The young age of primary RSV infection, immunosenescence of the elderly, the failure of natural RSV infection to generate long-term protective immunity, and the history of vaccine-enhanced disease present considerable challenges to development of a safe and effective RSV vaccine. The ability of TLR agonists to modulate immunity provides new strategies that may improve vaccine design. In particular, TLR agonists have been shown to stimulate mature immune responses in neonates [38
]. We therefore tested the ability of TLR7/8 and TLR9 agonists (1) to serve as adjuvants and modulate FI-RSV-enhanced disease immunization and (2) to serve as a potential therapeutic agent and alter pathogenesis during live RSV infection of naïve or immunized mice.
CpG has been proven an effective adjuvant in multiple models [22
] inducing more type 1 CD4+ and CD8+ T cells. We demonstrate that CpG administration during FI-RSV immunization decreased disease and induced more Th1-like immune responses, reducing the Th2 bias of RSV-specific immunity induced by immunization with FI-RSV or purified RSV proteins. While TLR7/8 agonists have been successfully used as adjuvants during immunization with purfied HIV-1 proteins [30
], this was not an effective strategy during FI-RSV immunization. This may be due to the complexity of alum-precipitated whole virus immunogen. It may be that the viral RNA present in FI-RSV saturated the TLR7 and TLR8 receptors in endosomal compartments. In contrast, administration of both TLR agonists tended to increase disease when given during live RSV challenge in the setting of pre-existing RSV immunity induced by FI-RSV priming. Addition of either TLR7/8 or TLR9 agonist during RSV infection enhanced disease severity as evidenced by weight loss () and higher levels of MIP-1α in the lung ( and ). Elevated levels of MIP-1α have previously been associated with severe illness in RSV-infected infants [41
] and in murine models of RSV and pneumonia virus of mice pathogenesis [42
]. Similarly, mice treated with TLR agonists during RSV infection often exhibited elevated levels of IFN-γ. This finding may suggest increased NK, NK T, and/or CD8+ T cell activity.
Administration of TLR agonists during RSV infection has the potential to broadly impact viral pathogenesis due to TLR expression not only on pulmonary dendritic cells [44
], but also on airway epithelium [45
] and NK cells present early during infection [47
]. Interactions between RSV and several TLRs have been demonstrated as reviewed [48
]. In the murine model of RSV pathogenesis NK T cells contribute to the activation and recruitment of virus-specific CD8+ T cells [49
], and the magnitude of IFN-γ-producing NK cells correlates with the subsequent level of CD8+ T cell recruitment [50
]. In addition, excessive numbers of RSV-specific CD8+ T cells result in severe disease during RSV infection [51
]. MIP-1α has been shown to enhance CTL survival and function [52
]. Thus, activation of dendritic cells, NK cells, and NK T cells and increased MIP-1α production induced by TLR agonists at the wrong time may amplify the virus-specific CD8+ T cell response, increasing immunopathology and disease. Similar factors may be involved in the enhanced pulmonary pathology observed after RSV challenge of cotton rats immunized with CpG and RSV F protein [24
]. RSV has been shown to result in prolonged retention of DCs in the lung [32
]. Thus, the two week interval between the second CpG-F intranasal immunization and RSV challenge may have resulted in altered T cell differentiation or recruitment, leading to severe pulmonary pathology. Additional analyses of the post-challenge immune responses would be informative in elucidating these mechanisms and contributing factors. While increased innate immunity has generally been associated with greater protection against RSV infection [20
] and adaptive immune responses have more often been associated with disease [55
], these data show innate responses can also contribute to immunopathology, depending upon the magnitude, timing, and composition. However, more extensive analyses are required to determine the precise basis for the enhanced disease severity observed here.
We also demonstrate that the genetic background of the host has consequences on the patterns of virus-specific immune responses, particularly when TLR treatment occurs during live RSV infection. While FI-RSV immunization uniformally predisposes for type 2 immune responses, the strain of mouse impacts the magnitude of the response as evidenced by at least twice as many eosinophils recruited to the lungs of FI-RSV-immunized RSV-challenged BALB/c mice compared to C57BL/6 mice [34
]. Though not tested, it is hypothesized that CpG would enhance induction of Th1 immunity in all strains of mice, but that selected strains (e.g. C57BL/6) would evidence greater Th2 to Th1 shifts than would strains such as BALB/c. Studies demonstrate differences in early innate responses of particular strains induce more rapid type I responses, e.g. C57BL/6 mice have more NK cells while A/J mice have low NK cell activity [56
] and 129 mice have greater numbers of pDCs [57
]). Any one or a combination of these factors may then impact the induction of T cell and cytokine responses and result in different patterns of responsiveness in TLR treatment of various mouse strains.
These studies show CpG, but not TLR7/8 agonist, is an effective adjuvant capable of modulating FI-RSV-induced immune responses immunization, demonstrating a level of specificity in the ability of TLR agonists to function as vaccine adjuvants. Importantly, we demonstrate that the timing of immune interventions is of critical importance in RSV pathogenesis. While we have not identified the specific components of the immune responses resulting in increased disease, these data underscore that a precarious balance of RSV immunity exists and must be maintained to elicit immunoprotection without concomitant immunopathology.