Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.     

Pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of CD4+ T cells.

In previous studies, it was observed that children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency during subsequent natural RSV infection than did controls. During earlier efforts to develop an animal model of this phenomenon, enhanced pulmonary histopathology was observed after intranasal RSV challenge of FI-RSV-immunized cotton rats. Progress in understanding the immunologic basis for these observations has been hampered by the lack of reagents useful in manipulating the immune response of the cotton rat. This problem prompted us to reinvestigate the characteristics of immunity to RSV in the mouse. In the present studies, extensive pulmonary histopathology was observed in FI-RSV-immunized or RSV-infected BALB/c mice upon RSV challenge, and studies to determine the relative contributions of CD4+ or CD8+ T cells to this process were undertaken. Mice previously immunized with FI-RSV or infected with RSV were depleted of CD4+, CD8+, or both T-cell subsets immediately prior to RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels and into alveolar spaces was quantified. The magnitude of infiltration at each anatomic site in previously FI-RSV-immunized or RSV-infected, nondepleted animals was similar, indicating that this is not a relevant model for enhanced disease. However, the effect of T-cell subset depletion on pulmonary histopathology following RSV challenge was very different between the two groups. Depletion of CD4+ T cells completely abrogated pulmonary histopathology in FI-RSV-immunized mice, whereas it had a much smaller effect on mice previously infected with RSV. FI-RSV-immunized or RSV-infected animals depleted of CD8+ T cells had only a modest reduction of pulmonary histopathology. In addition, RSV infection induced high levels of major histocompatibility complex class I-restricted cytotoxic T-cell activity, whereas FI-RSV immunization induced a low level. These data indicate that immunization with FI-RSV induces a cellular immune response different from that induced by RSV infection, which likely played a role in enhanced disease observed in infants and children.     

Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10.

In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.     

Generation of atypical pulmonary inflammatory responses in BALB/c mice after immunization with the native attachment (G) glycoprotein of respiratory syncytial virus.

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.     

Double-stranded RNA induces similar pulmonary dysfunction to respiratory syncytial virus in BALB/c mice

Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3(-/-) mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2-3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl(-) secretion in the lung or severe, lethal hypoxemia, both of which are features of influenza infection.     

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

Priming with a secreted form of the fusion protein of respiratory syncytial virus (RSV) promotes interleukin-4 (IL-4) and IL-5 production but not pulmonary eosinophilia following RSV challenge

The attachment (G) protein of respiratory syncytial virus (RSV) is synthesized as two mature forms: a membrane-anchored form and a smaller secreted form. BALB/c mice scarified with vaccinia virus (VV) expressing the secreted form develop a greater pulmonary eosinophilic influx following RSV challenge than do mice scarified with VV expressing the membrane-anchored form. To determine if a soluble form of an RSV protein was sufficient to induce eosinophilia following RSV challenge, a cDNA that encoded a secreted form of the fusion (F) protein of RSV was constructed and expressed in VV (VV-Ftm(-)). Splenocytes and lung lymphocytes from mice primed with VV-Ftm(-) produced significantly more of the Th2 cytokines interleukin-4 (IL-4) and IL-5 than did mice vaccinated with VV expressing either the native (membrane-anchored) form of the F protein or the G protein. Although mice scarified with VV-Ftm(-) developed a slight increase in the number of pulmonary eosinophils following RSV infection, the increase was not as great as that seen in VV-G-primed mice. Despite the increased IL-4 and IL-5 production and in contrast to mice primed with VV-G, mice primed with VV-Ftm(-) developed RSV-specific cytotoxic T lymphocytes (CTL) and maintained high levels of gamma interferon production. These data demonstrate that recombinant VV strains expressing soluble forms of RSV proteins induce immune responses that are more Th2-like. However, this change alone does not appear sufficient to induce vaccine-augmented disease in the face of active CD8(+) CTL populations.     

Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge

Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVDeltaG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVDeltaG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVDeltaG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVDeltaG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.     

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.     

Respiratory syncytial virus (RSV) G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with formalin-inactivated RSV

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.     

An adjuvanted respiratory syncytial virus fusion protein induces protection in aged BALB/c mice

Background

Respiratory Syncytial Virus (RSV) causes significant disease in the elderly, in part, because immunosenescence impairs protective immune responses to infection in this population. Despite previous and current efforts, there is no RSV vaccine currently licensed in infants or elderly adults. Adjuvanted RSV subunit vaccines have the potential to boost waning immune responses and reduce the burden of RSV disease in the elderly population.

Results

We used an aged BALB/c mouse model to evaluate immune responses to RSV Fusion (F) protein in the absence and presence of an alum adjuvant. We demonstrate that aged BALB/c mice immunized with alum-adjuvanted RSV F protein had significantly reduced lung viral titers at day 4 following challenge with wild-type (wt) RSV. Serum neutralizing antibody titers measured on day 27 correlated with protection in both young and aged vaccinated mice, although the magnitude of antibody titers was lower in aged mice. Unlike young mice, in aged mice, alum-adjuvanted RSV F did not induce lung T_H2-type cytokines or eosinophil infiltration compared to non-adjuvanted F protein following wt RSV challenge.

Conclusion

Our studies demonstrate that neutralizing anti-RSV antibody titers correlate with protection in both young and aged BALB/c mice vaccinated with RSV F protein vaccines. The F + alum formulation mediated greater protection compared to the non-adjuvanted F protein in both young and aged mice. However, while alum can boost F-specific antibody responses in aged mice, it does not completely overcome the reduced ability of a senescent immune system to respond to the RSV F antigen. Thus, our data suggest that a stronger adjuvant may be required for the prevention of RSV disease in immunosenescent populations, to achieve the appropriate balance of protective neutralizing antibodies and effective T_H1-type cytokine response along with minimal lung immunopathology.

     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4+ and CD8+ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4+ and CD8+ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust secondary Ab responses. Thus, immunity mediated by CD4+ and CD8+ T cells and Abs can be readily induced in mice by live RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs.     

Interleukin-4 production in BALB/c mice immunized with Anisakis simplex.

We investigated the interleukin (IL-4) levels in BALB/c mice immunized with Anisakis extract in single or multiple doses and in mice orally infected with a larva. From animals immunized maximum responses were obtained with the multiple doses with an only IL-4 peak. Conversely, in the mice inoculated with a larva per os, the IL-4 levels showed two peaks of different rates.     

Recombinant Sendai virus expressing the G glycoprotein of respiratory syncytial virus (RSV) elicits immune protection against RSV

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.     

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.     

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.