Mucosal and systemic immune correlates of viral control after SARS-CoV-2 infection challenge in seronegative adults.

Human infection challenge permits in-depth, early, and pre-symptomatic characterization of the immune response, enabling the identification of factors that are important for viral clearance. Here, we performed intranasal inoculation of 34 young adult, seronegative volunteers with a pre-Alpha severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. Of these participants, 18 (53%) became infected and showed an interferon-dominated mediator response with divergent kinetics between nasal and systemic sites. Peripheral CD4+ and CD8+ T cell activation and proliferation were early and robust but showed distinct kinetic and phenotypic profiles; antigen-specific T cells were largely CD38+Ki67+ and displayed central and effector memory phenotypes. Both mucosal and systemic antibodies became detectable around day 10, but nasal antibodies plateaued after day 14 while circulating antibodies continued to rise. Intensively granular measurements in nasal mucosa and blood allowed modeling of immune responses to primary SARS-CoV-2 infection that revealed CD8+ T cell responses and early mucosal IgA responses strongly associated with viral control, indicating that these mechanisms should be targeted for transmission-reducing intervention.

Viral emissions into the air and environment after SARS-CoV-2 human challenge: a phase 1, open label, first-in-human study.

BACKGROUND: Effectively implementing strategies to curb SARS-CoV-2 transmission requires understanding who is contagious and when. Although viral load on upper respiratory swabs has commonly been used to infer contagiousness, measuring viral emissions might be more accurate to indicate the chance of onward transmission and identify likely routes. We aimed to correlate viral emissions, viral load in the upper respiratory tract, and symptoms, longitudinally, in participants who were experimentally infected with SARS-CoV-2. METHODS: In this phase 1, open label, first-in-human SARS-CoV-2 experimental infection study at quarantine unit at the Royal Free London NHS Foundation Trust, London, UK, healthy adults aged 18-30 years who were unvaccinated for SARS-CoV-2, not previously known to have been infected with SARS-CoV-2, and seronegative at screening were recruited. Participants were inoculated with 10 50% tissue culture infectious dose of pre-alpha wild-type SARS-CoV-2 (Asp614Gly) by intranasal drops and remained in individual negative pressure rooms for a minimum of 14 days. Nose and throat swabs were collected daily. Emissions were collected daily from the air (using a Coriolis µ air sampler and directly into facemasks) and the surrounding environment (via surface and hand swabs). All samples were collected by researchers, and tested by using PCR, plaque assay, or lateral flow antigen test. Symptom scores were collected using self-reported symptom diaries three times daily. The study is registered with ClinicalTrials.gov, NCT04865237. FINDINGS: Between March 6 and July 8, 2021, 36 participants (ten female and 26 male) were recruited and 18 (53%) of 34 participants became infected, resulting in protracted high viral loads in the nose and throat following a short incubation period, with mild-to-moderate symptoms. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Viral RNA was detected in 63 (25%) of 252 Coriolis air samples from 16 participants, 109 (43%) of 252 mask samples from 17 participants, 67 (27%) of 252 hand swabs from 16 participants, and 371 (29%) of 1260 surface swabs from 18 participants. Viable SARS-CoV-2 was collected from breath captured in 16 masks and from 13 surfaces, including four small frequently touched surfaces and nine larger surfaces where airborne virus could deposit. Viral emissions correlated more strongly with viral load in nasal swabs than throat swabs. Two individuals emitted 86% of airborne virus, and the majority of airborne virus collected was released on 3 days. Individuals who reported the highest total symptom scores were not those who emitted most virus. Very few emissions occurred before the first reported symptom (7%) and hardly any before the first positive lateral flow antigen test (2%). INTERPRETATION: After controlled experimental inoculation, the timing, extent, and routes of viral emissions was heterogeneous. We observed that a minority of participants were high airborne virus emitters, giving support to the notion of superspreading individuals or events. Our data implicates the nose as the most important source of emissions. Frequent self-testing coupled with isolation upon awareness of first symptoms could reduce onward transmissions. FUNDING: UK Vaccine Taskforce of the Department for Business, Energy and Industrial Strategy of Her Majesty's Government.

Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge in young adults.

Since its emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused hundreds of millions of cases and continues to circulate globally. To establish a novel SARS-CoV-2 human challenge model that enables controlled investigation of pathogenesis, correlates of protection and efficacy testing of forthcoming interventions, 36 volunteers aged 18-29 years without evidence of previous infection or vaccination were inoculated with 10 TCID50 of a wild-type virus (SARS-CoV-2/human/GBR/484861/2020) intranasally in an open-label, non-randomized study (ClinicalTrials.gov identifier NCT04865237 ; funder, UK Vaccine Taskforce). After inoculation, participants were housed in a high-containment quarantine unit, with 24-hour close medical monitoring and full access to higher-level clinical care. The study's primary objective was to identify an inoculum dose that induced well-tolerated infection in more than 50% of participants, with secondary objectives to assess virus and symptom kinetics during infection. All pre-specified primary and secondary objectives were met. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Eighteen (~53%) participants became infected, with viral load (VL) rising steeply and peaking at ~5 days after inoculation. Virus was first detected in the throat but rose to significantly higher levels in the nose, peaking at ~8.87 log10 copies per milliliter (median, 95% confidence interval (8.41, 9.53)). Viable virus was recoverable from the nose up to ~10 days after inoculation, on average. There were no serious adverse events. Mild-to-moderate symptoms were reported by 16 (89%) infected participants, beginning 2-4 days after inoculation, whereas two (11%) participants remained asymptomatic (no reportable symptoms). Anosmia or dysosmia developed more slowly in 15 (83%) participants. No quantitative correlation was noted between VL and symptoms, with high VLs present even in asymptomatic infection. All infected individuals developed serum spike-specific IgG and neutralizing antibodies. Results from lateral flow tests were strongly associated with viable virus, and modeling showed that twice-weekly rapid antigen tests could diagnose infection before 70-80% of viable virus had been generated. Thus, with detailed characterization and safety analysis of this first SARS-CoV-2 human challenge study in young adults, viral kinetics over the course of primary infection with SARS-CoV-2 were established, with implications for public health recommendations and strategies to affect SARS-CoV-2 transmission. Future studies will identify the immune factors associated with protection in those participants who did not develop infection or symptoms and define the effect of prior immunity and viral variation on clinical outcome.

Risk factors, symptom reporting, healthcare-seeking behaviour and adherence to public health guidance: protocol for Virus Watch, a prospective community cohort study.

INTRODUCTION: The coronavirus (COVID-19) pandemic has caused significant global mortality and impacted lives around the world. Virus Watch aims to provide evidence on which public health approaches are most likely to be effective in reducing transmission and impact of the virus, and will investigate community incidence, symptom profiles and transmission of COVID-19 in relation to population movement and behaviours. METHODS AND ANALYSIS: Virus Watch is a household community cohort study of acute respiratory infections in England and Wales and will run from June 2020 to August 2021. The study aims to recruit 50 000 people, including 12 500 from minority ethnic backgrounds, for an online survey cohort and monthly antibody testing using home fingerprick test kits. Nested within this larger study will be a subcohort of 10 000 individuals, including 3000 people from minority ethnic backgrounds. This cohort of 10 000 people will have full blood serology taken between October 2020 and January 2021 and repeat serology between May 2021 and August 2021. Participants will also post self-administered nasal swabs for PCR assays of SARS-CoV-2 and will follow one of three different PCR testing schedules based on symptoms. ETHICS AND DISSEMINATION: This study has been approved by the Hampstead National Health Service (NHS) Health Research Authority Ethics Committee (ethics approval number 20/HRA/2320). We are monitoring participant queries and using these to refine methodology where necessary, and are providing summaries and policy briefings of our preliminary findings to inform public health action by working through our partnerships with our study advisory group, Public Health England, NHS and government scientific advisory panels.

Influenza A (H3) illness and viral aerosol shedding from symptomatic naturally infected and experimentally infected cases.

BACKGROUND: It has long been known that nasal inoculation with influenza A virus produces asymptomatic to febrile infections. Uncertainty persists about whether these infections are sufficiently similar to natural infections for studying human-to-human transmission. METHODS: We compared influenza A viral aerosol shedding from volunteers nasally inoculated with A/Wisconsin/2005 (H3N2) and college community adults naturally infected with influenza A/H3N2 (2012-2013), selected for influenza-like illness with objectively measured fever or a positive Quidel QuickVue A&B test. Propensity scores were used to control for differences in symptom presentation observed between experimentally and naturally infected groups. RESULTS: Eleven (28%) experimental and 71 (86%) natural cases shed into fine particle aerosols (P < .001). The geometric mean (geometric standard deviation) for viral positive fine aerosol samples from experimental and natural cases was 5.1E + 3 (4.72) and 3.9E + 4 (15.12) RNA copies/half hour, respectively. The 95th percentile shedding rate was 2.4 log10 greater for naturally infected cases (1.4E + 07 vs 7.4E + 04). Certain influenza-like illness-related symptoms were associated with viral aerosol shedding. The almost complete lack of symptom severity distributional overlap between groups did not support propensity score-adjusted shedding comparisons. CONCLUSIONS: Due to selection bias, the natural and experimental infections had limited symptom severity distributional overlap precluding valid, propensity score-adjusted comparison. Relative to the symptomatic naturally infected cases, where high aerosol shedders were found, experimental cases did not produce high aerosol shedders. Studying the frequency of aerosol shedding at the highest observed levels in natural infections without selection on symptoms or fever would support helpful comparisons.

Minimal transmission in an influenza A (H3N2) human challenge-transmission model within a controlled exposure environment.

Uncertainty about the importance of influenza transmission by airborne droplet nuclei generates controversy for infection control. Human challenge-transmission studies have been supported as the most promising approach to fill this knowledge gap. Healthy, seronegative volunteer 'Donors' (n = 52) were randomly selected for intranasal challenge with influenza A/Wisconsin/67/2005 (H3N2). 'Recipients' randomized to Intervention (IR, n = 40) or Control (CR, n = 35) groups were exposed to Donors for four days. IRs wore face shields and hand sanitized frequently to limit large droplet and contact transmission. One transmitted infection was confirmed by serology in a CR, yielding a secondary attack rate of 2.9% among CR, 0% in IR (p = 0.47 for group difference), and 1.3% overall, significantly less than 16% (p<0.001) expected based on a proof-of-concept study secondary attack rate and considering that there were twice as many Donors and days of exposure. The main difference between these studies was mechanical building ventilation in the follow-on study, suggesting a possible role for aerosols.

The Effect of Influenza Virus on the Human Oropharyngeal Microbiome.

BACKGROUND: Secondary bacterial infections are an important cause of morbidity and mortality associated with influenza infections. As bacterial disease can be caused by a disturbance of the host microbiome, we examined the impact of influenza on the upper respiratory tract microbiome in a human challenge study. METHODS: The dynamics and ecology of the throat microbiome were examined following an experimental influenza challenge of 52 previously-healthy adult volunteers with influenza A/Wisconsin/67/2005 (H3N2) by intranasal inoculation; 35 healthy control subjects were not subjected to the viral challenge. Serial oropharyngeal samples were taken over a 30-day period, and the V1-V3 region of the bacterial 16S ribosomal RNA sequences were amplified and sequenced to determine the composition of the microbiome. The carriage of pathogens was also detected. RESULTS: Of the 52 challenged individuals, 43 developed proven influenza infections, 33 of whom became symptomatic. None of the controls developed influenza, although 22% reported symptoms. The diversity of bacterial communities remained remarkably stable following the acquisition of influenza, with no significant differences over time between individuals with influenza and those in the control group. Influenza infection was not associated with perturbation of the microbiome at the level of phylum or genus. There was no change in colonization rates with Streptococcus pneumoniae or Neisseria meningitidis. CONCLUSIONS: The throat microbiota is resilient to influenza infection, indicating the robustness of the upper-airway microbiome.

Pasteurella multocida meningoencephalitis in an immunocompetent adult with multiple cat scratches.

We present the case of a 49-year-old woman admitted to our Acute Medical Unit with a 2-day history of fever, vomiting and confusion. The patient was alcohol dependent and had sustained several scratches from her pet cat, which her pet dog had licked. She deteriorated in the Emergency Department-developing high fever, worsening confusion and meningism. Blood cultures were taken and broad spectrum antibiotics commenced prior to CT scanning and diagnostic lumbar puncture. Blood cultures and CSF 16S ribosomal PCR confirmed a diagnosis of Pasteurella multocida bacteraemia and meningoencephalitis. The patient was successfully treated with 14 days of intravenous antibiotics. P multocida is a Gram-negative coccobacillus which frequently colonises the nasopharynx of animals; it is a recognised but very rare cause of meningoencephalitis in immunocompetent adults. This case highlights the need to consider P multocida infection in patients with prior animal contact, regardless of their immune status.

Routes of influenza transmission.

Remarkably little is known definitively about the modes of influenza transmission. Thus, important health policy and infection control issues remain unresolved. These shortcomings have been exposed in national and international pandemic preparedness activities over recent years. Indeed, WHO, CDC, ECDC and the U.S. Institute of Medicine have prioritised understanding the modes of influenza transmission as a critical need for pandemic planning. Studying influenza transmission is difficult; seasonality, unpredictable attack rates, role of environmental parameters such as temperature and humidity, numbers of participants required and confounding variables all present considerable obstacles to the execution of definitive studies. A range of investigations performed to date have failed to provide definitive answers and key questions remain. Reasons for this include the fact that many studies have not sought to investigate routes of transmission as a primary objective (instead, they have evaluated specific interventions) and that fieldwork in natural settings, specifically assessing the dynamics and determinants of transmission between humans, has been limited. The available evidence suggests that all routes of transmission (droplet, aerosol and contact) have a role to play; their relative significance will depend on the set of circumstances acting at a given time. Dictating the process are factors related to the virus itself, the host and the environment.

Use of a human influenza challenge model to assess person-to-person transmission: proof-of-concept study.

BACKGROUND: Influenza transmission in humans remains poorly understood. In particular, the relative contribution of contact, large droplet, and aerosol transmission is unknown. The aims of this proof-of-concept study were to determine whether an experimentally induced influenza infection is transmissible between humans and whether this would form a viable platform for future studies. METHODS: In a quarantine facility, healthy volunteers ("donors") were inoculated with A/Wisconsin/67/2005 (H3N2) influenza virus via intranasal drops. On study days 2 and 3 "recipient" volunteers were exposed to donors under close living conditions. Volunteers socialized for 30 hours during a 2-day period. Infection was confirmed by ≥1 positive results from polymerase chain reaction, virus culture, or serology. RESULTS: After inoculation, 4 of 9 donors developed symptoms consistent an influenza-like illness (ILI) and 7 of 9 were proven to be influenza-infected. After exposure, 4 of 15 recipients developed symptoms of ILI and 3 of 15 were proven to be infected. Serum collected within 2 days of study initiation indicated that 1 donor and 3 recipients were seropositive at study initiation. After adjustment for preexposure immunity, the overall secondary attack rate was 25% (3 of 12). CONCLUSIONS: Experimental human exposure studies offer an attractive potential method for answering outstanding questions related to influenza transmission and the evaluation of interventions to reduce it.

Potential role of human challenge studies for investigation of influenza transmission.

The importance of different routes of influenza transmission (including the role of bioaerosols) and the ability of masks and hand hygiene to prevent transmission remain poorly understood. Interest in transmission of influenza has grown as the effectiveness of prevention measures implemented during the 2009 H1N1 pandemic are questioned and as plans to better prepare for the next pandemic are debated. Recent studies of naturally infected patients have encountered difficulties and have fallen short of providing definitive answers. Human challenge studies with influenza virus date back to the 1918 pandemic. In more recent decades they have been undertaken to investigate the efficacy of antiviral agents and vaccines. Could experimental challenge studies, in which volunteers are deliberately infected with influenza virus, provide an alternative approach to the study of transmission? Here, we review the latest intervention studies and discuss the potential of challenge studies to address the remaining gaps in our knowledge.

The first once-daily single-tablet regimen for the treatment of HIV-infected patients.

Since the first description of AIDS cases over 25 years ago, great strides have been made in the treatment of HIV infection. The pursuit of new and novel therapies is ongoing, and a particular drive to simple regimens with low toxicities has been seen over the last few years. The arrival of the first once-daily single-tablet regimen is a noteworthy milestone. ATRIPLA (Bristol-Myers Squibb, Princeton, NJ, and Gilead Sciences, Foster City, CA, USA) is a combination of three drugs that have been well studied and are currently approved as first-line agents internationally. The studies that demonstrate the efficacy and safety of these drugs are reviewed as well as the potential drawbacks. Single-tablet regimens are likely to become the therapies of choice for those commencing antiretroviral treatment, and their place in today's management is discussed.

Pneumocystis pneumonia revisited: delayed diagnosis in an HIV infected individual with high blood and low lung CD4 T cell counts.

The peripheral blood CD4 count is a useful marker of immunological status in HIV infection. However, it makes up only 1% of total body CD4 cells [Parslow T. Lymphocytes and lymphoid tissue. In: Stites D, Terr A, Parslow D, editors. Basic and clinical immunology. Appleton and Lange; 1994. p. 22-40.] has a wide intra- and inter-individual variability [Turner BJ, Hecht FM, Ismail RB. CD4+ T-lymphocyte measures in the treatment of individuals infected with human immunodeficiency virus type 1. A review for clinical practitioners. Arch Intern Med 1994;154:1561-73.] and CD4 cell pathology is just one of the immune defects caused by HIV [Fauci S. Multifactorial nature of human immunodeficiency virus disease. Science 1993;262:1011-8.]. Thus a given value should always be interpreted within a specific clinical context. We describe an individual with Pneumocystis pneumonia (PCP) who was reviewed by HIV medical services several times before the correct diagnosis was made. On each occasion the possibility of PCP was discounted as he had a well-preserved blood CD4 count. Subsequent examination of his pulmonary T-lymphocyte subsets revealed marked CD4 lymphopenia.