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.

An inactivated multivalent influenza A virus vaccine is broadly protective in mice and ferrets.

Influenza A viruses (IAVs) present major public health threats from annual seasonal epidemics and pandemics and from viruses adapted to a variety of animals including poultry, pigs, and horses. Vaccines that broadly protect against all such IAVs, so-called "universal" influenza vaccines, do not currently exist but are urgently needed. Here, we demonstrated that an inactivated, multivalent whole-virus vaccine, delivered intramuscularly or intranasally, was broadly protective against challenges with multiple IAV hemagglutinin and neuraminidase subtypes in both mice and ferrets. The vaccine is composed of four ß-propiolactone-inactivated low-pathogenicity avian IAV subtypes of H1N9, H3N8, H5N1, and H7N3. Vaccinated mice and ferrets demonstrated substantial protection against a variety of IAVs, including the 1918 H1N1 strain, the highly pathogenic avian H5N8 strain, and H7N9. We also observed protection against challenge with antigenically variable and heterosubtypic avian, swine, and human viruses. Compared to control animals, vaccinated mice and ferrets demonstrated marked reductions in viral titers, lung pathology, and host inflammatory responses. This vaccine approach indicates the feasibility of eliciting broad, heterosubtypic IAV protection and identifies a promising candidate for influenza vaccine clinical development.

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.

Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model.

The conserved region of influenza hemagglutinin (HA) stalk (or stem) has gained attention as a potent target for universal influenza vaccines1-5. Although the HA stalk region is relatively well conserved, the evolutionarily dynamic nature of influenza viruses6 raises concerns about the possible emergence of viruses carrying stalk escape mutation(s) under sufficient immune pressure. Here we show that immune pressure on the HA stalk can lead to expansion of escape mutant viruses in study participants challenged with a 2009 H1N1 pandemic influenza virus inoculum containing an A388V polymorphism in the HA stalk (45% wild type and 55% mutant). High level of stalk antibody titers was associated with the selection of the mutant virus both in humans and in vitro. Although the mutant virus showed slightly decreased replication in mice, it was not observed in cell culture, ferrets or human challenge participants. The A388V mutation conferred resistance to some of the potent HA stalk broadly neutralizing monoclonal antibodies (bNAbs). Co-culture of wild-type and mutant viruses in the presence of either a bNAb or human serum resulted in rapid expansion of the mutant. These data shed light on a potential obstacle for the success of HA-stalk-targeting universal influenza vaccines-viral escape from vaccine-induced stalk immunity.

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections.

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1-2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10-15 years on therapy, there were as many as 3.5-5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.