Mucosal correlates of protection after influenza viral challenge of vaccinated and unvaccinated healthy volunteers.

The induction of systemic antibody titers against hemagglutinin has long been the main focus of influenza vaccination strategies, but mucosal immunity has also been shown to play a key role in the protection against respiratory viruses. By vaccinating and challenging healthy volunteers, we demonstrated that inactivated influenza vaccine (IIV) modestly reduced the rate of influenza while predominantly boosting serum antibody titers against hemagglutinin (HA) and HA stalk, a consequence of the low neuraminidase (NA) content of IIV and the intramuscular route of administration. The viral challenge induced nasal and serum responses against both HA and NA. Correlations between mucosal IgA and serum IgG against specific antigens were low, whether before or after challenge, suggesting a compartmentalization of immune responses. Even so, volunteers who developed viral shedding for multiple days had lower baseline titers across both systemic and mucosal compartments as compared to those with no shedding or a single day of shedding. Regression analysis showed that pre-challenge HA inhibition titers were the most consistent correlate of protection across clinical outcomes combining shedding and symptoms, with NA inhibition titers and HA IgG levels only predicting the duration of shedding. Despite the inclusion of data from multiple binding and functional antibody assays against HA and NA performed on both serum and nasal samples, multivariate models were unable to account for the variability in outcomes, emphasizing our imperfect understanding of immune correlates in influenza and the importance of refining models with assessments of innate and cellular immune responses.IMPORTANCEThe devastating potential of influenza has been well known for over 100 years. Despite the development of vaccines since the middle of the 20th century, influenza continues to be responsible for substantial global morbidity and mortality. To develop next-generation vaccines with enhanced effectiveness, we must synthesize our understanding of the complex immune mechanisms culminating in protection. Our study outlines the differences in immune responses to influenza vaccine and influenza infection, identifying potential gaps in vaccine-induced immunity, particularly at the level of the nasal mucosa. Furthermore, this research underscores the need to refine our imperfect models while recognizing potential pitfalls in past and future attempts to identify and measure correlates of protection.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Leveraging CTSA hubs for rapid, large-scale, high-impact research: A case study during a global public health emergency.

As the COVID-19 pandemic took hold in the USA in early 2020, it became clear that knowledge of the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among asymptomatic individuals could inform public health policy decisions and provide insight into the impact of the infection on vulnerable populations. Two Clinical and Translational Science Award (CTSA) Hubs and the National Institutes of Health (NIH) set forth to conduct a national seroprevalence survey to assess the infection's rate of spread. This partnership was able to quickly design and launch the project by leveraging established research capacities, prior experiences in large-scale, multisite studies and a highly skilled workforce of CTSA hubs and unique experimental capabilities at the NIH to conduct a diverse prospective, longitudinal observational cohort of 11,382 participants who provided biospecimens and participant-reported health and behavior data. The study was completed in 16 months and benefitted from transdisciplinary teamwork, information technology innovations, multimodal communication strategies, and scientific partnership for rigor in design and analytic methods. The lessons learned by the rapid implementation and dissemination of this national study is valuable in guiding future multisite projects as well as preparation for other public health emergencies and pandemics.

Sex Differences in Influenza: The Challenge Study Experience.

BACKGROUND: Preclinical animal studies and retrospective human studies suggest that adult females have worse outcomes from influenza than males. Prospective studies in humans are missing. METHODS: Data from 164 healthy volunteers who underwent influenza A/California/04/2009/H1N1 challenge were compiled to compare differences between sexes. Baseline characteristics, including hormone levels, hemagglutination inhibition (HAI) titers, neuraminidase inhibition (NAI) titers, and outcomes after challenge were compared. Linear and logistic regression models were built to determine significant predictor variables with respect to outcomes of interest. RESULTS: HAI titers were similar between the sexes, but NAI titers were higher in males than females at 4 weeks and 8 weeks postchallenge. Females were more likely to have symptoms (mean, 0.96 vs 0.80; P = .003) and to have a higher number of symptoms (median, 3 vs 4; P = .011) than males. Linear and logistic regression models showed that prechallenge NAI titers, but not HAI titers or sex hormone levels, were predictive of all shedding and symptom outcomes of interest. CONCLUSIONS: Females in our cohorts were more likely to be symptomatic and to have a higher number of symptoms than males. NAI titers predicted all outcomes of interest and may explain differential outcomes between the sexes.

Undiagnosed SARS-CoV-2 seropositivity during the first 6 months of the COVID-19 pandemic in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

Safety and Efficacy of CR6261 in an Influenza A H1N1 Healthy Human Challenge Model.

BACKGROUND: It is imperative to identify new targets for improved vaccines and therapeutics against influenza. One such target is the relatively conserved stalk region of the influenza A hemagglutinin (HA) surface protein. METHODS: We conducted a randomized, double-blind, phase 2, placebo-controlled trial of a monoclonal antibody that targets the HA stalk (CR6261) in a H1N1pdm09 healthy volunteer human challenge model. A single 50 mg/kg dose of CR6261 was infused 24 hours after challenge. The primary efficacy outcome was area under the curve (AUC) of viral RNA detection over time. RESULTS: Ninety-one healthy volunteers were randomized and underwent influenza challenge; 49 received CR6261 and 42 received placebo. CR6261 had no statistically significant effect on AUC (AUC, 48.56 log [copies/mL] × days, interquartile range [IQR], 202 vs AUC, 25.53 log [copies/mL] × days, IQR, 155; P = .315) and no clinically significant effect on influenza disease measures including number of symptoms, duration of symptoms, or inFLUenza Patient-Reported Outcome (FLU-PRO) scores. Preexisting anti-NA antibody titers were most predictive of reduced influenza disease. CR6261 reached a mean peak serum concentration of 1 × 106 ng/mL 15 minutes after infusion and a mean peak of 5.97 × 102 ng/mL in the nasal mucosa 2-3 days after infusion. CONCLUSIONS: The results of this study suggest that a monoclonal anti-stalk approach to prevent or treat influenza infection may be limited in efficacy. Future approaches should consider including and evaluating anti-stalk antibodies as part of a multifaceted strategy rather than as a stand-alone therapeutic. CLINICAL TRIALS REGISTRATION: NCT02371668.

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

BACKGROUND: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. METHODS: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual. FINDINGS: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63). INTERPRETATION: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. FUNDING: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

A Dose-finding Study of a Wild-type Influenza A(H3N2) Virus in a Healthy Volunteer Human Challenge Model.

BACKGROUND: The development of vaccines and therapeutics has relied on healthy volunteer influenza challenge studies. A validated human infection model with wild-type A(H1N1)pdm09 was reported previously. Our objective was to characterize a wild-type influenza A/Bethesda/MM1/H3N2 challenge virus in healthy volunteers. METHODS: Participants received a single dose of a cell-based, reverse-genetics, Good Manufacturing Practices-produced wild-type influenza A(H3N2)2011 virus intranasally and were isolated at the National Institutes of Health Clinical Center for ≥9 days. Dose escalation was performed from 104 to 107 TCID50 (50% tissue culture infectious dose). Viral shedding and clinical disease were evaluated daily. RESULTS: Of 37 participants challenged, 16 (43%) had viral shedding and 27 (73%) developed symptoms, with 12 (32%) participants experiencing mild to moderate influenza disease (MMID), defined as shedding and symptoms. Only participants receiving 106 and 107 TCID50 experienced MMID at 44% and 40%, respectively. Symptom severity peaked on day 3, whereas most viral shedding occurred 1-2 days after challenge. Only 10 (29%) participants had a ≥4-fold rise in hemagglutination inhibition antibody titer after challenge. CONCLUSIONS: The A/Bethesda/MM1/H3N2 challenge virus safely induced MMID in healthy volunteers, but caused less MMID than the A(H1N1)pdm09 challenge virus even at the highest dose. There was less detection of shedding though the incidence of symptoms was similar to A(H1N1)pdm09. Fewer serum anti-hemagglutinin (HA) antibody responses with less MMID indicate that preexisting immunity factors other than anti-HA antibody may limit shedding in healthy volunteers. This A/Bethesda/MM1/H3N2 challenge virus can be utilized in future studies to further explore pathogenesis and immunity and to evaluate vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02594189.