M2 Protein Of Respiratory Syncytial Virus Research Articles

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.

Vaccine containing G protein fragment and recombinant baculovirus expressing M2 protein induces protective immunity to respiratory syncytial virus.

PurposeRespiratory syncytial virus (RSV) can cause serious respiratory illnesses such as pneumonia, asthma, and bronchiolitis in infants and elderly or immunocompromised individuals. An RSV vaccine has yet to be developed; only prophylactic anti-RSV antibody is commercially available. So, we investigated whether our vaccine candidate is able to induce type 1 CD4+ T helper (Th1), CD8+ T-cell responses, and protective immunity without vaccine-enhanced disease (VED) against RSV.Materials and MethodsWe used RSV G protein fragment (Gcf A) with recombinant baculovirus capable of expressing the RSV M2 protein (Bac M2) as a vaccine candidate, and injected this vaccine (Gcf A/Bac M2) intramuscularly, and challenged with RSV intranasally into mice. Enzyme-linked immunosorbent assay, flow cytometry, plaque assay, and weight measurement were performed to confirm humoral immunity, cellular immunity, and protective immunity.ResultsThe Gcf A/Bac M2 formulation induced a stronger IgG response to Gcf A than Gcf A inoculation alone, and the ratio of IgG1/IgG2a indicated that the responses shifted predominantly to Th1. In addition, both RSV G-specific Th1 responses and RSV M2-specific CD8+ T-cell responses were induced, and G protein-associated eosinophilic infiltration was suppressed compared to the control group. Moreover, the Gcf A/Bac M2 group showed effective protection after an RSV challenge.ConclusionBac M2 could serve as a vaccine with intrinsic adjuvant activity, and the Gcf A/Bac M2 shows promise as a vaccine candidate for inducing protective immunity without inciting VED.

Memory Inflation Drives Tissue-Resident Memory CD8+ T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus.

Tissue-resident memory T (TRM) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, TRM cells protect against respiratory infections, but wane more quickly than TRM cells in other tissues. This lack of a sustained TRM population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8+ TRM cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8+ T cell population elicited by MCMV-M vaccination with a conventional CD8+ T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8+ TRM cells that waned rapidly, akin to the M2-specific CD8+ TRM cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8+ T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8+ T cells. Moreover, effective viral clearance mediated by M-specific CD8+ TRM cells was not affected by the coinduction of M2-specific CD8+ T cells. These data show that memory inflation is required for the maintenance of CD8+ TRM cells in the lungs after IN vaccination with MCMV.

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8+ T-cell immunity against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8+ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8+ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.

Enhancement of the CD8 + T cell response to a subdominant epitope of respiratory syncytial virus by deletion of an immunodominant epitope

Cytotoxic T lymphocytes (CTLs) are critical for the control of respiratory syncytial virus infection (RSV) in humans and mice. Recently, we identified a new H-2K d-restricted subdominant epitope in the respiratory syncytial virus M2 protein. In this study, we investigated if modification of anchor residues at positions 2 and 9 in the dominant M2 82–90 epitope in the M2 protein would alter the CTL epitope dominance hierarchy following immunization with plasmid DNA encoding M2 proteins. We showed that immunogenicity of the subdominant epitope M2 127–135 was enhanced when the anchor residues of the dominant epitope were mutated, suggesting that the immunodominant epitope induces a suppression of response to the subdominant epitope.

Modulation of the CD8+ T cell response to subdominant epitopes of respiratory syncytial virus by deletion of an immunodominant epitope (43.9)

Abstract Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. Recently, we identified a new H-2Kd- restricted subdominant epitope in the RSV M2 protein. This finding allowed us to study the frequency of T lymphocytes responding two H-2Kd-restricted epitopes, M282–90 and M2127–135, following RSV infection in both lymphoid and non-lymphoid tissues. The ratio of T lymphocytes during the peak CTL response to RSV infection that were specific for immunodominant or subdominant was approximately 3:1 in the spleen and 10:1 in the lung. This ratio was observed consistently in primary or secondary infection. In addition, we showed that the kinetics of response against the two epitopes was regulated differently; the response to the subdominant epitope was delayed compared to the dominant epitope. In further studies, we demonstrated that modification of residues at positions 2 and 9 in the dominant M282–90 epitope altered the CTL epitope dominance hierarchy following immunization with plasmid DNA encoding M2 or mutant M2 proteins. Immunogenicity of the subdominant epitope M2127–135 was enhanced when the anchor residues of the dominant epitope were mutated, suggesting that the immunodominant epitope induces a suppression of response to subdominant epitopes. Taken together, our findings have implications for understanding the immune response to RSV infection or vaccination and provide insight into methods for the rational design of vaccines that induce CD8+ T cells.

Functional impairment of CD8 T cells following acute virus infections (43.32)

Abstract Following acute respiratory syncytial virus (RSV) infection the virus-specific CD8 T cells from the spleen show a 1:1 correspondence between tetramer staining and in vitro cytokine production after peptide stimulation. In contrast, pulmonary virus-specific CD8 T cells are deficient in IFN-γ and TNF-α production and show impaired ex vivo cytotoxicity compared to CD8 T cells from the spleen. We have studied CD8 T cell functional impairment in viral infections outside the Paramyxoviridae family and show that similar dysregulation of pulmonary CD8 T cells also occurs after respiratory infections by lymphocytic choriomeningitis virus (LCMV) and by vaccinia virus expressing the RSV M2 protein (vvM2). In addition, LCMV and vvM2-specific CD8 T cells in the lungs demonstrate impaired degranulation relative to splenic CD8 T cells as measured by CD107a staining following ex vivo peptide stimulation. Importantly, we show here that pulmonary CD8 T cell IFN-γ and TNF-α production can be recovered by stimulating these cells in vitro using P815 cells pulsed with peptide as a source of exogenous antigen presenting cells (APCs). Our results indicate that the CD8 T cells in the lung are fully functional and that the apparent functional impairment may be caused by the APC milieu within the lung tissue.

Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis.

A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+ T-cell memory. After intranasal RSV challenge, accelerated CD8+ T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+ T-cell responses than those elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.

Nasal delivery of chitosan–DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice

Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract, is responsible for severe illness both in new born and young children and in elderly people. Due to complications associated with the use of the early developed vaccines, there is still a need for an effective vaccine against RSV. Most pathogens enter the body via mucosal surfaces and therefore vaccine delivery via routes such as the nasal, may well prove to be superior in inducing protective immune responses against respiratory viruses, since both local and systemic immunity can be induced by nasal immunisation. Previously we have shown that intradermal immunisation of a plasmid DNA encoding the CTL epitope from the M2 protein of RSV induced protective CTL responses. In the present study, the mucosal delivery of plasmid DNA formulated with chitosan has been investigated. Chitosan is a polysachharide consisting of copolymers of N-acetylglucosamine and glucosamine that is derived from chitin, a material found in the shells of crustacea. Intranasal immunisation with plasmid DNA formulated with chitosan induced peptide- and virus-specific CTL responses in BALB/c mice that were comparable to those induced via intradermal immunisation. Following RSV challenge of chitosan/DNA immunised mice, a significant reduction ( P<0.001) in the virus load was observed in the lungs of immunised mice compared to that in the control group. These results indicate the potential of immunisation with chitosan-formulated epitope-based vaccines via the intranasal route.

Interleukin-4 diminishes CD8(+) respiratory syncytial virus-specific cytotoxic T-lymphocyte activity in vivo.

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using an H-2K(d)-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct (51)Cr release assay and intracellular gamma interferon (IFN-gamma) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-gamma production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8(+) RSV-specific CTL responses in vivo.

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.

Immunization of mice with vaccinia virus-M2 recombinant induces epitope-specific and cross-reactive Kd-restricted CD8+ cytotoxic T cells.

The M2 protein of respiratory syncytial virus (RSV) is a protective antigen in H-2d, but not H-2b or H-2k mice. None of the other RSV proteins, excluding the surface glycoproteins that induce neutralizing antibodies, is protective in mice bearing these haplotypes. Thus, the M2 protein stands alone as a nonglycoprotein-protective antigen of RSV. The M2 protein is a target for murine Kd-restricted cytotoxic T lymphocytes (CTLs), and the resistance induced by infection with a vaccinia virus-RSV M2 (vac-M2) recombinant is mediated by CD8+ CTLs. Since the nonameric consensus sequence for H-2 Kd-restricted T-cell epitopes and the amino acid sequence of the M2 protein of subgroup A and B strains of RSV are known, the present study sought to identify the specific epitope(s) on the M2 protein recognized by CD8+ CTLs. This was done by examining the ability of four predicted Kd-specific motif peptides present in the M2 amino acid sequence of an RSV subgroup A strain to sensitize target cells for lysis by pulmonary or splenic CTLs obtained from mice infected with RSV or vac-M2. The following observations were made. First, two of the four peptides sensitized target cells for lysis by pulmonary or splenic CTLs induced by infection with either vac-M2 or RSV. Second, one of the two peptides, namely the 82-90 (M2) peptide, sensitized targets at a very low peptide concentration (10(-10) to 10(-12) M). Third, cold-target competition experiments revealed that the predominant CTL population induced by infection with vac-M2 or RSV recognized the 82-90 (M2) peptide, and this CTL population appeared to recognize the 71-79 (M2) peptide in a cross-reactive manner. Fourth, CTL recognition of targets sensitized with either the 71-79 (M2) or the 82-90 (M2) peptide was Kd restricted. Fifth, CTLs induced by infection with RSV subgroup A or B strains recognized the two M2 peptides. The findings suggest that the M2 protein of RSV contains an immunodominant Kd-restricted CTL epitope consisting of amino acid residues 82 to 90 (SYIGSINNI), which are shared by subgroup A and B RSVs.

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.

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.