Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections

Isolation of Non-Tuberculous Mycobacteria in Children Investigated for Pulmonary Tuberculosis

OBJECTIVE: To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community. METHODS: Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001–2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis. RESULTS: Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5–7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31–50), compared to 67% (95% CI 58–76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9–12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0–0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04). DISCUSSION: NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation

Maternal colonization with Streptococcus agalactiae and associated stillbirth and neonatal disease in coastal Kenya

Streptococcus agalactiae (group B streptococcus, GBS) causes neonatal disease and stillbirth, but its burden in sub-Saharan Africa is uncertain. We assessed maternal recto-vaginal GBS colonization (7,967 women), stillbirth and neonatal disease. Whole-genome sequencing was used to determine serotypes, sequence types and phylogeny. We found low maternal GBS colonization prevalence (934/7,967, 12%), but comparatively high incidence of GBS-associated stillbirth and early onset neonatal disease (EOD) in hospital (0.91 (0.25-2.3)/1,000 births and 0.76 (0.25-1.77)/1,000 live births, respectively). However, using a population denominator, EOD incidence was considerably reduced (0.13 (0.07-0.21)/1,000 live births). Treated cases of EOD had very high case fatality (17/36, 47%), especially within 24 h of birth, making under-ascertainment of community-born cases highly likely, both here and in similar facility-based studies. Maternal GBS colonization was less common in women with low socio-economic status, HIV infection and undernutrition, but when GBS-colonized, they were more probably colonized by the most virulent clone, CC17. CC17 accounted for 267/915 (29%) of maternal colonizing (265/267 (99%) serotype III; 2/267 (0.7%) serotype IV) and 51/73 (70%) of neonatal disease cases (all serotype III). Trivalent (Ia/II/III) and pentavalent (Ia/Ib/II/III/V) vaccines would cover 71/73 (97%) and 72/73 (99%) of disease-causing serotypes, respectively. Serotype IV should be considered for inclusion, with evidence of capsular switching in CC17 strains

RISK6, a 6-gene transcriptomic signature of TB disease risk, diagnosis and treatment response

Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for multiple applications: identifying individuals at risk of incident disease, as a screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment. RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven independent cohorts. Prognostic performance significantly exceeded that of previous signatures discovered in the same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and HIV-infected persons, assessed by area under the receiver-operating characteristic curve, exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or approached the benchmarks set out in World Health Organization target product profiles for non-sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by positron emission tomography, and tracked treatment response, demonstrating utility as treatment response biomarker, while predicting treatment failure prior to treatment initiation. Performance of the test in capillary blood samples collected by finger-prick was noninferior to venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field studies

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Influenza A virus challenge models in Cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (&gt; 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was subclinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000- fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections

Using mortuary and burial data to place COVID-19 in Lusaka, Zambia within a global context

Reported COVID-19 cases and associated mortality remain low in many sub-Saharan countries relative to global averages, but true impact is difficult to estimate given limitations around surveillance and mortality registration. In Lusaka, Zambia, burial registration and SARS-CoV-2 prevalence data during 2020 allow estimation of excess mortality and transmission. Relative to pre-pandemic patterns, we estimate age-dependent mortality increases, totalling 3,212 excess deaths (95% CrI: 2,104-4,591), representing an 18.5% (95% CrI: 13.0-25.2%) increase relative to pre-pandemic levels. Using a dynamical model-based inferential framework, we find that these mortality patterns and SARS-CoV-2 prevalence data are in agreement with established COVID-19 severity estimates. Our results support hypotheses that COVID-19 impact in Lusaka during 2020 was consistent with COVID-19 epidemics elsewhere, without requiring exceptional explanations for low reported figures. For more equitable decision-making during future pandemics, barriers to ascertaining attributable mortality in low-income settings must be addressed and factored into discourse around reported impact differences