TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity
Introduction
Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations due to viral infections and causes significant disease in the elderly and in bone marrow transplant recipients. Despite extensive efforts, no safe and effective vaccine or antiviral therapy is currently licensed for RSV. Passive prophylactic administration of the RSV-specific neutralizing antibody Synagis® to premature infants is the only approved intervention for RSV [1]. Development of RSV vaccines has been hampered by failed vaccine trials in which infants immunized with formalin-inactivated alum-precipitated RSV (FI-RSV) were not protected against subsequent RSV infection, but experienced more severe disease, often requiring hospitalization and resulting in two deaths. Despite use of molecularly cloned RSV immunogens it has been difficult to arrive at the right degree of attenuation for live candidate RSV vaccines that have sufficient immunogenicity and acceptable reactogenicity in all infants [2].
Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) serving as sentinel molecules for detection of pathogens expressing pathogen-associated pattern molecules (PAMPs) [3]. Several TLRs are of particular importance to induction of antiviral immunity and include TLR3, TLR7 and TLR8, and TLR9 [4]. While traditionally thought of as dendritic cell proteins, PRRs are also expressed on many cells of immune and non-immune function. Distribution of these TLRs and their impact on antiviral immunity has been recently reviewed [5], [6]. TLR–PAMP interactions at mucosal surfaces elicit cytokine production, recruiting dendritic cells to and initiating immune responses at sites of inflammation and infection. Thus, selective activation of individual TLRs through agonist binding has been hypothesized as a means to modulate and enhance immunity [7].
While severe RSV disease, induced by immunization, allergen sensitization in animal models, or primary infection in humans, has often been associated with the induction of type 2 T cell responses and eosinophilia, the complexity of RSV disease suggests there are multiple factors, of both viral and host origin, contributing to immunopathology [8], [9], [10], [11]. Interference with cytokine function and the use of adjuvants does not completely alleviate disease, but has modest effects or modulates only certain aspects of disease [12], [13], [14], [15], [16], [17], illustrating the intricacies of RSV pathogenesis. The multi-factorial nature of severe RSV disease led us to hypothesize that “targeted” activation of specific innate immune response pathways might more effectively induce protective and disease-sparing immunity than interference with a single component of adaptive immunity. We therefore investigated TLR agonists as adjuvants or immunotherapy for RSV. TLR7/8 and TLR9 agonists were used to modulate the immune responses induced by FI-RSV immunization either during the initial induction phase (at priming) or during the recall effector phase (at challenge), hypothesizing that administration of TLR agonists would boost the induction of type 1 primary immune responses to minimize generation of Th2 responses during immunization and to counterbalance Th2 memory responses during challenge. In particular, we hypothesized that augmenting relevant TLR7/8 signaling pathways may provide an immune advantage to the host since TLR7 and TLR8 recognize ssRNA, creating responses well-suited to control ssRNA viruses such as RSV. Natural RSV infection does not induce long-term protective immunity [18], [19]. We therefore evaluated the potential therapeutic use of TLR agonists to enhance RSV-specific immune responses during primary infection, hypothesizing that TLR-mediated signals might overcome the ability of RSV to inhibit type I IFN production [20], [21] and result in sustained immunity.
In the course of these studies, reports were published examining CpG effects on immune responses induced during immunization with purified RSV F or G [22], [23], [24] or with killed bovine RSV [25], [26] or during allergen sensitization [27]. Collectively, these studies show the presence of CpG during induction of immunity results in more Th1-associated immune responses and increases protection against RSV challenge. We confirm these studies using formalin-inactivated human RSV and expand upon them evaluating a broad array of cytokines and chemokines. Furthermore, we test the efficacy of TLR7/8 agonists as an adjuvant, examine the ability of CpG and TLR7/8 agonists given at challenge to modulate Th2 memory responses generated by FI-RSV, and evaluate the effects of these agonists when given during primary RSV infection. While FI-RSV will not be used as a vaccine in humans, it is a well-defined complex, multi-component immunogen that induces type 2 memory immune responses and provides a system for evaluating immunomodulatory strategies. These data support the potential for using TLR agonists during RSV immunization, even in the context of a more complex whole virus immunogen. However, the importance of timing and the specific TLR agonist used in a particular host will have to be cautiously evaluated due to the potential for TLR agonists to induce immunopathology rather than immunoprotection.
Section snippets
Viruses and TLR agonists
HEp-2 and Vero cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in Eagle's minimal essential media supplemented with 10% fetal calf serum, glutamine, and antibiotics (10% EMEM). A challenge stock of RSV A2 strain was prepared as previously described [28]. A formalin-inactivated alum-precipitated stock RSV (FI-RSV) was prepared in Vero cells as described [29]. CpG 1826 oligodeoxynucleotides (Class B) were purchased from Coley Pharmaceutical
Reduction in vaccine-enhanced RSV disease is dependent on TLR specificity and timing of administration
Mice were immunized with FI-RSV containing CpG, TLR7/8 agonist, or both. Statistically significant improvements in weight loss were observed when CpG was present during FI-RSV immunization, with either CpG alone (Fig. 1A, p < 0.05 relative to FI-RSV only controls on days 7–10 post-infection) or in combination with TLR7/8 agonist (days 5–10 post-infection). In contrast, TLR7/8 agonist alone had minimal impact. Administration of TLR agonists during RSV challenge of FI-RSV-immunized mice increased
Discussion
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
Acknowledgements
We wish to acknowledge the technical assistance of Saran Bao. This research was supported by the Intramural Research Program of the National Institutes of Health, NIAID, Vaccine Research Center.
Conflict of interest statement: The authors have no conflict of interest to report.
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