Vaccine-enhanced Disease Research Articles

TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity

Agonists for TLR7, TLR8, and TLR9 have been shown to enhance vaccine immunogenicity. We evaluated the impact of TLR activation on RSV disease in a murine model by administering TLR7/8 and TLR9 agonists during FI-RSV immunization or RSV infection. CpG administered during immunization reduced disease following challenge as evidenced by decreased lung pathology, illness, and cytokines. In marked contrast, TLR7/8 agonist had little impact. To evaluate potential therapeutic use, TLR agonists were administered during primary infection. Although type 2 cytokine responses decreased and type 1 cytokines and MIP-1-α/β increased, both TLR7/8 and TLR9 agonists increased clinical symptoms and pulmonary inflammation when administered during primary infection. Thus, TLR9-induced signaling during FI-RSV immunization reduced vaccine-enhanced disease whereas immunostimulatory properties of TLR agonists enhanced disease severity when used during RSV infection. Immunomodulation elicited by TLR9 agonist confirms the adjuvant potential of TLR agonists during RSV immunization. However, in contrast to work done with HIV-1 vaccines, the inability of TLR7/8 agonist to boost type 1 vaccine-induced RSV immunity demonstrates pathogen–TLR specificity. These data reveal that the timing of administration of immunomodulatory agents is critical. Furthermore, these data underscore that amplification of anti-viral immune responses may result in immunopathology rather than immune-mediated protection.

Fas Ligand Is Required for the Development of Respiratory Syncytial Virus Vaccine-Enhanced Disease

Children immunized with a formalin-inactivated respiratory syncytial virus (RSV) vaccine experienced enhanced disease and exhibited pulmonary eosinophilia upon natural RSV infection. BALB/c mice immunized with either formalin-inactivated RSV or a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein develop extensive pulmonary eosinophilia after RSV challenge that mimics the eosinophilic response observed in the children during the 1960s vaccine trials. Fas ligand (FasL) is a major immune effector molecule that can contribute to the clearance of respiratory viruses. However, the role of FasL in the development of RSV vaccine-enhanced disease has not been elucidated. RSV challenge of vacvG-immunized gld mice, that lack functional FasL, results in diminished systemic disease as well as pulmonary eosinophilia. The magnitude of the secondary RSV G-specific CD4 T cell response was diminished in gld mice as compared with wild-type controls. Furthermore, we show that CD4 T cells isolated after RSV challenge of vacvG-immunized gld mice exhibit enhanced expression of Annexin V and caspase 3/7 indicating that FasL is important for either the survival or the expansion of virus-specific secondary effector CD4 T cells. Taken together, these data identify a previously undefined role for FasL in the accumulation of secondary effector CD4 T cells and the development of RSV vaccine-enhanced disease.

The Number of Respiratory Syncytial Virus (RSV)-Specific Memory CD8 T Cells in the Lung Is Critical for Their Ability to Inhibit RSV Vaccine-Enhanced Pulmonary Eosinophilia

Children that were administered a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine experienced enhanced respiratory disease, including pulmonary eosinophilia, after contracting a natural RSV infection. RSV vaccine-enhanced disease can be mimicked in BALB/c mice immunized with either FI-RSV or with a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein. We have recently demonstrated that memory CD8 T cells directed against the RSV immunodominant M2(82-90) epitope inhibit the development of pulmonary eosinophilia in either vacvG- or FI-RSV-immunized mice by reducing the total number of Th2 cells in the lung after RSV challenge. In this study, we show that memory CD8 T cells specific to a subdominant epitope within the RSV fusion (F) protein fail to inhibit the development of pulmonary eosinophilia after RSV challenge of mice previously co-immunized with vacvF and with either vacvG or FI-RSV. We observed that the inability of RSV F(85)-specific memory CD8 T cells to inhibit the development of pulmonary eosinophilia was largely due to an inadequate total number of F(85)-specific memory CD8 T cells in the lung at early times after RSV challenge. Increasing the number of F(85)-specific memory CD8 T cells after immunization grants these cells the ability to inhibit RSV vaccine-enhanced pulmonary eosinophilia. Moreover, we demonstrate that RSV-specific memory CD8 T cells, when present in sufficient numbers, inhibit the production of the Th2-associated chemokines CCL17 and CCL22. Taken together, these results indicate that RSV-specific memory CD8 T cells may alter the trafficking of Th2 cells and eosinophils into the lung.

Cutting Edge: Eosinophils Do Not Contribute to Respiratory Syncytial Virus Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) infection of BALB/c mice previously immunized with a recombinant vaccinia virus (vacv) expressing the attachment (G) protein of RSV (vacvG) results in pulmonary eosinophilia, which mimics the response of formalin-inactivated RSV-vaccinated children, as well as increased weight loss, clinical illness, and enhanced pause (Penh). We show that RSV infection of eosinophil-deficient mice previously immunized with vacvG results in the development of increased weight loss, clinical illness, and Penh similar to that in wild-type controls. These measures of RSV vaccine-enhanced disease are dependent upon STAT4. Interestingly, neither IL-12 nor IL-23, the two most common STAT4-activating cytokines, proved necessary for the development of disease. We demonstrate that IFN-gamma, which is produced following STAT4 activation, contributes to clinical illness and increased Penh, but not weight loss. Our results have important implications for future RSV vaccine design, suggesting that enhancing a Th1 response may exacerbate disease.

Overcoming T-Cell-Mediated Immunopathology to Achieve Safe Respiratory Syncytial Virus Vaccination

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. Premature infants, immunocompromised individuals, and the elderly exhibit an increased risk for the development of severe disease after RSV infection. Currently, there is not a safe and effective RSV vaccine available, in part due to our incomplete understanding of how severe immunopathology was induced following RSV infection of children previously immunized with a formalin-inactivated RSV vaccine. Much of our current understanding of RSV vaccine-enhanced disease can be attributed to the establishment of multiple mouse models of RSV vaccination. Studies analyzing the RSV-specific immune response in mice have clearly demonstrated that both CD4 and CD8 memory T cells contribute to RSV-induced immunopathology. In this review we will focus our discussion on data generated from the mouse models of RSV immunization that have advanced our understanding of how virus-specific T cells mediate immunopathology and RSV vaccine-enhanced disease.

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.

Early Responding CD8+ T cells are Critical in the Resolution of Pulmonary Eosinophilia in a Murine Model of Respiratory Syncytial Virus Vaccine-enhanced Disease

Respiratory syncytial virus (RSV) vaccine-enhanced disease in mice can be generated by vaccinating mice with a vaccinia virus vector containing the RSV G protein followed by intranasal RSV infection. This process is characterized by a potent memory CD4+ T cell response with type II features including an enhanced pulmonary eosinophil infiltrate without the simultaneous induction of a memory CD8+ T lymphocyte (mCD8+) response. In this study, we investigate how and when the generation of a mCD8+ T cell response may affect the development of pulmonary eosinophilia and the progression of vaccine-enhanced disease.

CD8 T Cells Inhibit Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-gamma-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.

Understanding respiratory syncytial virus (RSV) vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide. In addition, RSV causes serious disease in elderly and immune compromised individuals. RSV infection of children previously immunized with a formalin-inactivated (FI)-RSV vaccine is associated with enhanced disease and pulmonary eosinophilia that is believed to be due to an exaggerated memory Th2 response. As a consequence, there is currently no licensed RSV vaccine and detailed studies directed towards prevention of vaccine-associated disease are a critical first step in the development of a safe and effective vaccine. The BALB/c mouse model of RSV infection faithfully mimics the human respiratory disease. Mice previously immunized with either FI-RSV or a recombinant vaccinia virus (vv) that expresses the attachment (G) glycoprotein exhibit extensive lung inflammation and injury, pulmonary eosinophilia, and enhanced disease following challenge RSV infection. CD4 T cells secreting Th2 cytokines are necessary for this response because their depletion eliminates eosinophilia. Intriguing recent studies have demonstrated that RSV-specific CD8 T cells can inhibit Th2-mediated pulmonary eosinophilia in vvG-primed mice by as yet unknown mechanisms. Information gained from the animal models will provide important information and novel approaches for the rational design of a safe and efficacious RSV vaccine.

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.

The TLR4 agonist, monophosphoryl lipid A, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease

Formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) induces a poorly understood immunopathological response that leads to disease enhancement upon RSV infection of vaccinees. In the cotton rat model, inclusion of monophosphoryl lipid A (MPL) in the FI-RSV formulation was found to mitigate the lung pathology associated with vaccine-enhanced disease. Here we report that the protective effect of MPL on FI-RSV vaccine-enhanced disease is associated with a dramatic reduction in levels of Th1- and Th2-type cytokines and chemokines normally elicited in response to RSV challenge. Our data illustrate the complexity of proinflammatory response elicited by FI-RSV vaccination and RSV infection and the potential importance of MPL in modifying this response.

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.

Animal Models for Studying Respiratory Syncytial Virus Infection and Its Long Term Effects on Lung Function

Human respiratory syncytial virus (hRSV) infection causes a spectrum of illnesses ranging from mild infection to life-threatening bronchiolitis and respiratory failure. Human studies on the pathogenesis of RSV infection are invaluable, but animal models permit advances with the use of experimental strategies that would be inappropriate in human studies. We review the advantages and disadvantages of various animal models for the study of hRSV infection. No animal model of hRSV infection replicates the complete spectrum of disease severity seen in humans. Available models differ in their ability to incorporate genetic technology and to allow the study of immunity, vaccine efficacy and treatment interventions. Although hRSV establishes disease in primates, this advantage is outweighed by the impracticalities and cost of using such models. The study of bovine RSV infection in calves is appealing because of parallels with human disease. Among rodent models, BALB/c mice have helped delineate the mechanisms underlying vaccine-enhanced RSV disease, and cotton rats have been used for preclinical testing. The single major disadvantage of studying hRSV in rodent models is the limited extent to which this host-restricted human pneumovirus replicates in mouse lung tissue. The rodent-specific Pneumovirus pathogen, pneumonia virus of mice, causes an infection that mirrors severe bronchiolitis and pneumonia in infants infected with RSV, including robust virus replication with profound inflammation. The recent development of the pneumonia virus of mice model has permitted exploration of the mechanisms of severe Pneumovirus disease in vivo with the use of sophisticated genetic tools and genetically manipulated mouse strains.

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

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

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

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

Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with Lot 100 or a newly prepared reference vaccine

A formalin-inactivated respiratory syncytial virus vaccine was used to immunize infants in the mid-1960s; when these children later were naturally infected by the virus they developed markedly accentuated disease, and two died. For the present work, a new batch of vaccine was prepared using the original formula. Administration of either the old or new vaccines resulted in enhanced lesions in immunized cotton rats subsequently challenged with live virus, although administration of the vaccine reduced virus replication by 90%. Animals primed with formalin-inactivated virus and challenged developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which appear to be specific markers for the vaccine enhancement. The newly prepared vaccine appears suitable as a reference standard for studying the mechanism of vaccine-enhanced disease caused by this virus. Additionally, we reviewed the lesions in the lungs of the two humans who died with the vaccine-enhanced disease in 1967, and found that they were similar to, but more severe than those seen in the cotton rats.

Immunopathogenesis of vaccine-enhanced RSV disease

Inducing a strong immune response is an essential aim of vaccination. Although immune responses to virus infections are usually protective, they can also be harmful. The best-documented examples of an immune response increasing disease severity are with dengue, measles and respiratory syncytial virus infections. In the 1960s, administration of formalin-inactivated, tissue culture grown RSV (FI-RSV) was found to induce strong ELISA binding but poor virus-neutralising antibody. Infants given this ‘lot 100’ vaccine appeared to exhibit an increased rate of RSV infection during subsequent natural RSV outbreaks. Although it has not been possible to exactly delineate the cause of disease enhancement in man, animal models strongly suggest that it was due to strong (and perhaps unbalanced) T cell priming rather than infection-enhancing or sensitising antibody. In animal models, enhanced disease can result from over-exuberant T cell priming which recruits an abundant inflammatory infiltrate in the lung (the nature of which depends on the patterns of cytokines and chemokines produced). Formalin-treated RSV vaccination has been linked specifically to the induction of Th2 cells, which make IL-4 and IL-5 and induce a strong pulmonary eosinophilic response. The vaccine dosing regime and the interval between vaccination and challenge can be critical to the induction of protection or pathology. Defining the correlates of protection and disease enhancement in man is critical to the rational development of effective and protective vaccines against RSV.

Animal models of respiratory syncytial virus infection.

Over the past two decades, animal models of respiratory syncytial virus (RSV) infection have been developed using primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters. Use of these models has shed light on the mechanisms of vaccine-enhanced disease seen in clinical trials of a formalin-inactivated RSV vaccine and has provided a means for testing efficacy and safety of candidate prophylactic and therapeutic strategies. The development of multiple animal models has coincided with the realization that RSV disease in humans is a multifaceted disease whose clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status, and concurrent disease within subpopulations. There is no single human subpopulation in whom all forms of RSV disease manifest, nor is there a single animal model that duplicates all forms of RSV disease. The choice of an experimental model will be governed by the specific manifestation of disease to be studied.

T cell source of type 1 cytokines determines illness patterns in respiratory syncytial virus-infected mice.

Manipulation of the cytokine microenvironment at the time of vaccination can influence immune responses to remote challenge, providing a strategy to study the molecular pathogenesis of respiratory syncytial virus (RSV) vaccine-enhanced disease in the mouse model. Although treatment with antibody against IL-4 or recombinant IL-12 (rIL-12) at the time of formalin-inactivated RSV vaccination induced a similar shift in the pattern of cytokine mRNA expression upon live virus challenge, anti-IL-4 treated mice had increased CD8+ cytotoxic T lymphocyte activity and reduced illness compared with rIL-12-treated mice. To define effector mechanisms responsible for these patterns, CD4+ and/or CD8+ T lymphocytes were selectively depleted in vivo at the time of RSV challenge. In rIL-12-treated mice, CD4+ lymphocytes made the largest contribution to IFN-gamma mRNA, RSV clearance, and illness, while in anti-IL-4 treated mice, CD8+ lymphocytes were the major effector. The effector responsible for virus clearance also mediated illness, suggesting that efficiency of virus clearance determined disease expression. These results demonstrate that the phenotype of effector cells involved in the immune response to virus challenge may be a more important determinant of disease than patterns of cytokine expression classically assigned to Th1 and Th2 lymphocytes.

Determinants and kinetics of cytokine expression patterns in lungs of vaccinated mice challenged with respiratory syncytial virus

The development of a successful respiratory syncytial virus (RSV) vaccine will be advanced by an improved understanding of the pathogenesis of natural disease and vaccine-enhanced illness. Using a murine model, we have examined cytokine message expression and cytokine secretion in lungs of mice primed with killed or live antigens and challenged with RSV. Stable cytokine mRNA expression was achieved if the prime-challenge interval was 2 weeks. The pattern of expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) mRNA was established by day 4 after challenge and was maintained at least through day 12, and was not affected by the concentration of priming immunogen or virus challenge. An enzyme-linked immunospot assay demonstrated that CD4+ T cells were responsible for the production of IL-4, while many cell types secreted IFN-γ. These experiments begin to define the kinetics of cytokine expression and phenotypes of cytokine-producing cells following RSV infection, supporting previous findings that suggested aberrant infiltration of CD4+ T lymphocytes and excessive IL-4 secretion may play a role in the vaccine-enhanced disease associated with RSV.