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.     

Immunogenicity of recombinant measles vaccine expressing fusion protein of respiratory syncytial virus in cynomolgus monkeys

Respiratory syncytial virus (RSV) is a common cause of respiratory infections in infants. Effective vaccines are currently being sought, but no vaccine is thus far available. In our previous study, recombinant AIK‐C measles vaccine expressing the RSV fusion protein (MVAIK/RSV/F) was developed and protective immunity against RSV demonstrated in cotton rats. In the present study, the immunogenicity and protective effects were investigated in three cynomolgus monkeys immunized with MVAIK/RSV/F. Neutralizing test antibodies against RSV were detected and no infectious virus was recovered from the lungs of monkeys immunized with MVAIK/RSV/F after challenge. MVAIK/RSV/F has the potential to inhibit RSV infection.

     

Effectiveness of enteric immunization in the development of secretory immunoglobulin A response and the outcome of infection with respiratory syncytial virus.

Cotton rats were immunized via intranasal, intradermal, or enteric routes with respiratory syncytial virus (RSV) or a live recombinant vaccinia virus expressing the RSV F glycoprotein (vaccinia F). The animals were tested for the appearance of RSV-specific antibody responses in the serum, bronchoalveolar lavage, and nasal wash after immunization and for virus replication 4 days after intranasal challenge with RSV. RSV antibody response in the serum and respiratory tract was demonstrated in all immunization groups and was significantly increased after intranasal challenge with RSV. Immunoglobulin A (IgA) antibody response in bronchoalveolar lavage fluid after intranasal or enteric immunization was two- to threefold higher than that after intradermal immunization. Nasal-wash IgA antibody response was not significantly different among three immunization groups, although mean antibody titer was the highest in intranasal immunization group. Complete resistance to replication of RSV challenge was observed in the lungs of cotton rats immunized by the intranasal or enteric routes, whereas a low level of replication was detected in the lungs of rats immunized intradermally. Enteric or intradermal immunization conferred partial protection to the upper respiratory tract, but complete protection of the upper respiratory tract was observed in the intranasal immunization group. These observations suggest that while enteric immunization is quite effective in inducing antibody responses in the respiratory tract, the magnitude of antiviral immunity induced in the respiratory tract after intranasal immunization may be superior to that observed after enteric immunization.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

Neonatal respiratory syncytial virus infection: role of transplacentally and breast milk-acquired antibodies.

The effect of transplacentally and breast milk-acquired antibodies on respiratory syncytial virus infection was studied in neonatal and 2-month-old cotton rats. Adult female rats infected intranasally with live virus regularly produced virus-specific antibodies in the serum, colostrum, and breast milk. By using foster feeding techniques, we showed that both transplacentally and breast milk-acquired antibodies were effective in reducing the replication of respiratory syncytial virus in the lungs of neonatal animals when they were challenged with live virus via the nasal route at 3 days of age. However, the protection provided by these antibodies was rather brief. There was no difference in the replication of respiratory syncytial virus in the lungs of 2-month-old animals that were delivered and nursed by seropositive (immunized) or seronegative (control) cotton rats.     

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.     

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.     

Enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactiva ted virus.

Cotton rats previously inoculated with Formalin-inactivated respiratory syncytial virus (RSV) were challenged intranasally with live RSV to induce an enhancement of RSV disease similar to that observed after the administration of Formalin-inactivated RSV vaccine to human infants 20 years ago. Within 24 h after infection with RSV, cotton rats developed pulmonary lesions that reached a maximum by day 4. Histologically, the lesions resembled an experimental pulmonary Arthus reaction. An action of Formalin on RSV appears to be responsible for this effect, because live virus or virus heated in the absence of Formalin did not induce enhanced immunopathology. Selected epitopes on the fusion (F) or attachment (G) or both RSV surface glycoproteins that are involved in inducing neutralizing antibodies were modified to reduce or ablate their antigenicity. However, other epitopes on the F or G or both glycoproteins were not ablated by Formalin, because cotton rats inoculated parenterally with a Formalin-inactivated virus developed a high level of F and G antibodies measurable by an enzyme-linked immunosorbent assay. At this time, the effect of Formalin on RSV cannot be localized to either the F or G glycoprotein of RSV.     

Human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats

Human metapneumovirus (hMPV) is a recently described paramyxovirus that is a major cause of upper and lower respiratory infection in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We previously reported the development of the cotton rat model of hMPV infection and pathogenesis (J. V. Williams et al., J. Virol. 79:10944-10951, 2005). We report here the immunogenicity of an hMPV fusion (F) protein in this model. We constructed DNA plasmids that exhibited high levels of expression of hMPV F in mammalian cells (DNA-F). These constructs were used to develop a novel strategy to produce highly pure, soluble hMPV F protein lacking the transmembrane domain (FDeltaTM). We then immunized cotton rats at 0 and 14 days with either control vector, DNA-F alone, DNA-F followed by FDeltaTM protein, or FDeltaTM alone. All groups were challenged intranasally at 28 days with live hMPV. All three groups that received some form of hMPV F immunization mounted neutralizing antibody responses and exhibited partial protection against virus shedding in the lungs compared to controls. The FDeltaTM-immunized animals showed the greatest degree of protection (>1,500-fold reduction in lung virus titer). All three immunized groups showed a modest reduction of nasal virus shedding. Neither evidence of a Th2-type response nor increased lung pathology were present in the immunized animals. We conclude that sequence-optimized hMPV F protein protects against hMPV infection when delivered as either a DNA or a protein vaccine in cotton rats.     

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

Modulation of Antibody Responses against Gnathostoma spinigerum in Mice Immunized with Crude Antigen Formulated in CpG Oligonucleotide and Montanide ISA720

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.     

A Protective and Safe Intranasal RSV Vaccine Based on a Recombinant Prefusion-Like Form of the F Protein Bound to Bacterium-Like Particles

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.     

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.     

Experimental animal model for analyzing immunobiological responses following vaccination with formalin‐inactivated respiratory syncytial virus

Formalin‐inactivated respiratory syncytial virus (FI‐RSV) vaccine was developed in the 1960s. However, this vaccine does not prevent infection in RSV‐naïve recipients and has the paradoxical effect of increasing the severity of RSV illness following natural infection, which has been a major obstacle to developing RSV vaccines. Several experimental animal models for determining the cause of the severe symptoms in FI‐RSV recipients have been developed. In the present study, cotton rats immunized with FI‐RSV were challenged with RSV and histopathological findings and recovery of infectious virus were studied. Copy numbers of mRNA of Th1 and Th2 cytokines were measured in lung tissues to gain better understanding of their immune responses. Infiltration of inflammatory cells and prominent interstitial pneumonitis were observed in the FI‐RSV group, as was induction of mRNA of Th2 cytokines such as IL‐4, IL‐10, IL‐13 and RANTES. Rats immunized with recombinant measles virus expressing the RSV F protein (MVAIK/RSV/F) and those treated with anti‐RSV mAb (palivizumab) showed very mild interstitial pneumonitis. Amounts of mRNA of IL‐1β, IFN‐γ and IL‐4 were higher in the MVAIK/RSV/F group. Administration of palivizumab before RSV challenge decreased the severity of interstitial pneumonitis in the FI‐RSV group. FI‐RSV induced skewed Th2 responses, resulting in severe inflammatory responses.     

Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.     

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.     

Preexposure to CpG Protects against the Delayed Effects of Neonatal Respiratory Syncytial Virus Infection

Severe respiratory viral infection in early life is associated with recurrent wheeze and asthma in later childhood. Neonatal immune responses tend to be skewed toward T helper 2 (Th2) responses, which may contribute to the development of a pathogenic recall response to respiratory infection. Since neonatal Th2 skewing can be modified by stimulation with Toll-like receptor (TLR) ligands, we investigated the effect of exposure to CpG oligodeoxynucleotides (TLR9 ligands) prior to neonatal respiratory syncytial virus (RSV) infection in mice. CpG preexposure was protective against enhanced disease during secondary adult RSV challenge, with a reduction in viral load and an increase in Th1 responses. A similar Th1 switch and reduction in disease were observed if CpG was administered in the interval between neonatal infection and challenge. In neonates, CpG pretreatment led to a transient increase in expression of major histocompatibility complex class II (MHCII) and CD80 on CD11c-positive cells and gamma interferon (IFN-γ) production by NK cells after RSV infection, suggesting that the protective effects may be mediated by antigen-presenting cells (APC) and NK cells. We conclude that the adverse effects of early-life respiratory viral infection on later lung health might be mitigated by conditions that promote TLR activation in the infant lung.     

Systemic and mucosal immunity to respiratory syncytial virus induced by recombinant Streptococcus gordonii surface-displaying a domain of viral glycoprotein G

A conserved fragment comprising amino acid residues 130-230 of the G glycoprotein of human respiratory syncytial virus subtype A was expressed in the commensal bacterium Streptococcus gordonii. Recombinant streptococci displaying the G domain at the cell surface were used to immunize mice via both parenteral and mucosal routes. Subcutaneous immunization induced respiratory syncytial virus-specific serum immunoglobin G (IgG) capable of partially controlling virus replication in the lungs. Intranasal immunization with live bacteria stimulated the production of IgA against both the whole virus and the G domain in serum and bronchoalveolar fluid. Upon challenge, immunized animals had significantly lower virus titres in the lungs than the controls. Our results show for the first time that the G domain-expressing S. gordonii strain elicits both systemic and mucosal immunity that reduced respiratory syncytial virus replication in the lungs of mice.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.