Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8⁺ T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

Influenza virus infection of neonates poses a major health concern, often resulting in severe disease and hospitalization. At present, vaccines for this at-risk population are lacking. Thus, development of an effective vaccine is an urgent need. In this study, we have used an innovative nonhuman primate neonate challenge model to test the efficacy of a novel TLR 7/8 agonist R848-conjugated influenza virus vaccine. The use of the intact virus represents a step forward in conjugate vaccine design because it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high-level virus-specific Ab- and cell-mediated responses in neonates that result in increased virus clearance and reduced lung pathology postchallenge compared with the nonadjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting that combinations of TLR that provide increased efficacy must be determined empirically. These data support further exploration of this new conjugate influenza vaccine approach as a platform for use in the at-risk neonate population.

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 six day old AGM were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild type or mutant flagellin (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following 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-specific T cells following challenge compared to infants vaccinated with m229 adjuvant. Finally, infants vaccinated with IPR8+flagellin exhibited reduced pathology in the lungs following challenge compared to those that received IPR8+m229. This study provides the first evidence of flagellin mediated enhancement of vaccine responses in nonhuman primate neonates.

Summary: Impaired immune responsiveness is a significant barrier to vaccination of neonates. By way of example, the low seroconversion observed following influenza vaccination has led to restriction of its use to infants over 6 months of age, leaving younger infants vulnerable to infection. Our previous studies using a non‐human primate neonate model demonstrated that the immune response elicited following vaccination with inactivated influenza virus could be robustly increased by inclusion of the Toll‐like receptor agonist flagellin or R848, either delivered individually or in combination. When delivered individually, R848 was found to be the more effective of the two. To gain insights into the mechanism through which these adjuvants functioned in vivo, we assessed the initiation of the immune response, i.e. at 24 hr, in the draining lymph node of neonate non‐human primates. Significant up‐regulation of co‐stimulatory molecules on dendritic cells could be detected, but only when both adjuvants were present. In contrast, R848 alone could increase the number of cells in the lymph node, presumably through enhanced recruitment, as well as B‐cell activation at this early time‐point. These changes were not observed with flagellin and the dual adjuvanted vaccine did not promote increases beyond those observed with R848 alone. In vitro studies showed that R848 could promote B‐cell activation, supporting a model wherein a direct effect on neonate B‐cell activation is an important component of the in vivo potency of R848 in neonates.