Immune induction and modulation in mice following immunization with DNA encoding F protein of respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the principal agents of bronchiolitis and pneumonia in young children. Thus, there is a strong need to make a safe and effective vaccine against the RSV infection. DNA immunization is very effective at inducing both cellular and humoral immune responses. In this study, we inserted the RSV-F gene into expression vectors, pcDNA3.1 and pQE. These constructs were transformed into C2C12 and E. coli M15 cells, respectively. The expression of the RSV-F protein was confirmed by SDS-PAGE, followed by Western blot analyses. The immunization of pcDNA3.1-RSV-F elicited both anti-RSV-F titer in mouse sera and CTL activities with mouse splenocytes. Especially, the co-administration of IL-4, or the GM-CSF gene with the RSV-F gene construct, enhanced the production of anti-RSV-F Ab. However, this enhancement disappeared by the simultaneous injection of the Th1 and Th2 type cytokine genes. The CTL activities were affected by the co-delivery of the IFN-gamma gene, but not by Th2-type cytokines.     

Engineering,Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus

A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1β, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/W(v) mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.     

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.     

Interleukin-27 inhibits vaccine-enhanced pulmonary disease following respiratory syncytial virus infection by regulating cellular memory responses

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in young children. In the 1960s, infants vaccinated with formalin-inactivated RSV developed a more severe disease characterized by excessive inflammatory immunopathology in lungs upon natural RSV infection. The fear of causing the vaccine-enhanced disease (VED) is an important obstacle for development of safe and effective RSV vaccines. The recombinant vaccine candidate G1F/M2 immunization also led to VED. It has been proved that cellular memory induced by RSV vaccines contributed to VED. Interleukin-27 (IL-27) and IL-23 regulate Th1, Th17, and/or Th2 cellular immune responses. In this study, mice coimmunized with pcDNA3-IL-27 and G1F/M2 were fully protected and, importantly, did not develop vaccine-enhanced inflammatory responses and immunopathology in lungs after RSV challenge, which was correlated with moderate Th1-, suppressed Th2-, and Th17-like memory responses activated by RSV. In contrast, G1F/M2- or pcDNA3-IL-23+G1F/M2-immunized mice, in which robust Th2- and Th17-like memory responses were induced, developed enhanced pulmonary inflammation and severe immunopathology. Mice coimmunized with G1F/M2 and the two cytokine plasmids exhibited mild inflammatory responses as well as remarkable Th1-, suppressed Th2-, and Th17-like memory responses. These results suggested that Th1-, Th2-, and Th17-like memory responses and, in particular, excessive Th2- and Th17-like memory responses were closely associated with VED; IL-27 may inhibit VED following respiratory syncytial virus infection by regulating cellular memory responses.     

IL-12-independent Th1-type immune responses to respiratory viral infection: requirement of IL-18 for IFN-gamma release in the lung but not for the differentiation of viral-reactive Th1-type lymphocytes

We demonstrated that IL-12 was induced during primary or secondary pulmonary adenoviral infection in wild-type (wt) mice. However, cellular responses were not compromised in the lungs of IL-12-/- mice. The level of IFN-gamma in the lung was similar in wt and IL-12-/- mice during pulmonary viral infection. Upon Ag stimulation in vitro, lymphocytes from draining lymph nodes or spleen of infected IL-12-/- mice released large amounts of IFN-gamma, but not IL-4, which were comparable to those released by wt lymphocytes. Furthermore, a predominantly IgG2a response to adenoviral infection was unimpaired in IL-12-/- mice. These significant anti-adenoviral Th1-type responses in IL-12-/- mice led to an efficient clearance of virus-infected cells in the lung. Whether IL-18 was involved in IL-12-independent anti-adenoviral immune responses was investigated. Abrogation of endogenous IL-18 by an Ab resulted in diminished IFN-gamma release and lymphocytic infiltrate in the lung during adenoviral infection. Nevertheless, the development of lymphocytes of the Th1 phenotype was unimpaired in the absence of both IL-12 and IL-18. In contrast to their intact ability to mount Th1-type responses to viral infection, IL-12-/- mice suffered impaired Th1-type immune responses to pulmonary mycobacterial infection. Our findings suggest that IL-12, although induced, is not required for Th1-type responses to respiratory viral infection, in contrast to mycobacterial infection. IL-18 is required for the optimal release of IFN-gamma in the lung during viral infection, but is not required for the generation of virus-reactive Th1-type lymphocytes. Th1 differentiation during respiratory adenoviral infection may involve molecules different from IL-12 or IL-18.     

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.     

Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes

We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct resulted in: 1) simultaneous CTL induction against all nine CTL epitopes despite their varying MHC binding affinities; 2) CTL responses that were equivalent in magnitude to those induced against a lipopeptide known be immunogenic in humans; 3) induction of memory CTLs up to 4 mo after a single DNA injection; 4) higher epitope-specific CTL responses than immunization with DNA encoding whole protein; and 5) a correlation between the immunogenicity of DNA-encoded epitopes in vivo and the in vitro responses of specific CTL lines against minigene DNA-transfected target cells. Examination of potential variables in minigene construct design revealed that removal of the PADRE Th cell epitope or the signal sequence, and changing the position of selected epitopes, affected the magnitude and frequency of CTL responses. Our results demonstrate the simultaneous induction of broad CTL responses in vivo against multiple dominant HLA-restricted epitopes using a minigene DNA vaccine and underline the utility of HLA transgenic mice in development and optimization of vaccine constructs for human use.     

A self MHC class II beta-chain peptide prevents diabetes in nonobese diabetic mice

We explored T cell responses to the self class II MHC (I-Ag7) beta-chain-derived peptides in diabetic and prediabetic nonobese diabetic (NOD) mice. We found that one of these immunodominant epitopes of the beta-chain of I-Ag7 molecule, peptide 54-76, could regulate autoimmunity leading to diabetes in NOD mice. T cells from prediabetic young NOD mice do not respond to the peptide 54-76, but T cells from diabetic NOD mice proliferated in response to this peptide. T cells from older nondiabetic mice or mice protected from diabetes do not respond to this peptide, suggesting a role for peptide 54-76-specific T cells in pathogenesis of diabetes. We show that this peptide is naturally processed and presented by the NOD APCs to self T cells. However, the peptide-specific T cells generated after immunization of young mice regulate autoimmunity in NOD mice by blocking the diabetogenic cells in adoptive transfer experiments. The NOD mice immunized with this peptide are protected from both spontaneous and cyclophosphamide-induced insulin-dependent diabetes mellitus. Immunization of young NOD mice with this peptide elicited T cell proliferation and production of Th2-type cytokines. In addition, immunization with this peptide induced peptide-specific Abs of IgG1 isotype that recognized native I-Ag7 molecule on the cell surface and inhibited the T cell proliferative responses. These results suggest that I-Abetag7(54-76) peptide-reactive T cells are involved in the pathogenesis of diabetes. However, immunization with this peptide at young age induces regulatory cells and the peptide-specific Abs that can modulate autoimmunity in NOD mice and prevent spontaneous and induced diabetes.     

Modulation of Protease Activated Receptor 1 Influences Human Metapneumovirus Disease Severity in a Mouse Model

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus

Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-alpha) and IFN-gamma responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.     

Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus

Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

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.     

Role for innate IFNs in determining respiratory syncytial virus immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1(-/-) mice that are incapable of generating Th1 CD4(+) cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-alphabetaR (IFN-alphabetaR(-/-)), the IFN-gammaR (IFN-gammaR(-/-)), or both receptors (IFN-alphabetagammaR(-/-)). Although both the IFN-gammaR(-/-) and the IFN-alphabetagammaR(-/-) animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8(+) T cells could be detected in lungs of IFN-alphabetagammaR(-/-) animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4(+) Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.     

Infection with Human Metapneumovirus Predisposes Mice to Severe Pneumococcal Pneumonia

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes respiratory tract infections. Prior clinical studies have highlighted the importance of respiratory viruses, such as influenza virus, in facilitating secondary bacterial infections and increasing host immunopathology. The objective of the present work was to evaluate the effects of initial viral infection with hMPV or influenza A virus followed by Streptococcus pneumoniae superinfection 5 days later in a murine model. Both groups of superinfected mice demonstrated significant weight loss (mean of 15%) and higher levels of airway obstruction (mean enhanced pause value of 2.7) compared to those of mice infected with hMPV, influenza virus, or pneumococcus alone. Bacterial counts increased from 5 × 10² CFU/lung in mice infected with pneumococcus only to 10⁷ and 10⁹ CFU/lung in mice with prior infections with hMPV and influenza A virus, respectively. A more pronounced interstitial and alveolar inflammation correlated with higher levels of inflammatory cytokines and chemokines such as interleukin-1α (IL-1α), IL-1β, IL-6, IL-12, monocyte chemotactic protein 1, macrophage inflammatory protein 1α, KC, and granulocyte colony-stimulating factor, as well as greater expression of Toll-like receptor 2 (TLR2), TLR6, TLR7, and TLR13 in the lungs of superinfected animals compared to results for single infections, with similar immunological effects seen in both coinfection models. Prior infection with either hMPV or influenza A virus predisposes mice to severe pneumococcus infection.     

The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 x 10(2) PFU/gram tissue (C3H mice) to greater than 10(5) PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 x 10(5) PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.     

Expression of interleukin-4 by recombinant respiratory syncytial virus is associated with accelerated inflammation and a nonfunctional cytotoxic T-lymphocyte response following primary infection but not following challenge with wild-type virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.