Formalin-inactivated Vaccine Research Articles

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.

Differentiation of Feline Immunodeficiency Virus Vaccination, Infection, or Vaccination and Infection in Cats

Serodiagnosis of feline immunodeficiency virus (FIV) is complicated by the use of a formalin-inactivated whole-virus FIV vaccine. Cats respond to immunization with antibodies indistinguishable from those produced during natural infection by currently available diagnostic tests, which are unable to distinguish cats that are vaccinated against FIV, infected with FIV, or both. An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide will distinguish uninfected from infected cats, regardless of vaccination status. Blood samples were evaluated from uninfected unvaccinated cats (n = 73 samples), uninfected FIV-vaccinated cats (n = 89), and FIV-infected cats (n = 102, including 3 from cats that were also vaccinated). The true status of each sample was determined by virus isolation. Plasma samples were tested for FIV antibodies by a commercial FIV diagnostic assay and an experimental discriminant ELISA. All samples from uninfected cats were correctly identified by the discriminant ELISA (specificity 100%). Of the samples collected from FIV-infected cats, 99 were correctly identified as FIV-infected (sensitivity 97.1%). With the exception of viral isolation, the discriminant ELISA is the most reliable assay for diagnosis of FIV. A practical strategy for the diagnosis of FIV infection would be to use existing commercial FIV antibody assays as screening tests. Negative results with commercial assays are highly reliable predictors for lack of infection. Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results are likely to represent infection.

Separation of pulmonary and systemic aspects of respiratory syncytial virus vaccine‐enhanced disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Prior vaccination with a formalin‐inactivated (FI) RSV vaccine resulted in enhanced morbidity and mortality upon natural RSV infection that was characterized by an extensive pulmonary mononuclear cell infiltrate and eosinophilia. Vaccination of BALB/c mice with a recombinant vaccinia virus expressing the attachment protein of RSV (vvG) results in pulmonary eosinophilia mimicking the response of the FI‐RSV vaccine recipients. The G protein elicits a CD4 T cell response that is comprised of a mixture of Th1 and Th2 cells which predominately express the VÎ214 T cell receptor. We have utilized STAT6‐ and STAT4‐deficient mice to examine the role of IL‐4/IL‐13 and IL‐12 signaling in mediating individual aspects of RSV vaccine enhanced‐disease. We demonstrate that STAT6 is necessary for the development of pulmonary eosinophilia, but that systemic disease is unaltered in the absence of STAT6. In contrast, we find that STAT4 is necessary for systemic disease but not for pulmonary eosinophilia. In both STAT6‐ and STAT4‐deficient mice, the magnitude of inflammation remained unaltered. Our results suggest that RSV vaccine‐enhanced pulmonary injury and systemic disease are mediated by distinct pathways. This work was supported by the NIH (to SMV) and the American Heart Association (to EMC).

The relative contribution of antibody and CD8 + T cells to vaccine immunity against West Nile encephalitis virus

West Nile virus (WNV) is a mosquito borne, neurotropic flavivirus that causes a severe central nervous system (CNS) infection in humans and animals. Although commercial vaccines are available for horses, none is currently approved for human use. In this study, we evaluated the efficacy and mechanism of immune protection of two candidate WNV vaccines in mice. A formalin-inactivated WNV vaccine induced higher levels of specific and neutralizing antibodies compared to a DNA plasmid vaccine that produces virus-like particles. Accordingly, partial and almost complete protection against a highly stringent lethal intracranial WNV challenge were observed in mice 60 days after single dose immunization with the DNA plasmid and inactivated virus vaccines, respectively. In mice immunized with a single dose of DNA plasmid or inactivated vaccine, antigen-specific CD8(+) T cells were induced and contributed to protective immunity as acquired or genetic deficiencies of CD8(+) T cells lowered the survival rates. In contrast, in boosted animals, WNV-specific antibody titers were higher, survival rates after challenge were greater, and an absence of CD8(+) T cells did not appreciably affect mortality. Overall, our experiments suggest that in mice, both inactivated WNV and DNA plasmid vaccines are protective after two doses, and the specific contribution of antibody and CD8(+) T cells to vaccine immunity against WNV is modulated by the prime-boost strategy.

Efficacies of inactivated vaccines against betanodavirus in grouper larvae ( Epinephelus coioides) by bath immunization

Betanodavirus is the pathogen of viral nervous necrosis (VNN) disease that has caused mass mortality among many species of marine fish at larval stage. In this study, the efficacy of inactivated betanodavirus was evaluated by bath-immunization and bath-challenge of orange-spotted grouper (Epinephelus coioides) at early larval stage. Two kinds of chemicals were used for inactivation of the virus, and the relative percent survival (RPS) values of 0.4mM binary ethylenimine (BEI)-inactivated vaccine was revealed to be 79-95, higher than that of 0.1-0.2% formalin-inactivated vaccines (39-43). Three lengths of bath immunization time were tested, and 20 min immersion of BEI-inactivated betanodavirus at a concentration of 10(6)TICD(50)/ml was sufficient to induce high protection (RPS > 75). Protection of the BEI-inactivated vaccine was evaluated at different time post immunization, and the peak of protection was observed 30 days post vaccination, and retained for at least 3 months. The efficacies of formalin-inactivated vaccines with or without encapsulation were compared, and the result revealed that the efficacy of formalin-inactivated vaccine could be significantly improved by nano-encapsulation (RPS = 85). All these data strongly suggested that bath immunization with nano-encapsulated formalin-inactivated or BEI-inactivated betanodavirus vaccines is an effective strategy to protect grouper larvae against VNN.

IL-13 Is Required for Eosinophil Entry into the Lung during Respiratory Syncytial Virus Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.

Measles vaccines

Measles is caused by infection with measles virus (MV), a negative strand RNA virus in the Morbillivirus genus of the Paramyxoviridae family. Measles is a highly infectious disease of humans spread by the respiratory route and characterized by fever and rash. Important complications include secondary infections associated with MV-induced immune suppression and the neurological disease post-infectious encephalomyelitis. The virus was first isolated in 1954 paving the way for development of the vaccines that have played an essential role in decreasing the worldwide morbidity and mortality due to measles. One of the first vaccines was a formalin-inactivated vaccine that provided only short-lived protection from infection and primed for a more severe disease, atypical measles. This vaccine was withdrawn. The other early vaccine was a live attenuated vaccine (LAV) developed by passage of the original isolate of Edmonston virus through cells in culture, primarily chicken cells. LAV was reactogenic and often given along with immune globulin. Further passage of the Edmonston virus resulted in further attenuation and the well-tolerated vaccines in common use today. LAV is generally given between 9 and 15 months of age. Seroconversion at 9 months is about 85% and at 12 months is about 95%. At younger ages seroconversion is hampered by the presence of maternal antibody and the immaturity of the immune system. The R0 (numbers of people in a susceptible population that will be infected by one person with the disease) for MV is 15-20 and interruption of endemic transmission of MV in a population requires that >95% of the population is immune. A second dose is necessary to achieve this level and can be given either as a part of a routine immunization program or through periodic mass vaccination campaigns. Research toward improved measles vaccines has focused on development of a vaccine that could be given before 6 months of age, needle-less delivery and heat stability. Several new recombinant vaccines expressing MV proteins have demonstrated induction of protective immunity in macaques and are in various stages of development.

Intranasal immunization of mice with a formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG oligodeoxynucleotides and polyphosphazenes results in enhanced protection

As respiratory syncytial virus (RSV) targets the mucosal surfaces of the respiratory tract, induction of both systemic and mucosal immunity will be critical for optimal protection. In this study, the ability of an intranasally delivered, formalin-inactivated bovine RSV (FI-BRSV) vaccine co-formulated with CpG oligodeoxynucleotides (ODN) and polyphosphazenes (PP) to induce systemic and mucosal immunity, as well as protection from BRSV challenge, was evaluated. Intranasal immunization of mice with FI-BRSV formulated with CpG ODN and PP resulted in both humoral and cell-mediated immunity, characterized by enhanced production of BRSV-specific serum IgG, as well as increased gamma interferon and decreased interleukin-5 production by in vitro-restimulated splenocytes. These mice also developed mucosal immune responses, as was evident from increased production of BRSV-specific IgG and IgA in lung-fragment cultures. Indeed, the increases in serum and mucosal IgG, and in particular mucosal IgA and virus-neutralizing antibodies, were the most critical differences observed between FI-BRSV formulated with both CpG ODN and PP in comparison to formulations with CpG ODN, non-CpG ODN or PP individually. Finally, FI-BRSV/CpG/PP was the only formulation that resulted in a significant reduction in viral replication upon BRSV challenge. Co-formulation of CpG ODN and PP is a promising new vaccine platform technology that may have applications in mucosal immunization in humans.

Bovine respiratory syncytial virus infection: immunopathogenic mechanisms

Bovine respiratory syncytial virus (BRSV) causes severe respiratory disease in young cattle. Much like the human respiratory syncytial virus, BRSV induces immunomodulation in the infected host, favoring a Th2 response. Several groups have demonstrated IgE responses to BRSV proteins during infection and particularly in response to vaccination with formalin-inactivated vaccine in the field and experimentally. Newer vaccine modalities that favor a shift to Th1 cytokine production have provided promising results. Infection with BRSV is a major contributor to the multi-pathogen disease, bovine respiratory disease complex. This review stresses the unique immunomodulatory aspects of BRSV infection, vaccination and its interaction with the host's immune system.

Complement Protein C1q Inhibits Antibody-Dependent Enhancement of Flavivirus Infection in an IgG Subclass-Specific Manner

Severe dengue virus infection can occur in humans with pre-existing antibodies against the virus. This observation led to the hypothesis that a subneutralizing antibody level in vivo can increase viral burden and cause more severe disease. Indeed, antibody-dependent enhancement of infection (ADE) in vitro has been described for multiple viruses, including the flaviviruses dengue virus and West Nile virus. Here, we demonstrate that the complement component C1q restricts ADE by anti-flavivirus IgG antibodies in an IgG subclass-specific manner in cell culture and in mice. IgG subclasses that avidly bind C1q induced minimal ADE in the presence of C1q. These findings add a layer of complexity for the analysis of humoral immunity and flavivirus infection.

Severe Acute Respiratory Syndrome Coronavirus Infection in Vaccinated Ferrets

Background. Development of vaccines to prevent severe acute respiratory syndrome (SARS) is limited by the lack of well-characterized animal models. Previous vaccine reports have noted robust neutralizing antibody and inflammatory responses in ferrets, resulting in enhanced hepatitis. Methods. We evaluated the humoral immune response and pathological end points in ferrets challenged with the Urbani strain of SARS-associated coronavirus (SARS-CoV) after having received formalin-inactivated whole-virus vaccine or mock vaccine. Results. Humoral responses were observed in ferrets that received an inactivated virus vaccine. Histopathological findings in lungs showed that infection of ferrets produced residual lung lesions not seen in both mock and vaccinated ferrets. SARS-CoV infection demonstrated bronchial and bronchiolar hyperplasia and perivascular cuffing in ferret lung tissue, as seen previously in infected mice. No evidence of enhanced disease was observed in any of the ferrets. All of the ferrets cleared the virus by day 14, 1 week earlier if vaccinated. Conclusions. The vaccine provided mild immune protection to the ferrets after challenge; however, there was no evidence of enhanced liver or lung disease induced by the inactivated whole-virus vaccine. The ferret may provide another useful model for evaluating SARS vaccine safety and efficacy.

Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is an important cause of acute respiratory tract disease. In the 1960s, vaccination with formalin-inactivated paramyxovirus preparations – respiratory syncytial virus (RSV) and measles virus (MV) – resulted in predisposition for enhanced disease upon natural infection. We have produced a formalin-inactivated hMPV preparation (FI-hMPV), which was used to immunize young cynomolgus macaques. Six days after challenge FI-hMPV-primed monkeys had developed eosinophilic bronchitis and bronchiolitis, indicative of a hypersensitivity response. This study indicates that formalin-inactivated hMPV vaccines have the same propensity to predispose for immune-mediated disease as inactivated RSV and MV vaccines.

Field effectiveness of vaccination against tick-borne encephalitis

Tick-borne encephalitis (TBE) is a vaccine-preventable disease caused by a flavivirus (TBE virus) that is endemic in many European countries and large parts of Central and Eastern Asia. In Europe, highly purified formalin-inactivated whole virus vaccines are in widespread use, but the vaccination coverage differs significantly between countries with TBE endemicity. Austria presents an exceptional situation because 88% of the total population have a history of TBE vaccination, with 58% being regularly vaccinated within the recommended schedule. In this study, we investigated the field effectiveness of TBE vaccination in Austria for the years 2000–2006 in different age groups on the basis of the documented numbers of hospitalized cases in unvaccinated and vaccinated people and the sizes of these population groups as revealed by representative inquiries. We show that the overall effectiveness in regularly vaccinated persons is about 99% with no statistically significant difference between age groups. It is at least as high after the first two vaccinations, i.e. before the completion of the basic vaccination scheme by a third vaccination, but is significantly lower (about 95%) in those with a record of irregular vaccination. Our data confirm the excellent performance of TBE vaccine under field conditions and provide evidence that, in Austria, about 2800 cases were prevented by vaccination in the years 2000–2006.

The absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines

Early in the development of respiratory syncytial virus (RSV) vaccines severe disease occurred in children after receipt of formalin-inactivated RSV vaccine. Continuing efforts to develop an appropriately attenuated and immunogenic live RSV vaccine have given opportunities to assure that live vaccines are safe through surveillance of children after vaccination. In the present study, the rate of RSV-associated upper respiratory tract illness in 388 children was lower in RSV vaccinated children than in controls (14% versus 20% in a 6–24 month old group and 16% versus 25% in infants). Additionally, there was no evidence that vaccination predisposed to more severe lower respiratory tract illness. Thus infection with a series of live attenuated RSV vaccines did not result in enhanced disease upon infection with wild type RSV. The impact of RSV during this surveillance will inform the design of future efficacy studies with RSV vaccines.

Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains

A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1–3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

Past, present and future of RSV and PIV vaccines and anti-RSV antibodies for the protection of humans against RSV

The human respiratory syncytial virus (RSV) is a human pathogen that infects infants, children under 2 years of age and elderly people, causing a lower respiratory tract infection, while evading the host's adaptive immune response. This review summarises the efforts to develop a safe vaccine and synthetic antibodies for immunisation of infants and children at risk. The first formalin-inactivated human RSV vaccine, FI-RSV, was developed and tested during the 1960s in infants and children. The results of this human trial revealed that control children, who were not vaccinated, recovered from RSV infection, while the vaccinated children required hospitalisation and two infants died. These results led to attempts to develop a cold-passage, temperature-sensitive (cpts) attenuated RSV vaccine. This vaccine was tested in small laboratory animals, monkeys and chimpanzees, and finally in children. The results of the human trial (published in 2005) revealed that the cpts vaccine was over-attenuated. RSV recombinants and parainfluenza virus recombinants developed in additional studies were tested in small laboratory animals. The unsuccessful attempts to produce an RSV vaccine led to the development of humanised anti-RSV mouse monoclonal antibodies (palivizumab) that were approved by the FDA for passive immunisation of infants and children at risk of aggravated RSV disease. Recent studies with formalin-inactivated bovine RSV vaccine formulated with the adjuvant monophosphoryl lipid A (MPL) protected newborn calves against RSV challenge. It is suggested that an apathogenic attenuated RSV deletion mutant, formulated with MPL and devoid of the viral genes that result in evasion of the human adaptive immune response, may be a useful vaccine.

Immunogenicity and efficacy of recombinant RSV-F vaccine in a mouse model

RSV vaccine development has constraints due to safety issues encountered by formalin-inactivated FI-RSV vaccines. A desirable vaccine should induce Th(1) responses and a strong mucosal immunity to provide complete protection from RSV infection. In the present paper, we developed and evaluated a mucosal vaccine against RSV in a mouse model. The antigenic regions corresponding to residues 412-524 of RSV-F protein were amplified by RT-PCR and cloned into a vector containing the ctxA(2)B gene of the cholera toxin. The recombinant protein was expressed in E. coli and properties of the recombinant protein were analyzed by SDS-PAGE, Western blot and G(M1)-ELISA. The purified recombinant protein (rRF-412) was used to immunize BALB/c mice intranasally. The results from our studies show that the rRF-412 immunogen induced mucosal (IgA) and systemic antibody (IgG, IgG1, IgG2a, and IgG2b) responses which neutralized RSV. The IgG1/IgG2a ratios indicated a Th(1)-biased antibody response. The Th(1) (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th(2) (IL-10, IL-4 and IL-5) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. Similar to the antibody response, we observed that the rRF-412 immunogen induced a mixed Th(1)/Th(2) cytokine immune response with a Th(1)-bias response. Serum antibodies were capable of neutralizing RSV and mice immunized with rRF-412 were significantly protected from live RSV challenge. Our data provides evidence that the rRF-412 immunogen may be a potential mucosal vaccine candidate against RSV.

Human metapneumovirus: Enhanced pulmonary disease in cotton rats immunized with formalin-inactivated virus vaccine and challenged

Cotton rats (Sigmodon hispidus) are susceptible to the recently discovered human metapneumovirus (hMPV), an agent closely related to human respiratory syncytial virus. Since certain respiratory syncytial virus vaccines can induce enhanced disease upon viral challenge, we have done similar experiments with hMPV in cotton rats. Young adult cotton rats were vaccinated with a formalin-inactivated preparation of hMPV strain C-85473, or with a mock preparation of the vaccine on day 0 and again on day 28. All animals were challenged intranasally on day 49 with 10(7) TCID50 of the same hMPV strain. Animals were sacrificed on days 4, 7, and 10 post-challenge and lungs were removed for viral quantitation, histopathology, and cytokine mRNA expression analysis (interferon-gamma (IFN-gamma) and interleukin-4 (IL-4)). Although the vaccinated animals showed almost complete protection from viral replication in the lungs (<10(2.0) TCID50 per gram), there was a dramatic increase in the lung pathology, particularly the interstitial pneumonitis and alveolitis with elevated serum neutralizing antibody titer prior to challenge. Cytokine profiles were distinctive from those observed during primary infection and re-infection. The data raise safety concerns for hMPV vaccine preparations.

Respiratory Syncytial Virus Vaccine-Enhanced Pulmonary Eosinophilia Requires IL-13, IL-4Rα, and STAT6 (96.6)

Abstract Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. A formalin-inactivated RSV vaccine administered to children in the 1960′s led to enhanced morbidity and mortality upon natural RSV infection. Histological analysis of the lungs upon autopsy revealed pulmonary eosinophilia. BALB/c mice primed with a recombinant vaccinia virus expressing the attachment (G) protein of RSV (vacG) develop pulmonary eosinophilia and vaccine-enhanced disease mimicking the response of the vaccinated children. Although the G protein elicits a mixed Th1 and Th2 response in these mice, the Th2 cells are known to be necessary for the induction of pulmonary eosinophilia. Here we have investigated role of individual Th2 cytokines in mediating RSV vaccine-enhanced disease. Our work shows that IL-13, the IL-4 receptor α chain (IL-4Rα), and STAT6, are required for the development of pulmonary eosinophilia in vacG-primed mice challenged with RSV whereas IL-4 is not required. We have found that IL-4, IL-13, IL-4Rα, and STAT6 do not contribute to weight loss or clinical illness whereas STAT4 is necessary. Lung histology and viral titers have also been examined in IL-13-, IL-4-, IL-4/13-, and IL-4Rα-deficient mice. Our results demonstrate a critical role for IL-13 in driving RSV vaccine-induced pulmonary eosinophilia.

C57Bl/6 mice are protected from respiratory syncytial virus (RSV) challenge and IL-5 associated pulmonary eosinophilic infiltrates following intranasal immunization with Protollin-eRSV vaccine

The protective efficacy of an intranasal (IN) Protollin-eRSV vaccine has recently been demonstrated in the RSV-susceptible BALB/c mouse model. Here, we report the safety, immunogenicity and efficacy of Protollin-eRSV vaccine in the relatively resistant C57Bl/6 mouse model. C57Bl/6 mice immunized IN with either two or three doses of Protollin-eRSV produced significant systemic and mucosal RSV-specific antibodies. Mice immunized with the Protollin vaccine displayed polarized Th1 responses with augmented IFNγ/IL-5 ratios in RSV-restimulated lung and spleen cell preparations compared with animals that received antigen alone. The Protollin-eRSV immunized C57Bl/6 mice were fully protected against challenge without eosinophilic pulmonary pathology observed in the animals immunized with the formalin-inactivated RSV vaccine. This new model will permit us to dissect the respective roles of the TLR2 and TLR4 ligands contained in the vaccine using TLR knock-out animals established on the C57Bl/6 background.