The administration of intranasal live attenuated influenza vaccine induces changes in the nasal microbiota and nasal epithelium gene expression profiles

Abstract

Background: Viral infections such as influenza have been shown to predispose hosts to increased colonization of the respiratory tract by pathogenic bacteria and secondary bacterial pneumonia. To examine how viral infections and host antiviral immune responses alter the upper respiratory microbiota, we analyzed nasal bacterial composition by 16S ribosomal RNA (rRNA) gene sequencing in healthy adults at baseline and at 1 to 2 weeks and 4 to 6 weeks following instillation of live attenuated influenza vaccine or intranasal sterile saline. A subset of these samples was submitted for microarray host gene expression profiling.

Results: We found that live attenuated influenza vaccination led to significant changes in microbial community structure, diversity, and core taxonomic membership as well as increases in the relative abundances of Staphylococcus and Bacteroides genera (both p < 0.05). Hypergeometric testing for the enrichment of gene ontology terms in the vaccinated group reflected a robust up-regulation of type I and type II interferon-stimulated genes in the vaccinated group relative to controls. Translational murine studies showed that poly I:C administration did in fact permit greater nasal Staphylococcus aureus persistence, a response absent in interferon alpha/beta receptor deficient mice.

Conclusions: Collectively, our findings demonstrate that although the human nasal bacterial community is heterogeneous and typically individually robust, activation of a type I interferon (IFN)-mediated antiviral response may foster the disproportionate emergence of potentially pathogenic species such as S. aureus.

Trial registration: This study was registered with Clinicaltrials.gov on 11/3/15, NCT02597647 .

Fig. 1

Measures of diversity in LAIV and control groups, by visit as ascertained by hypervariable regions V3–V5 sequencing. a demonstrates calculated tail statistics at all time points for either group (LAIV visit 1 to 2 p = 0.020; LAIV visit 2 to 3 p = 0.017). b demonstrates Shannon indices for all time points (LAIV visit 1 to 2 p = 0.014; LAIV visit 2 to 3 p = 0.049). (*p < 0.05 by Wilcoxon signed-rank testing)

Fig. 2

Multidimensional scaling plots demonstrating genus-level dissimilarities by Bray-Curtis indices between the first two visits in the control (a) and LAIV (b) groups. Data points from the first visits are in blue, while the second visits are in red. Circles represent respective centroid locations. V1 = visit 1, V2 = visit 2

Fig. 3

Ubiquity-ubiquity plot depicting the change in ubiquities for selected taxa using operational taxonomic units (OTUs) across the range of relative abundances between visits 1 and 2 in the LAIV group as ascertained by hypervariable regions V3–V5 sequencing. Only OTUs with a relative abundance greater than 0.5 % with a change in ubiquity greater than 20 % were included in this plot. OTUs that aligned to the right of the diagonal were more ubiquitous during visit 1, while those to the left were more ubiquitous at visit 2. The taxonomic classifications labeled are the closest, most confident taxonomic assignment that could be given to the OTU in question

Fig. 4

Absolute changes in relative abundance in the top five changed taxa in the control (a) and LAIV (b) groups as ascertained by hypervariable regions V3–V5 sequencing (in the LAIV group, Staphylococcus p = 0.048, Bacteroides p = 0.013). (*p < 0.05 by Wilcoxon signed-rank testing)

Fig. 5

A heat map of log₂ gene expression for the most differentially expressed genes in the LAIV group, selected by thresholds of p value <0.01 and log₂ fold changes greater than 0.7 between visits. The left hand column shows selected gene ontology associations from the hypergeometric GO/BP analysis as described. Given significant overlap between gene ontology terms, representative groups were chosen to represent as many unique and non-overlapping GO/BP terms as possible

Fig. 6

Gene set enrichment analysis for type I (a) and type II (b) interferon-stimulated genes in our expression sets. These figures demonstrate a probability density function for each gene, the center of which is marked by a vertical line on each x-axis. Distributions that lie predominantly to the right of the 0 mark are more likely up-regulated, while those to the left are not. Peaked and therefore narrower functions are represented by green colors, while broader distributions will tend towards the color blue. The sum of these functions, corrected for inter-gene correlation, is represented by a thick black distribution for each group. The LAIV group is shown on the upright graph, and the control group is drawn in the opposite direction for comparison. The LAIV group showed significant gene set enrichment in both types I and II interferon sets, when compared with the control group (p < 0.05)

Fig. 7

MRSA colonization is type I interferon-dependent. a Wild type (WT) C57Bl/6 mice, type I IFN receptor knockout (IFNAR KO), and type II IFN receptor knockout mice (IFNGR KO) underwent intranasal instillation of MRSA (6.7 × 10⁷ CFU). Twenty-four hours later, nasal lavage was performed using 200 μl of sterile saline for enumeration of CFU. b wild type, IFNAR, and IFNGR knockout mice were administered intranasal poly I:C (50 mcg) daily for 3 days, followed by i.n. MRSA (6.7 × 10⁷ CFU). Twenty-four hours later, nasal lavage was performed for enumeration of CFU. (**adjusted p < 0.01 by Kruskal-Wallis ANOVA with Dunn correction for multiple comparisons)