Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3 −/− mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.

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. Here 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 as it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high level virus-specific antibody and cell mediated responses in neonates that result in increased virus clearance and reduced lung pathology following challenge compared to the non-adjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting 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 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...

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Respiratory infection of young infants results in increased morbidity and mortality compared to infection of adults. In spite of the significance of this health issue, our understanding of the immune response elicited in infants, especially in the respiratory tract is highly limited. We developed a nonhuman primate model to probe the virus-specific antibody response in infants following infection with influenza virus. Infection of infants resulted in more pulmonary damage and higher viral loads compared to adults. While the systemic IgG antibody response was similar in infant and adult animals, the response in the upper respiratory tract of the infant was compromised. This lower response was associated with an increased prevalence of Treg cells and low levels of BALT. These data suggest a defect in the ability to produce effective virus-specific antibody responses at the local infection site is a contributor to increased pulmonary damage in the at-risk infant population.

Streptococcus pneumoniae (pneumococcus) is both a widespread nasal colonizer and a leading cause of otitis media, one of the most common diseases of childhood. Pneumococcal phase variation influences both colonization and disease and thus has been linked to the bacteria's transition from colonizer to otopathogen. Further contributing to this transition, coinfection with influenza A virus has been strongly associated epidemiologically with the dissemination of pneumococci from the nasopharynx to the middle ear. Using a mouse infection model, we demonstrated that coinfection with influenza virus and pneumococci enhanced both colonization and inflammatory responses within the nasopharynx and middle ear chamber. Coinfection studies were also performed using pneumococcal populations enriched for opaque or transparent phase variants. As shown previously, opaque variants were less able to colonize the nasopharynx. In vitro, this phase also demonstrated diminished biofilm viability and epithelial adherence. However, coinfection with influenza virus ameliorated this colonization defect in vivo. Further, viral coinfection ultimately induced a similar magnitude of middle ear infection by both phase variants. These data indicate that despite inherent differences in colonization, the influenza A virus exacerbation of experimental middle ear infection is independent of the pneumococcal phase. These findings provide new insights into the synergistic link between pneumococcus and influenza virus in the context of otitis media.

Infection with influenza A virus (IAV) 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. Here we show that coinfection of mice with influenza virus followed by the non-invasive 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 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 interferon γ exhibited a decreased potential for co-production of tumor necrosis factor α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node (MLN) was minimal compared to that present in the lung, supporting a model of negative regulation in the tissue harboring high pathogen burden. These results elucidate a new aspect of immune regulation as a result of entry of a coinfecting pathogen, modulation of ongoing adaptive immune responses in the lung. These findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens.