The respiratory tract is a major portal for pathogens. The bronchoalveolar macrophage, positioned at the mucosal surface, recognizes "pathogen associated molecular patterns (PAMPs)" through "pattern recognition receptors (PRRs)." A family of mammalian PRRs, "Toll-like receptors" (TLRs), are transmembrane signaling molecules that respond to diverse PAMPs. Gram negative lipopolysaccharide (LPS) stimulates cells through TLR4 to elicit a strongly proinflammatory pattern of gene expression, resulting in a "Th1-type" cytokine milieu. Respiratory syncytial virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and young children worldwide, and has recently been attributed to increased morbidity and mortality in the elderly and immunosuppressed. The RSV fusion (F) protein is also a TLR4 agonist. Prophylactic administration of anti-F antibodies to high-risk infants is highly protective. In a failed clinical trial in the 1960's, a formalin-inactivated RSV (FI-RSV) vaccine led to exacerbated RSV disease, findings we have recapitulated in the cotton rat (S. hispidus), assessed by pulmonary histopathology and airway hyperreactivity. During the first cycle of this grant, we identified cyclooxygenase-2 and prostanoids as key therapeutic targets for RSV-induced lung pathology. We found that vaccination of cotton rats with the original FI-RSV used in the failed trials, newly formulated with a non-toxic adjuvant and TLR4 agonist, monophosphoryl lipid A (MPL), suppressed FI-RSV vaccine-enhanced disease by blunting the mixed Th1- and Th2-type "cytokine storm" that is elicited upon RSV infection of vaccinated subjects. In vitro, purified F protein activation of NF-?B and IL-8 secretion in HEK293T cells is TLR4-, MD-2-, and CD14-dependent, and transfectants that express TLR4 proteins with one or both of two single nucleotide polymorphisms (SNPs), previously associated with LPS-hyporesponsiveness, were significantly less responsive to purified RSV F protein, under conditions of equal TLR expression. Importantly, we identified a highly significant overrepresentation of these TLR4 SNPs in DNA samples derived from a case series of high-risk infants and children with documented RSV infection. These data strongly support our overarching hypothesis that TLR4 plays a central role in the innate immune response to RSV and imply that initial engagement of TLR4 is required for development of a protective, adaptive immune response, rather than a pathological one. This proposal details innovative experimental approaches that will (i) lead to development a safe and effective RSV F protein subunit vaccine, (ii) lead to development of therapeutic intervention strategies based on a characterization of the interaction of F protein with the TLR4/MD-2/CD14 complex and examine the role(s) of TLR4 signaling in RSV infection/protection. It is expected that at the completion of this grant, we will have identified strategies that may lead to development of a RSV vaccine and new therapeutics for mitigating the pathologic host response to RSV.

Public Health Relevance

Toll-like receptor 4 (TLR4) detects many microbial structures and host danger signals during the early phases of the host immune response to infection. Respiratory Syncytial Virus (RSV) expresses a protein called F protein that stimulates cells using TLR4. We have recently shown that a previously failed vaccine can be made safe and partially protective by including a TLR4-activating agent in the vaccine. Our proposal seeks to test a new subunit vaccine and to identify new ways to treat RSV infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Innate Immunity and Inflammation Study Section (III)
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Kim, Sonnie
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Sigmovir Biosystems, Inc.
United States
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Blanco, Jorge C G; Boukhvalova, Marina S; Pletneva, Lioubov M et al. (2014) A recombinant anchorless respiratory syncytial virus (RSV) fusion (F) protein/monophosphoryl lipid A (MPL) vaccine protects against RSV-induced replication and lung pathology. Vaccine 32:1495-500
Shirey, Kari Ann; Lai, Wendy; Scott, Alison J et al. (2013) The TLR4 antagonist Eritoran protects mice from lethal influenza infection. Nature 497:498-502
Shirey, K A; Lai, W; Pletneva, L M et al. (2013) Role of the lipoxygenase pathway in RSV-induced alternatively activated macrophages leading to resolution of lung pathology. Mucosal Immunol :
Boukhvalova, Marina S; Sotomayor, Talia B; Point, Ryan C et al. (2010) Activation of interferon response through toll-like receptor 3 impacts viral pathogenesis and pulmonary toll-like receptor expression during respiratory syncytial virus and influenza infections in the cotton rat Sigmodon hispidus model. J Interferon Cytokine Res 30:229-42
Shirey, K A; Pletneva, L M; Puche, A C et al. (2010) Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R alpha-, TLR4-, and IFN-beta-dependent. Mucosal Immunol 3:291-300
Blanco, Jorge C G; Boukhvalova, Marina S; Shirey, Kari Ann et al. (2010) New insights for development of a safe and protective RSV vaccine. Hum Vaccin 6:482-92
Boukhvalova, Marina S; Prince, Gregory A; Blanco, Jorge C G (2009) The cotton rat model of respiratory viral infections. Biologicals 37:152-9
Pletneva, Lioubov M; Haller, Otto; Porter, David D et al. (2008) Induction of type I interferons and interferon-inducible Mx genes during respiratory syncytial virus infection and reinfection in cotton rats. J Gen Virol 89:261-70
Awomoyi, Agnes A; Rallabhandi, Prasad; Pollin, Toni I et al. (2007) Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children. J Immunol 179:3171-7
Yim, Kevin C; Cragin, Rose P; Boukhvalova, Marina S et al. (2007) Human metapneumovirus: enhanced pulmonary disease in cotton rats immunized with formalin-inactivated virus vaccine and challenged. Vaccine 25:5034-40

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