RSV-specific CD8 Research Articles

Increased innate immune activation induces protective RSV-specific lung-resident memory T cells in neonatal mice

Young infants frequently experience respiratory tract infections, yet vaccines designed to provide mucosal protection are lacking. Localizing pathogen-specific cellular and humoral immune responses to the lung could provide improved immune protection. We used a well-characterized murine model of respiratory syncytial virus (RSV) to study the development of lung-resident memory T cells (TRM) in neonatal compared to adult mice. We demonstrated that priming with RSV during the neonatal period failed to retain RSV-specific clusters of differentiation (CD8) TRM 6weeks post infection, in contrast to priming during adulthood. The reduced development of RSV-specific TRM was associated with poor acquisition of two key markers of tissue residence: CD69 and CD103. However, by augmenting both innate immune activation and antigen exposure, neonatal RSV-specific CD8 T cells increased expression of tissue-residence markers and were maintained in the lung at memory time points. Establishment of TRM correlated with more rapid control of the virus in the lungs upon reinfection. This is the first strategy to effectively establish RSV-specific TRM in neonates providing new insight into neonatal memory T cell development and vaccine strategies.

Age upon RSV infection and epitope-specificity influence the memory differentiation of antiviral T cells

Abstract Background Despite the high rate of respiratory syncytial virus (RSV) infection and severe symptoms in infants, subsequent infections occur, indicating impaired mucosal immune memory at early life. CD8 T cells are associated with reduced disease in human adult challenge trials and murine models, demonstrating their protective roles. However, since primary RSV infection occurs in infancy, a better understanding of the memory differentiation of RSV-specific respiratory CD8 T cells in neonates is required. Methods CB6F1/J mice were infected at 7, 14, 25 days of life or adulthood to characterize the RSV-specific memory T-cell development. Cells from mediastinal lymph nodes (MLN) and lungs were analyzed by flow cytometry at 7, 11, 14, and 40 days post RSV infection (dpi) after staining with fluorescent antibodies and tetramers specific for M and M2 epitopes in RSV. Results RSV-specific CD8 T cell responses were overall lower in neonates from peak to memory phase, but differ by epitope. At 40dpi, M-specific CD8 T cells preferentially differentiated to central-memory (CM) and resided in the MLN in both adult and neonatal mice, whereas M2-specific CD8 T cells preferentially differentiated to effector-memory (EM) and resided in the lungs in adult but not neonatal mice. Importantly, lung tissue resident memory cell differentiation was limited in early life and increased with age upon infection. Conclusion Our results indicate that the magnitude and memory development of RSV epitope-specific CD8 T cells differ by tissue and age at infection, suggesting that age-associated mucosal immune factors contribute to the development of RSV-specific memory T cells. This work was supported by funding through the NIAID R01AI154619 (A.M.W.M.). Supplemental support provided by USUHS Department of Pediatrics grant PED-86-3658 (A.M.W.M.).

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (TRM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69+CD103- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M282 epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 TRM markers. Surprisingly, the CD69+CD103+ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to TRM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.

Immunological and Inflammatory Biomarkers of Susceptibility and Severity in Adult Respiratory Syncytial Virus Infections.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in young infants. However, it is also a significant pathogen in older adults. Validated biomarkers of RSV disease severity would benefit diagnostics, treatment decisions, and prophylactic interventions. This review summarizes knowledge of biomarkers for RSV disease in adults. A literature review was performed using Ovid Medline, Embase, Global health, Scopus, and Web of Science for articles published 1946-October 2016. Nine articles were identified plus 9 from other sources. From observational studies of natural infection and challenge studies in volunteers, biomarkers of RSV susceptibility or disease severity in adults were: (1) lower anti-RSV neutralizing antibodies, where neutralizing antibody (and local IgA) may be a correlate of susceptibility/severity; (2) RSV-specific CD8+ T cells in bronchoalveolar lavage fluid preinfection (subjects with higher levels had less severe illness); and (3) elevated interleukin-6 (IL-6), IL-8, and myeloperoxidase levels in the airway are indicative of severe infection. Factors determining susceptibility to and severity of RSV disease in adults have not been well defined. Respiratory mucosal antibodies and CD8+ T cells appear to contribute to preventing infection and modulation of disease severity. Studies of RSV pathogenesis in at-risk populations are needed.

Simultaneous Administration of Recombinant Measles Viruses Expressing Respiratory Syncytial Virus Fusion (F) and Nucleo (N) Proteins Induced Humoral and Cellular Immune Responses in Cotton Rats.

We previously reported that recombinant measles virus expressing the respiratory syncytial virus (RSV) fusion protein (F), MVAIK/RSV/F, induced neutralizing antibodies against RSV, and those expressing RSV-NP (MVAIK/RSV/NP) and M2-1 (MVAIK/RSV/M2-1) induced RSV-specific CD8+/IFN-γ+ cells, but not neutralizing antibodies. In the present study, MVAIK/RSV/F and MVAIK/RSV/NP were simultaneously administered to cotton rats and immune responses and protective effects were compared with MVAIK/RSV/F alone. Sufficient neutralizing antibodies against RSV and RSV-specific CD8+/IFN-γ+ cells were observed after re-immunization with simultaneous administration. After the RSV challenge, CD8+/IFN-γ+ increased in spleen cells obtained from the simultaneous immunization group in response to F and NP peptides. Higher numbers of CD8+/IFN-γ+ and CD4+/IFN-γ+ cells were detected in lung tissues from the simultaneous immunization group after the RSV challenge. No detectable RSV was recovered from lung homogenates in the immunized groups. Mild inflammatory reactions with the thickening of broncho-epithelial cells and the infiltration of inflammatory cells were observed in lung tissues obtained from cotton rats immunized with MVAIK/RSV/F alone after the RSV challenge. No inflammatory responses were observed after the RSV challenge in the simultaneous immunization groups. The present results indicate that combined administration with MVAIK/RSV/F and MVAIK/RSV/NP induces humoral and cellular immune responses and shows effective protection against RSV, suggesting the importance of cellular immunity.

Vaccination with RSV M209-223 peptide promotes a protective immune response associated with reduced pulmonary inflammation

Respiratory syncytial virus (RSV) is the most common etiologic agent in severe infections of the lower respiratory tract in children with a high mortality rate. However, there are still no licensed vaccines for RSV. In this study, we investigated a putative vaccine based on M209-223 peptide. Mice vaccinated with M209-223 peptide expanded M209-223-specific effector CD4+ T cells upon infection. Vaccination resulted in increased numbers of regulatory T cells (Treg) and Th1 cells, and decreased numbers of Th2 cells. In addition, vaccination with M209-223 peptide, protected mice from infection and prevented lung inflammation, leading to increase in IL-10 and IFN-γ production by lung CD4+ T cells. Treg depletion with anti-CTLA4 antibodies abrogated protection induced by peptide vaccination. Our results support vaccination with M209-223 peptide as an important strategy to generate protection, both systemic and local, by memory RSV-specific CD4+ T cells in mice. Contrarily to inactivated RSV particles, M209-223 peptide vaccination is capable of not only promoting viral clearance, but also reducing inflammatory processes in lungs upon infection.

T-Cell Responses in Adults During Natural Respiratory Syncytial Virus Infection.

The pathogenesis of respiratory syncytial virus (RSV) in older adults may be due to age-related T-cell immunosenescence. Thus, we evaluated CD4 and CD8 T-cell responses during RSV infection in adults across the age spectrum. Peripheral blood mononuclear cells collected during RSV infection in adults, age 26-96 years, were stimulated with live RSV and peptide pools representing F, M, NP, and G proteins and analyzed by flow cytometry. There were no significant age-related differences in frequency of CD4+ T cells synthesizing interferon (IFN)γ, interleukin (IL)2, IL4, IL10, or tumor necrosis factor (TNF)α or in CD8+IFNγ+ T cells. IL4+CD4+ T-cell numbers were low, as were IL13 and IL17 responses. However, in univariate analysis, CD4 T-cell IFNγ, IL2, IL4, IL10, and TNFα responses and CD8+IFNγ+ T cells were significantly increased with more severe illness requiring hospitalization. In multivariate analysis, viral load was also associated with increased T-cell responses. We found no evidence of diminished RSV-specific CD4 or CD8 T-cell responses in adults infected with RSV. However, adults with severe disease seemed to have more robust CD4 and CD8 T-cell responses during infection, suggesting that disease severity may have a greater association with T-cell responses than age.

Single-cycle respiratory syncytial virus infection induces robust adaptive immune responses and reduces disease severity in mice

Abstract Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. However, despite extensive efforts, there remains no licensed RSV vaccine. Recently, a recombinant infectious RSV strain lacking the gene for the matrix (M) protein (M-null RSV) was developed as a potential vaccine candidate. The M protein is required for reassembly of the virion following infection and gene expression within a host cell. Infection with M-null RSV produces viral proteins but does not result in the generation of infectious virus progeny resulting in a single-cycle infection. To determine its immunogenicity in vivo, mice were infected with either M-null RSV or a wild-type (WT) RSV. M-null RSV-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared to mice given WT RSV. Remarkably, M-null RSV infection induced CD4 and CD8 T cell responses in the lung and airways of similar magnitude to that of a WT RSV infection at acute time-points. Germinal center B cell numbers and serum antibody levels were also similar between M-null and WT RSV-infected mice. M-null RSV infection also generated robust memory immune responses. A substantial proportion of B cells, CD4 T cells, and CD8 T cells remained within the lung tissue of M-null RSV-infected mice 30 days post-infection. Importantly, RSV-specific memory CD8 T cells expressing CD69 and CD103, the canonical markers of tissue resident memory, were also generated by M-null RSV infection. Given the reduced disease severity and robust immune responses developed following single-cycle infection, M-null RSV may be an effective vaccine candidate.

Memory CD8 T cells mediate severe immunopathology following respiratory syncytial virus infection.

Memory CD8 T cells can provide protection from re-infection by respiratory viruses such as influenza and SARS. However, the relative contribution of memory CD8 T cells in providing protection against respiratory syncytial virus (RSV) infection is currently unclear. To address this knowledge gap, we utilized a prime-boost immunization approach to induce robust memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. Unexpectedly, RSV infection of mice with pre-existing CD8 T cell memory led to exacerbated weight loss, pulmonary disease, and lethal immunopathology. The exacerbated disease in immunized mice was not epitope-dependent and occurred despite a significant reduction in RSV viral titers. In addition, the lethal immunopathology was unique to the context of an RSV infection as mice were protected from a normally lethal challenge with a recombinant influenza virus expressing an RSV epitope. Memory CD8 T cells rapidly produced IFN-γ following RSV infection resulting in elevated protein levels in the lung and periphery. Neutralization of IFN-γ in the respiratory tract reduced morbidity and prevented mortality. These results demonstrate that in contrast to other respiratory viruses, RSV-specific memory CD8 T cells can induce lethal immunopathology despite mediating enhanced viral clearance.

Immunostimulation during primary neonatal RSV infection alters memory response.

Abstract Respiratory syncytial virus (RSV) is recognized as a leading cause of acute viral illness in neonates worldwide. However, those with balanced Th1/Th2 responses, as a result of higher IFNγ concentrations, have milder disease and better clinical outcomes. The present study examined the effect of IFNγ administration during primary, neonatal RSV infection on innate and T cell responses during primary and secondary RSV exposure. During primary infection, neonates treated with IFNγ had increased alveolar macrophage (AM) expression of CD80, MHCII, and scavenger receptor A. Subsequent CD8 T cell recruitment and activation was reduced in this group compared to RSV controls, however the RSV-specific CD8 T cell response was enhanced. Upon secondary infection, neutrophil and monocyte recruitment was boosted and expression of CD86 and MHCII was significantly increased on AMs with a correlative decline in CD73 expression compared to primary infection controls. In mice previously infected with RSV, T cell recruitment and activation was significantly increased. However, only those mice that were treated with IFNγ during primary infection showed improved CD8 T cell specificity following secondary RSV exposure. Viral lung titers were also significantly reduced in groups previously exposed to RSV but only IFNγ treatment during primary infection demonstrated significant improvements in airway resistance following methacholine challenge. These results suggest that immunostimulation with IFNγ during primary neonatal RSV is associated with increased innate cell activation and improved CD8 T cell specificity. These cellular changes are associated with enhanced viral clearance and improved airway function.

Distinct innate signaling and activation of dendritic cells during the neonatal period determines their functional response to respiratory syncytial virus infection

Abstract Dendritic cells (DCs) are among the first to respond to infection, providing the critical link between innate and adaptive immunity. Neonates often have weak innate immune responses that can limit adaptive immunity and contribute to increased susceptibility and more severe infection with intracellular pathogens, such as respiratory syncytial virus (RSV). We have previously shown differential migration and defective activation of neonatal murine respiratory CD103+ DCs, which is associated with diminished RSV-specific CD8 T cell responses in neonatal mice. In order to define the age-dependent regulation of respiratory DC function, we have developed a neonatal mouse model of RSV to track antigen presentation via infection with an ovalbumin-expressing RSV and an antibody against H2kb-OVA257–264. We found adequate transfer of RSV-derived OVA257–264 by neonatal CD103+ DCs to the dLN, suggesting poor CD8 T cell responses are not due to limited antigen presentation. However, neonatal CD103+ DCs expressing H2kb-OVA257–264 failed to upregulate CD80/86, which is critical to T cell stimulation. RNA-seq analysis of H2kb-OVA257–264+ CD103+ DCs implicated defective type I IFN signaling in neonates that may influence reduced activation. In support of this, while we find type I IFN to be diminished in the neonatal lung, treatment of neonates with IFN-α failed to restore the activation of neonatal H2kb-OVA257–264+ CD103+DCs. Our data demonstrate a distinct developmental regulation of innate immunity in early life and identify type I IFN signaling as contributing to defective DC activation during RSV infection in neonates. Defining age-dependent mechanisms controlling immunity may lead to intelligently designed vaccines for this age group.

Pulmonary Dendritic Cell Subsets Shape the Respiratory Syncytial Virus-Specific CD8+ T Cell Immunodominance Hierarchy in Neonates.

Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8+ T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8+ T cell immunodominance hierarchy to two RSV-derived epitopes, KdM282-90 and DbM187-195, was determined by the age at infection. To determine whether age-dependent RSV-specific CD8+ T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like KdM282-90 CD8+ T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b+ and CD103+ dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the KdM282-90 CD8+ T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8+ T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.

Differential antigen presentation and transport by neonatal and adult dendritic cell subsets during Respiratory Syncytial Virus (RSV) infection

Abstract Dendritic cells (DCs) are critical for the induction of anti-viral CD8+ T cells. Young infants mount poor antiviral responses contributing to increased disease susceptibility and severity compared with acquisition later in life. Using a mouse model of respiratory syncytial virus (RSV), we have previously shown differences in the number of DCs that can carry antigen from the lung to the lung-draining lymph node (dLN) during infection between adults and neonates, and this is associated with age-dependent differences in RSV-specific CD8+ T cell responses. However whether adult or neonatal DCs more readily present antigen for T cell engagement is unknown. In this study, we used an ovalbumin-expressing RSV (RSV-OVA) to track antigen presentation through early infection. In the lung, expression of MHCI-bound OVA (MHCI-OVA) was highest on adult CD103+ DCs; while in the dLN a greater number of neonatal CD103+ DCs expressed MHCI-OVA. Quantification of the migration-associated chemokine receptor 7 identified increased expression on neonatal MHCI-OVA-expressing CD103+ DCs compared to adults, suggesting adults may more readily present antigen but neonatal CD103+ DCs were more apt to migrate upon antigen acquisition. However, neonates failed to induce an OVA-specific T cell response in the lung in contrast to adults. Together our data indicate that in neonates MHCI-OVA+ CD103+ DCs were more proficient at migration resulting in a greater number of MHCI-OVA-presenting CD103+ DCs in the dLN;- however, they failed to induce an OVA-specific T cell response. Thus antigen abundance was not the primary determinant of CD8+ T cell induction during RSV infection in neonates, suggesting that alternate mechanism(s) limit their DC-mediated T cell responses.

Memory CD8 T cells in RSV infection: friend or foe?

Abstract Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. CD8 T cells play a critical role in mediating viral clearance following an acute RSV infection. However the role of memory CD8 T cells in providing protection against RSV remains understudied. To generate high magnitude CD8 T cell memory in the absence of CD4 T cell memory and antibodies, we immunized naïve mice with dendritic cells pulsed with an RSV-derived peptide followed by a boost with a recombinant Listeria monocytogenes expressing the same RSV-derived epitope. Memory CD8 T cells significantly reduced viral titers following RSV challenge, but did so at the expense of increased airway dysfunction, weight loss, and mortality compared to controls. Importantly, the severe immunopathology and mortality observed was specific to the context of an RSV infection, as prime-boosted mice challenged with a recombinant influenza virus expressing the same RSV-derived epitope did not exhibit enhanced disease. The induction of a pro-inflammatory cytokine storm mediated by TNF-α and IFN-γ was observed in the serum of prime-boosted mice following RSV challenge. Additionally, RSV-specific memory CD8 T cells produced large amounts of IFN-γ locally within the lung, and adoptive transfer of wild-type but not IFN-γ-deficient memory CD8 T cells resulted in enhanced airway dysfunction and weight loss. Our results indicate that memory CD8 T cells are able to mediate protection against RSV infection. However, memory CD8 T cells acting alone in the absence of antibodies and memory CD4 T cells induce significant immunopathology and mortality through the induction of a systemic pro-inflammatory cytokine storm and local IFN-γ production.

T cell responses are elicited against Respiratory Syncytial Virus in the absence of signalling through TLRs, RLRs and IL-1R/IL-18R.

Pattern recognition receptors (PRRs) and cytokine receptors are key players in the initiation of immune responses to infection. PRRs detecting viral RNA, such as toll like receptor (TLR)-3, -7/8, and RIG-I like receptors (RLRs; RIG-I and MDA-5), as well as cytokine receptors such as interleukin 1 receptor (IL-1R), have been implicated in responses to RNA viruses that infect the airways. The latter includes respiratory syncytial virus (RSV), a human pathogen that can cause severe lower respiratory tract infections, especially in infants. To evaluate the collective contribution of PRRs and IL-1R signalling to RSV immunity, we generated Myd88/Trif/Mavs−/− mice that are deficient in signalling by all TLRs, RLRs and IL-1R, as well as other cytokine receptors such as IL-18 receptor. Early production of pro-inflammatory mediators and lung infiltration by immune cells were completely abrogated in infected Myd88/Trif/Mavs−/− mice. However, RSV-specific CD8+ T cells were elicited and recruited into the lungs and airways. Consistent with these findings, Myd88/Trif/Mavs−/− mice survived RSV infection but displayed higher viral load and weight loss. These data highlight an unappreciated level of redundancy in pathways that couple innate virus sensing to adaptive immunity, providing the host with remarkable resilience to infection.

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

STAT4 deficiency fails to induce lung Th2 or Th17 immunity following primary or secondary respiratory syncytial virus (RSV) challenge but enhances the lung RSV-specific CD8+ T cell immune response to secondary challenge.

Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4-/- mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4-/- mice and used STAT1-/- mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4-/- mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4-/- mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4-/- mice compared to WT mice. Following secondary challenge, WT and STAT4-/- mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4-/- mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.

DNGR-1 is dispensable for CD8+ T-cell priming during respiratory syncytial virus infection.

During respiratory syncytial virus (RSV) infection CD8(+) T cells both assist in viral clearance and contribute to immunopathology. CD8(+) T cells recognize viral peptides presented by dendritic cells (DCs), which can directly present viral antigens when infected or, alternatively, "cross-present" antigens after endocytosis of dead or dying infected cells. Mouse CD8α(+) and CD103(+) DCs excel at cross-presentation, in part because they express the receptor DNGR-1 that detects dead cells by binding to exposed F-actin and routes internalized cell debris into the cross-presentation pathway. As RSV causes death in infected epithelial cells, we tested whether cross-presentation via DNGR-1 is necessary for CD8(+) T-cell responses to the virus. DNGR-1-deficient or wild-type mice were intranasally inoculated with RSV and the magnitude of RSV-specific CD8(+) T-cell induction was measured. We found that during live RSV infection, cross-presentation via DNGR-1 did not have a major role in the generation of RSV-specific CD8(+) T-cell responses. However, after intranasal immunization with dead cells infected with RSV, a dependence on DNGR-1 for RSV-specific CD8(+) T-cell responses was observed, confirming the ascribed role of the receptor. Thus, direct presentation by DCs may be the major pathway initiating CD8(+) T-cell responses to RSV, while DNGR-1-dependent cross-presentation has no detectable role.

Vaccination with the RSV fusion protein formulated with a combination adjuvant induces long-lasting protective immunity

Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a Toll-like receptor (TLR) agonist and an immunostimulatory peptide in a carrier system (ΔF/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The ΔF was formulated with triple adjuvant (TriAdj) containing either polyinosinic : polycytidylic acid (polyI : C) or cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) and administered intranasally to mice. One year after the second vaccination all mice were challenged with RSV. Both ΔF/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus-neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN-γ- and IL-5-secreting cells in the spleen, the immune response was slightly T-helper cell type 1 (Th1)-biased. This was confirmed by the presence of F85-93-specific CD8(+) effector T cells in the lungs of both ΔF/TriAdj(polyI : C)- and ΔF/TriAdj(CpG)-immunized mice. Both ΔF/TriAdj formulations induced RSV-specific CD8(+) T cells. However, ΔF/TriAdj(polyI : C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8(+) effector memory T cells in lungs and CD8(+) central memory T cells in spleen and lymph nodes than ΔF/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the ΔF/TriAdj formulations, demonstrating that the duration of immunity induced with these vaccines is at least one year.

Genetic Vaccine for Respiratory Syncytial Virus Provides Protection Without Disease Potentiation

Respiratory syncytial virus (RSV) is a major cause of infectious lower respiratory disease in infants and the elderly. As there is no vaccine for RSV, we developed a genetic vaccine approach that induced protection of the entire respiratory tract from a single parenteral administration. The approach was based on adenovirus vectors derived from newly isolated nonhuman primate viruses with low seroprevalence. We show for the first time that a single intramuscular (IM) injection of the replication-deficient adenovirus vectors expressing the RSV fusion (F0) glycoprotein induced immune responses that protected both the lungs and noses of cotton rats and mice even at low doses and for several months postimmunization. The immune response included high titers of neutralizing antibody that were maintained ≥ 24 weeks and RSV-specific CD8+ and CD4+ T cells. The vectors were as potently immunogenic as a human adenovirus 5 vector in these two key respiratory pathogen animal models. Importantly, there was minimal alveolitis and granulocytic infiltrates in the lung, and type 2 cytokines were not produced after RSV challenge even under conditions of partial protection. Overall, this genetic vaccine is highly effective without potentiating immunopathology, and the results support development of the vaccine candidate for human testing.