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

Abstract

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.

Keywords: correlates of protection; human challenge; influenza; influenza vaccines; mucosal immunity.

Fig 1

Clinical outcomes after challenge demonstrating duration (days) of shedding (A), duration (days) of symptoms (B), total number of symptoms (C), and FLU-PRO (D) scores, which standardize symptom severity based on number of symptoms and duration with higher numbers signifying more severe illness. Individual values represented by dots for unvaccinated participants in blue and vaccinated participants in yellow. Horizontal lines represent medians and first and third quartiles. P-values from Wilcoxon rank sum tests comparing unvaccinated and vaccinated participants shown, with P < 0.05 considered statistically significant.

Fig 2

Antibody titers after vaccination (phase 1) and H1N1 viral challenge (phase 2) as tested by HA inhibition (HAI) assays (A), ELISA for nasal anti-HA IgA (B), and ELISA for serum anti-HA IgG (C). In phase 1, day 0 represents baseline titers on the day of vaccination. Subsequently, in phase 2, day −1 represents baseline titers prior to viral challenge on day 0. Individual values represented by dots for unvaccinated participants in blue and vaccinated participants in yellow. Horizontal lines represent medians and first and third quartiles. Vertical lines represent minimum and maximum non-outliers. P-values from Wilcoxon rank sum tests, adjusted for multiple comparisons by Holm’s method, comparing unvaccinated and vaccinated participants shown, with P < 0.05 considered statistically significant. Important non-significant (N.S.) comparisons also shown.

Fig 3

Antibody titers after vaccination (phase 1) and H1N1 viral challenge (phase 2) as tested by NAI assays (A), ELISA for nasal anti-NA IgA (B), and ELISA for serum anti-NA IgG (C). In phase 1, day 0 represents baseline titers on the day of vaccination. Subsequently, in phase 2, day −1 represents baseline titers prior to viral challenge on day 0. Individual values represented by dots for unvaccinated participants in blue and vaccinated participants in yellow. Horizontal lines represent medians and first and third quartiles. Vertical lines represent minimum and maximum non-outliers. P-values from Wilcoxon rank sum tests, adjusted for multiple comparisons by Holm’s method, comparing unvaccinated and vaccinated participants shown, with P < 0.05 considered statistically significant. Important N.S. comparisons also shown.

Fig 4

Antibody titers after vaccination (phase 1) and H1N1 viral challenge (phase 2) as tested by ELISA for nasal anti-HA stalk IgA (A) and ELISA for serum anti-HA stalk IgG (B). In phase 1, day 0 represents baseline titers on the day of vaccination. Subsequently, in phase 2, day −1 represents baseline titers prior to viral challenge on day 0. Individual values represented by dots for unvaccinated participants in blue and vaccinated participants in yellow. Horizontal lines represent medians and first and third quartiles. Vertical lines represent minimum and maximum non-outliers. P-values from Wilcoxon rank sum tests, adjusted for multiple comparisons by Holm’s method, comparing unvaccinated and vaccinated participants shown, with P < 0.05 considered statistically significant. Important N.S. comparisons also shown.

Fig 5

Heatmaps of Spearman rank correlation coefficients between serum and nasal antibody titers. Numbers within squares and square colors represent rho values, with blue indicating positive and red representing negative correlations. Green asterisks indicate statistically significant comparisons after the Bonferroni adjustment. Black squares represent comparisons left untested for scientific or statistical reasons.