Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.     

Gamma interferon-dependent protection of the mouse upper respiratory tract following parenteral immunization with a respiratory syncytial virus G protein fragment

The protective mechanisms induced in the mouse upper respiratory tract (URT) after intraperitoneal immunization with G2Na, a recombinant respiratory syncytial virus (RSV) G protein fragment (amino acid residues 130 to 230), were investigated. This protection was recently shown to be mediated by CD4(+) T cells and to be critically dependent on the cysteines and amino acids 193 and 194 (H. Plotnicky-Gilquin, A. Robert, L. Chevalet, J.-F. Haeuw, A. Beck, J.-Y. Bonnefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power, J. Virol. 74:3455-3463, 2000). On G2Na, we identified a domain (amino acid residues 182 to 198) responsible for the T-helper-cell activity. This region coincided with a peptide designed AICK (residues 184 to 198) which includes the previously identified murine and human T-helper-cell epitope on the native G protein (P. W. Tebbey, M. Hagen, and G. E. Hancock, J. Exp. Med. 188:1967-1972, 1998). Immunization with AICK, in alum or complete Freund's adjuvant, significantly reduced nasal RSV titers in normal BALB/c mice. However, although lung protection was induced, in contrast to the case with live RSV, neither AICK nor G2Na was able to prevent nasal infection in gamma interferon (IFN-gamma)-knockout mice. Anti-IFN-gamma neutralizing antibodies partially inhibited URT protection after administration to G2Na-immunized BALB/c mice. Furthermore, while purified CD4(+) T cells from BALB/c mice immunized with G2Na or AICK significantly reduced lung and nasal infection of naive recipient mice after adoptive transfer, the cells from IFN-gamma-knockout mice had no effect. Together, these results demonstrated for the first time that the T-helper-cell epitope of RSV G protein induces URT protection in mice after parenteral immunization through a Th1-type, IFN-gamma-dependent mechanism.     

Efficient lung recruitment of respiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.     

Differential role of gamma interferon in inhibiting pulmonary eosinophilia and exacerbating systemic disease in fusion protein-immunized mice undergoing challenge infection with respiratory syncytial virus

Secondary exposure to respiratory syncytial virus (RSV) can lead to immunopathology and enhanced disease in vaccinated individuals. Vaccination with individual RSV proteins influences the type of secondary RSV-specific immune response that develops upon challenge RSV infection, as well as the extent of immunopathology. RSV-specific memory CD4 T cells can directly contribute to immunopathology through their cytokine production. Immunization of BALB/c mice with a recombinant vaccinia virus (vv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia upon RSV challenge, whereas immunization of mice with a vv expressing the fusion (F) protein does not. We analyzed the CD4 T-cell response to an I-E^d-restricted CD4 T-cell epitope within the F protein of RSV corresponding to amino acids 51 to 66 in an effort to better understand the similarities and differences in the immune response elicited by the G versus the F protein. Vaccination with the G protein induces a mixture of RSV G-specific Th1 and Th2 cells with a restricted T-cell receptor repertoire. In contrast, we demonstrate here that immunization with the F protein elicits a broad repertoire of RSV F-specific CD4 T cells that predominantly exhibit a Th1 phenotype. However, in the absence of gamma interferon (IFN-γ), RSV F_51-66-specific CD4 T cells secreted interleukin-5, and mice developed pulmonary eosinophilia after RSV challenge. IFN-γ-deficient mice exhibited decreased weight loss compared to wild-type controls, suggesting that IFN-γ exacerbates systemic disease. These data demonstrate that IFN-γ can have both beneficial and detrimental effects during a secondary RSV infection.     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.     

CD8 T cells inhibit respiratory syncytial virus (RSV) vaccine-enhanced disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-gamma-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.     

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.     

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.     

Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.     

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.     

Cytotoxic T cells specific for a single peptide on the M2 protein of respiratory syncytial virus are the sole mediators of resistance induced by immunization with M2 encoded by a recombinant vaccinia virus.

We have studied the immunobiology of respiratory syncytial virus (RSV), a major cause of respiratory tract morbidity in children. As part of these studies, it was previously found that immunization of BALB/c (H-2d) mice with a recombinant vaccinia virus (rVV) which encoded the M2 protein of RSV provided complete protection against infection with RSV. This protection was transient and associated with M2-specific CD8+ T-cell (TCD8+) responses. In this study, we used two approaches to demonstrate that expression of an H-2Kd-restricted nonameric peptide (Ser Tyr Ile Gly Ser Ile Asn Asn Ile) corresponding to M2 residues 82 to 90 is necessary and sufficient to induce protective TCD8+ responses. First, infection of mice with an rVV which encoded the peptide M2Met82-90 induced levels of primary pulmonary TCD8+ and resistance to RSV challenge equivalent to that induced by infection with an rVV which expressed the complete M2 protein. Second, elimination of peptide binding to Kd by the replacement of Tyr with Arg at amino acid position 83 of the full-length protein completely abrogated the ability of an rVV-expressing full-length M2 to induce either M2-specific TCD8+ responses or resistance to RSV infection. These findings demonstrate that the M2(82-90) peptide is the sole determinant of immunity induced in BALB/c mice by the M2 protein and that a remarkably high level of transient resistance to infection with pulmonary virus is associated with TCD8+ responses to a single determinant.     

MyD88-mediated instructive signals in dendritic cells regulate pulmonary immune responses during respiratory virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.     

The respiratory syncytial virus G protein conserved domain induces a persistent and protective antibody response in rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130-230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 μg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.     

Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses. Here, a recombinant replication-deficient adenovirus-based vaccine, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 130 to 230) engineered by codon optimization and tandem repetition for higher-level expression, was constructed and evaluated for its potential as an RSV vaccine in a murine model. A single intranasal immunization with rAd/3xG provided potent protection against RSV challenge which lasted for more than 10 weeks. Strong mucosal immunoglobulin A responses were also induced by a single intranasal immunization but not by intramuscular or oral administration of rAd/3xG. Interestingly, neither gamma interferon- nor interleukin-4-producing CD4 T cells directed to I-E^d-restricted epitope were detected in the lungs of rAd/3xG-immune mice upon challenge, whereas priming with vaccinia virus expressing RSV G (vvG) elicited strong Th1/Th2 mixed CD4 T-cell responses. Lung eosinophilia and vaccine-induced weight loss were significantly lower in the rAd/3xG-immune group than in the vvG-primed group. Together, our data demonstrate that a single intranasal administration of rAd/3xG elicits beneficial protective immunity and represents a promising vaccine regimen against RSV infection.     

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.     

Venezuelan equine encephalitis virus replicon particles encoding respiratory syncytial virus surface glycoproteins induce protective mucosal responses in mice and cotton rats

Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K^d-restricted CD8⁺ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.     

Visualization and characterization of respiratory syncytial virus F-specific CD8(+) T cells during experimental virus infection

CTL play a major role in the clearance of respiratory syncytial virus (RSV) during experimental pulmonary infection. The fusion (F) glycoprotein of RSV is a protective Ag that elicits CTL and Ab response against RSV infection in BALB/c mice. We used the strategy of screening a panel of overlapping synthetic peptides corresponding to the RSV F protein and identified an immunodominant H-2K(d)-restricted epitope (F(85-93); KYKNAVTEL) recognized by CD8(+) T cells from BALB/c mice. We enumerated the F-specific CD8(+) T cell response in the lungs of infected mice by flow cytometry using tetramer staining and intracellular cytokine synthesis. During primary infection, F(85-93)-specific effector CD8(+) T cells constitute approximately 4.8% of pulmonary CD8(+) T cells at the peak of the primary response (day 8), whereas matrix 2-specific CD8(+) T cells constituted approximately 50% of the responding CD8(+) T cell population in the lungs. When RSV F-immune mice undergo a challenge RSV infection, the F-specific CD8(+) T cell response is accelerated and dominates, whereas the primary response to the matrix 2 epitope in the lungs is reduced by approximately 20-fold. In addition, we found that activated F-specific effector CD8(+) T cells isolated from the lungs of RSV-infected mice exhibited a lower than expected frequency of IFN-gamma-producing CD8(+) T cells and were significantly impaired in ex vivo cytolytic activity compared with competent F-specific effector CD8(+) T cells generated in vitro. The significance of these results for the regulation of the CD8(+) T cell response to RSV is discussed.     

Effect of chemokine receptor CX3CR1 deficiency in a murine model of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C chemokine motif to the host chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1(+) natural killer, CD11b(+), and RB6-8C5(+) polymorphonuclear cell trafficking to the lung and reduced IFNγ production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C-CX3CR1 interaction in the innate immune response to RSV infection.     

The attachment (G) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both Th1 and Th2 CD4+ T cell responses

BALB/c mice immunized with a vaccinia virus expressing the attachment (G) glycoprotein of respiratory syncytial virus (RSV) develop a virus-specific CD4(+) T cell response that consists of a mixture of Th1 and Th2 CD4(+) T cells following intranasal infection with live RSV. Recent work has shown that both Th1 and Th2 CD4(+) T cells are elicited to a single region comprising aa 183-197 of the G protein. To more precisely define the CD4(+) T cell epitope(s) contained within this region, we created a panel of amino- and carboxyl-terminal truncated as well as single alanine-substituted peptides spanning aa 183-197. These peptides were used to examine the ex vivo cytokine response of memory effector CD4(+) T cells infiltrating the lungs of G-primed RSV-infected mice. Analysis of lung-derived memory effector CD4(+) T cells using intracellular cytokine staining and/or ELISA of effector T cell culture supernatants revealed a single I-E(d)-restricted CD4(+) T cell epitope with a core sequence mapping to aa 185-193. In addition, we examined the T cell repertoire of the RSV G peptide-specific CD4(+) T cells and show that the CD4(+) T cells directed to this single immunodominant G epitope use a restricted range of TCR Vss genes and predominantly express Vss14 TCR.     

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.