, 2020, residual specimens were sampled from people undergoing general pathology testing (all ages), women attending antenatal screening (20-39 years), and blood donors (20-69 years) based on the Australian population's age and geographic distributions. Specimens were tested by Wantai total SARS-CoV-2-antibody assay. Seroprevalence estimates adjusted for test performance were produced. The SARS-CoV-2 antibody-positive specimens were characterized with microneutralization assays. RESULTS: Of 11 317 specimens (5132 general pathology; 2972 antenatal; 3213 blood-donors), 71 were positive for SARS-CoV-2-specific antibodies. Seroprevalence estimates were 0.47% (95% credible interval [CrI], 0.04%-0.89%), 0.25% (CrI, 0.03%-0.54%), and 0.23% (CrI, 0.04%-0.54%), respectively. No seropositive specimens had neutralizing antibodies. CONCLUSIONS: Australia's seroprevalence was extremely low (<0.5%) after the only national COVID-19 wave thus far. These data and the subsequent limited community transmission highlight the population's naivety to SARS-CoV-2 and the urgency of increasing vaccine-derived protection.

Trend in Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Serology One Year After Mild and Asymptomatic Coronavirus Disease 2019 (COVID-19): Unpacking Potential Bias in Seroprevalence Studies.

A key aim of serosurveillance during the coronavirus disease 2019 (COVID-19) pandemic has been to estimate the prevalence of prior infection, by correcting crude seroprevalence against estimated test performance for polymerase chain reaction (PCR)-confirmed COVID-19. We show that poor generalizability of sensitivity estimates to some target populations may lead to substantial underestimation of case numbers.

Evaluation of dried blood spots for hepatitis B and D serology and nucleic acid testing.

Areas with the highest burden of hepatitis B virus (HBV) infection are often low-middle-income countries with limited access to diagnosis due to isolation, affordability, and/or feasibility. Dried blood spots (DBSs) provide an alternative for remote areas where collection and transportation of serum is impractical. In this study, the application of DBS for serological and molecular detection of HBV and hepatitis D virus (HDV) was evaluated. Hepatitis B surface antigen was detected in 87 of 91 (95.6%) DBS. Seventeen of 21 (81%) had detectable HBeAg and 52 of 71 (73.2%) were anti-HBe positive. Anti-HD was detectable in 11 of 12 (91.6%) spiked control DBS after an initial failure to detect in patient DBS. HBV DNA was detected from 50 of 70 (71.4%) DBS with serum loads greater than 200 IU/mL in an in-house assay and 18 of 24 (75%) DBS with loads exceeding 389 IU/mL in a commercial assay. Using linear regression, HBV DNA loads from DBS were able to predict serum loads in 46 of 50 (92%) samples to within 1 log of actual serum load. HDV RNA was detected in 42 of 47 (89%) DBS with serum levels greater than 7200 IU/mL. DBSs are recommended for diagnosis of HBV, monitoring, and detection of high loads in pregnant women where peripheral blood testing remains unfeasible. Detection of HDV RNA from DBS may prove useful in endemic areas.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity.

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Evaluation of 6 Commercial SARS-CoV-2 Serology Assays Detecting Different Antibodies for Clinical Testing and Serosurveillance.

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.