Inclusion of Flagellin during Vaccination against Influenza Enhances Recall Responses in Nonhuman Primate Neonates

Abstract

Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four-to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates. Influenza virus remains one of the leading causes of morbidity and mortality worldwide. Infants less than 6 months of age are particularly vulnerable to development of severe disease following infection (1). Diseases associated with influenza virus infection in children include otitis media, pneumonia, myositis, and croup. While oseltamivir (Tamiflu), one of the two FDA-approved antiinfluenza drugs, can be used in infants aged 2 weeks and older, concerns exist due to the potential for adverse effects, drug resistance, and limited effectiveness in young infants (2).Currently, there are three approved approaches for vaccination against influenza in the United States: intramuscular (i.m.) administration of inactivated influenza virus, intramuscular administration of recombinant hemagglutinin (HA) proteins, and intranasal administration of a live attenuated influenza virus (LAIV). The first is approved for use in individuals aged 6 months and older, the second for use in individuals aged 18 to 49 years, and the last for use in healthy individuals aged 2 to 49 years. Thus, none are approved for use in the vulnerable neonate population. While the lack of approval for the use of these vaccines in the very young may reflect some safety concerns, a principal factor is the poor immune responses elicited in human neonates (3,4).Previous studies, while limited, have shown that an initia...

A Novel R848-Conjugated Inactivated Influenza Virus Vaccine Is Efficacious and Safe in a Neonate Nonhuman Primate Model

“…The increases observed with R848 are significantly greater than those observed in our previous study in which the utility of flagellin alone was assessed (43). Thus, the novel approach of conjugation of R848 to the virion studied here results in a superior vaccine.…”

“…1B). This level was not higher than that observed in our previous study of vaccination with flagellin as the sole adjuvant (43). Thus, the increase in CRP appears to be mediated predominantly by flagellin.…”

“…To assess candidate vaccines for the ability to elicit robust immune responses in young infants, we developed an African green monkey model of neonate vaccination (43). This serves as a tractable model for the human infant as it provides a period of infancy of adequate length to effectively evaluate prime boost strategies.…”

“…In some cases we also employed a combination adjuvant approach by including the soluble TLR5 agonist flagellin (flg), which was shown in our previous studies to possess some potential to enhance both T and B cell recall responses, i.e. the effect of flagellin was restricted to boosting (43). Control animals received inactivated PR8 together with an inactive flagellin construct (m229, non-adjuvanted control) or PBS (non-vaccinated control).…”

“…This approach was chosen as it is a direct measure of the ability of the antibody to prevent infection. The assay was validated against the standard HAI assay, finding that it provided similar relative results (43). The 2-way repeated measures ANOVA model found a significant day by group interaction and as such pairwise comparisons among groups were performed separately on each day.…”