Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Abstract

Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.

Keywords: Human nasal epithelial cells; Influenza; Innate immune response; Live attenuated influenza vaccine (LAIV).

Figure 1

Attenuation of Infectious LAIV Virus Production during Infection of hNEC cultures at 33°C. hNEC cultures were infected with wild type (WT) and an antigenically-matched cold adapted (CA) LAIV strain at an MOI of 10 in triplicate at 33°C. Virus titers in apical supernatants were measured by calculating the TCID₅₀/ml in MDCK cells at the indicated hours post infection (HPI). The inoculum (labeled input) was back tittered in parallel. The limit of detection is shown by the horizontal thin dotted line. Data are expressed as mean + SEM. *** p<.001 (n=7 different donors for hNECs).

Figure 2

Increased Expression of Pattern Recognition Receptors during LAIV Infection of hNEC cultures. Total RNA from WT and LAIV infected hNECs at 24HPI was analyzed for expression of RIG-I (A) and TLR-3 (B) by real time qRT-PCR and compared using the ΔΔ Ct method. Data are represented as the fold change in expression from Mock infected cultures. *p<.05 (n=7 different donors for hNECs).

Figure 3

hNEC cultures Infected with LAIV Express an Augmented Antiviral Innate Immune Response. Total RNA from hNECs collected at 24 hours post infection with WT and CA viruses was analyzed for the expression of antiviral innate immune gene expression including Interferon-β (A), IRF-7 (B), MxA (C), and STAT-1 (D) by real time qRT-PCR and compared using the ΔΔ Ct method. Data are represented as the fold change in expression from Mock infected cultures. *p<.05 (n=7 different donors for hNEC).

Figure 4

LAIV infected Nasal Epithelial Cell Cultures Contain Greater Levels of Viral RNA. Total RNA from WT and LAIV infected hNEC cultures collected at 24HPI were analyzed for the presence of viral RNA using qRT-PCR with primers specific for the Influenza M-segment. Levels of viral RNA were compared using the ΔΔ Ct method and represent the fold-change in expression relative to a WT-infected sample. p<.05 (n=7 different donors for hNEC).

Figure 5

WT and LAIV-Infected hNEC cultures Result in a Differential Chemokine Response. Apical and basolateral supernatants were collected 24hours after hNEC infection with WT and LAIV viruses and analyzed for the presence of IL-8 (A and B) and IP-10 (C and D) by ELISA and are displayed as fold change in expression relative to Mock-infected cultures. *p<.05 (n=7 different donors for hNEC).