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.

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.).

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.

Hepatitis B vaccine co-administration influences the heterologous effects of neonatal BCG vaccination in a sex-differential manner.

INTRODUCTION: Bacille Calmette-Guérin (BCG) and hepatitis B (HBV) vaccines are frequently given concomitantly at birth. Neonatal BCG vaccination induces off-target immunological effects. Whether HBV vaccine has immunomodulatory effects is unknown. As off-target effects might vary when vaccines are given simultaneously, this randomised controlled trial aimed to evaluate the influence of neonatal vaccination with BCG and/or HBV on heterologous immune responses. METHODS: A total of 185 neonates in Australia were randomised to receive either neonatal BCG-Denmark vaccine, HBV vaccine, both (BCG + HBV group), or none (No vaccine group). In-vitro responses to heterologous stimulants were assessed 7 days after vaccination. The influence of (i) randomisation group and (ii) sex on interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), and tumour necrosis factor-alpha (TNF-α) responses was analysed using linear regression. RESULTS: Overall, BCG vaccination alone or with HBV co-administration reduced IFN-γ and MCP-1 responses to heterologous stimulants. HBV vaccination alone did not alter heterologous cytokine responses. In general, males produced more IFN-γ and TNF-α than females. We observed a sex-differential effect in relation to the influence of HBV co-administration on the effect of BCG on heterologous responses. Compared with males in the No vaccine group, males in the BCG + HBV group had lower IFN-γ and MCP-1 responses. In contrast, compared with females in the No vaccine group, females in the BCG group had higher IFN-γ response and lower MCP-1 responses. CONCLUSION: Neonatal BCG vaccination resulted in lower cytokine responses to unrelated pathogens. HBV co-administration did not have a significant impact on responses overall but influenced the heterologous effects of neonatal BCG vaccination in a sex-differential manner.

Neonatal rotavirus vaccine (RV3-BB) immunogenicity and safety in a neonatal and infant administration schedule in Malawi: a randomised, double-blind, four-arm parallel group dose-ranging study.

BACKGROUND: Rotavirus vaccines reduce rotavirus-related deaths and hospitalisations but are less effective in high child mortality countries. The human RV3-BB neonatal G3P[6] rotavirus vaccine administered in a neonatal schedule was efficacious in reducing severe rotavirus gastroenteritis in Indonesia but had not yet been evaluated in African infants. METHODS: We did a phase 2, randomised, double-blind, parallel group dose-ranging study of three doses of oral RV3-BB rotavirus vaccine in infants in three primary health centres in Blantyre, Malawi. Healthy infants less than 6 days of age with a birthweight 2·5 to 4·0 kg were randomly assigned (1:1:1:1) into one of four treatment groups: neonatal vaccine group, which included high-titre (1·0 × 107 focus-forming unit [FFU] per mL), mid-titre (3·0 × 106 FFU per mL), or low-titre (1·0 × 106 FFU per mL); and infant vaccine group, which included high-titre (1·0 × 107 FFU per mL) using a computer generated code (block size of four), stratified by birth (singleton vs multiple). Neonates received their three doses at 0-5 days to 10 weeks and infants at 6-14 weeks. Investigators, participant families, and laboratory staff were masked to group allocation. Anti-rotavirus IgA seroconversion and vaccine take (IgA seroconversion and stool shedding) were evaluated. Safety was assessed in all participants who received at least one dose of vaccine or placebo. The primary outcome was the cumulative IgA seroconversion 4 weeks after three doses of RV3-BB in the neonatal schedule in the high-titre, mid-titre, and low-titre groups in the per protocol population, with its 95% CI. With the high-titre group as the active control group, we did a non-inferiority analysis of the proportion of participants with IgA seroconversion in the mid-titre and low-titre groups, using a non-inferiority margin of less than 20%. This trial is registered at ClinicalTrials.gov (NCT03483116). FINDINGS: Between Sept 17, 2018, and Jan 27, 2020, 711 participants recruited were randomly assigned into four treatment groups (neonatal schedule high titre n=178, mid titre n=179, low titre n=175, or infant schedule high titre n=179). In the neonatal schedule, cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed in 79 (57%) of 139 participants in the high-titre group, 80 (57%) of 141 participants in the mid-titre group, and 57 (41%) of 138 participants in the low-titre group and at 18 weeks in 100 (72%) of 139 participants in the high-titre group, 96 (67%) of 143 participants in the mid-titre group, and 86 (62%) of 138 of participants in the low-titre. No difference in cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed between high-titre and mid-titre groups in the neonatal schedule (difference in response rate 0·001 [95%CI -0·115 to 0·117]), fulfilling the criteria for non-inferiority. In the infant schedule group 82 (59%) of 139 participants had a cumulative IgA seroconversion 4 weeks after three doses of RV3-BB at 18 weeks. Cumulative vaccine take was detected in 483 (85%) of 565 participants at 18 weeks. Three doses of RV3-BB were well tolerated with no difference in adverse events among treatment groups: 67 (39%) of 170 participants had at least one adverse event in the high titre group, 68 (40%) of 172 participants had at least one adverse event in the mid titre group, and 69 (41%) of 169 participants had at least one adverse event in the low titre group. INTERPRETATION: RV3-BB was well tolerated and immunogenic when co-administered with Expanded Programme on Immunisation vaccines in a neonatal or infant schedule. A lower titre (mid-titre) vaccine generated similar IgA seroconversion to the high-titre vaccine presenting an opportunity to enhance manufacturing capacity and reduce costs. Neonatal administration of the RV3-BB vaccine has the potential to improve protection against rotavirus disease in children in a high-child mortality country in Africa. FUNDING: Bill & Melinda Gates Foundation, Australian Tropical Medicine Commercialisation Grant.

Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow.

Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.

Neonatal BCG Vaccination Reduces Interferon-γ Responsiveness to Heterologous Pathogens in Infants From a Randomized Controlled Trial.

BACKGROUND: BCG vaccination has beneficial nonspecific (heterologous) effects that protect against nonmycobacterial infections. We have previously reported that BCG vaccination at birth alters in vitro cytokine responses to heterologous stimulants in the neonatal period. This study investigated heterologous responses in 167 infants in the same trial 7 months after randomization. METHODS: A whole-blood assay was used to interrogate in vitro cytokine responses to heterologous stimulants (killed pathogens) and Toll-like receptor (TLR) ligands. RESULTS: Compared to BCG-naive infants, BCG-vaccinated infants had increased production of interferon gamma (IFN-γ) and monokine induced by gamma interferon (MIG) (CXCL9) in response to mycobacterial stimulation and decreased production of IFN-γ in response to heterologous stimulation and TLR ligands. Reduced IFN-γ responses were attributable to a decrease in the proportion of infants who mounted a detectable IFN-γ response. BCG-vaccinated infants also had increased production of MIG (CXCL9) and interleukin-8 (IL-8), and decreased production of IL-10, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß, the pattern of which varied by stimulant. IL-1Ra responses following TLR1/2 (Pam3CYSK4) stimulation were increased in BCG-vaccinated infants. Both sex and maternal BCG vaccination status influenced the effect of neonatal BCG vaccination. CONCLUSIONS: BCG vaccination leads to changes in IFN-γ responsiveness to heterologous stimulation. BCG-induced changes in other cytokine responses to heterologous stimulation vary by pathogen.

Can colony-forming unit testing be used to extend the shelf life of BCG vaccines?

A recent shortage in supply of Bacille Calmette-Guérin (BCG), the live attenuated vaccine given to protect against tuberculosis (TB) caused major disruption to global vaccination programs. In this study, we assessed whether quantification of viable bacteria, could be used to inform the use of the BCG vaccine beyond its manufacturer-assigned expiration date. The viability of a single batch of BCG-Denmark was tested in three independent laboratories. There was high inter-vial and inter-laboratory variability in viability counts, however all three laboratories detected a decrease in BCG viability over time. Despite this, there was no difference in the rate of BCG scar formation in infants who were vaccinated with this batch of BCG before and after its manufacturer-assigned expiration date. This study demonstrates the potential for using BCG viability counts to guide the use of BCG vaccine beyond the manufacturer-assigned expiration date.

Innate immune responses following Kawasaki disease and toxic shock syndrome.

The pathogenesis of Kawasaki disease (KD) remains unknown and there is accumulating evidence for the importance of the innate immune system in initiating and mediating the host inflammatory response. We compared innate immune responses in KD and toxic shock syndrome (TSS) participants more than two years after their acute illness with control participants to investigate differences in their immune phenotype. Toxic shock syndrome shares many clinical features with KD; by including both disease groups we endeavoured to explore changes in innate immune responses following acute inflammatory illnesses more broadly. We measured the in vitro production of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-1 receptor antagonist (IL-1ra), and IL-10 following whole blood stimulation with toll-like receptor and inflammasome ligands in 52 KD, 20 TSS, and 53 control participants in a case-control study. Analyses were adjusted for age, sex, and unstimulated cytokine concentrations. Compared to controls, KD participants have reduced IL-1ra production in response to stimulation with double stranded RNA (geometric mean ratio (GMR) 0.37, 95% CI 0.15, 0.89, p = 0.03) and increased IL-6 production in response to incubation with Lyovec™ (GMR 5.48, 95% CI 1.77, 16.98, p = 0.004). Compared to controls, TSS participants have increased IFN-γ production in response to peptidoglycan (GMR 4.07, 95% CI 1.82, 9.11, p = 0.001), increased IL-1ß production to lipopolysaccharide (GMR 1.64, 95% CI 1.13, 2.38, p = 0.01) and peptidoglycan (GMR 1.61, 95% CI 1.11, 2.33, p = 0.01), and increased IL-6 production to peptidoglycan (GMR 1.45, 95% CI 1.10, 1.92, p = 0.01). Years following the acute illness, individuals with previous KD or TSS exhibit a pro-inflammatory innate immune phenotype suggesting a possible underlying immunological susceptibility or innate immune memory.