Respiratory Syncytial Virus Vaccine-enhanced Disease Research Articles

A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

A critical role of Gas6/Axl signal in allergic airway responses during RSV vaccine-enhanced disease.

Respiratory syncytial virus (RSV) is a common virus that causes lower respiratory infections across a wide range of ages. A licensed RSV vaccine is not available because vaccination with formalin-inactivated RSV (FI-RSV) and the subsequent RSV infection cause not only insufficient induction of neutralizing antibodies but also severe allergic airway responses, termed FI-RSV vaccine-enhanced disease (FI-RSV VED). However, the underlying mechanism has not been identified, although a Th2-biased immune response is known to be a hallmark of this disease. Our previous studies have shown that growth arrest-specific 6 (Gas6)/Axl signaling leads to Th2-biased immune responses during fungus-induced allergic airway inflammation. Here, we show that Gas6/Axl signaling also leads to FI-RSV VED and partially identify the mechanism in mice. Inhibiting Gas6/Axl signaling using Gas6-deficient mice, neutralizing antibodies, and a specific inhibitor of Axl attenuated allergic airway hyperresponsiveness, including airway inflammation, goblet cell hyperplasia, and Th2 cytokine production, in addition to increasing interferon-γ levels and the production of RSV-neutralizing IgG2a in FI-RSV VED. Gas6 was produced in lymph nodes during immunization with FI-RSV. Lymph node cells derived from immunized mice produced high levels of Gas6 and Th2 cytokines, but not IFN-γ, after restimulation with RSV. Finally, we found that dendritic cells stimulated with RSV-glycoprotein (G protein) produced Gas6 and that Axl signaling suppressed DC maturation and the induction of IL-12 production by the toll-like receptor 4 agonist RSV-fusion protein. Taken together, these results indicate that RSV-G protein-induced Gas6/Axl signaling causes allergic airway responses during FI-RSV VED.

Innate and adaptive cellular phenotypes contributing to pulmonary disease in mice after respiratory syncytial virus immunization and infection

Respiratory syncytial virus (RSV) is the major leading cause of infantile viral bronchiolitis. However, cellular phenotypes contributing to the RSV protection and vaccine-enhanced disease remain largely unknown. Upon RSV challenge, we analyzed phenotypes and cellularity in the lung of mice that were naïve, immunized with formalin inactivated RSV (FI-RSV), or re-infected with RSV. In comparison with naïve and live RSV re-infected mice, the high levels of eosinophils, neutrophils, plasmacytoid and CD11b+ dendritic cells, and IL-4+ CD4+ T cells were found to be contributing to pulmonary inflammation in FI-RSV immune mice despite lung viral clearance. Alveolar macrophages appeared to play differential roles in protection and inflammation upon RSV infection of different RSV immune mice. These results suggest that multiple innate and adaptive immune components differentially contribute to RSV disease and inflammation.

RSV vaccine-enhanced disease is orchestrated by the combined actions of distinct CD4 T cell subsets.

There is no currently licensed vaccine for respiratory syncytial virus (RSV) despite being the leading cause of lower respiratory tract infections in children. Children previously immunized with a formalin-inactivated RSV (FI-RSV) vaccine exhibited enhanced respiratory disease following natural RSV infection. Subsequent studies in animal models have implicated roles for CD4 T cells, eosinophils and non-neutralizing antibodies in mediating enhanced respiratory disease. However, the underlying immunological mechanisms responsible for the enhanced respiratory disease and other disease manifestations associated with FI-RSV vaccine-enhanced disease remain unclear. We demonstrate for the first time that while CD4 T cells mediate all aspects of vaccine-enhanced disease, distinct CD4 T cell subsets orchestrate discrete and specific disease parameters. A Th2-biased immune response, but not eosinophils specifically, was required for airway hyperreactivity and mucus hypersecretion. In contrast, the Th1-associated cytokine TNF-α was necessary to mediate airway obstruction and weight loss. Our data demonstrate that individual disease manifestations associated with FI-RSV vaccine-enhanced disease are mediated by distinct subsets of CD4 T cells.

Dual proinflammatory and antiviral properties of pulmonary eosinophils in respiratory syncytial virus vaccine-enhanced disease.

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.

The role of non-viral antigens in the cotton rat model of respiratory syncytial virus vaccine-enhanced disease

In the 1960s, infant immunization with a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine candidate caused enhanced respiratory disease (ERD) following natural RSV infection. Because of this tragedy, intensive effort has been made to understand the root causes of how the FI-RSV vaccine induced a pathogenic response to subsequent RSV infection in vaccinees. A well-established cotton rat model of FI-RSV vaccine-enhanced disease has been used by numerous researchers to study the mechanisms of ERD. Here, we have dissected the model and found it to have significant limitations for understanding FI-RSV ERD. This view is shaped by our finding that a major driver of lung pathology is cell-culture contaminants, although FI-RSV immunization and RSV challenge serve as co-factors to exacerbate disease. Specifically, non-viral products from the vaccine and challenge preparations that are devoid of RSV give rise to alveolitis, which is considered a hallmark of FI-RSV ERD in the cotton rat model. Although FI-RSV immunization and RSV challenge promote more severe alveolitis, they also drive stronger cellular immune responses to non-viral antigens. The severity of alveolitis is associated with T cells specific for non-viral antigens more than with T cells specific for RSV. These results highlight the limitations of the cotton rat ERD model and the need for an improved animal model to evaluate the safety of RSV vaccine candidates.

Biological challenges and technological opportunities for respiratory syncytial virus vaccine development.

Respiratory syncytial virus (RSV) is an important cause of respiratory disease causing high rates of hospitalizations in infants, significant morbidity in children and adults, and excess mortality in the elderly. Major barriers to vaccine development include early age of RSV infection, capacity of RSV to evade innate immunity, failure of RSV-induced adaptive immunity to prevent reinfection, history of RSV vaccine-enhanced disease, and lack of an animal model fully permissive to human RSV infection. These biological challenges, safety concerns, and practical issues have significantly prolonged the RSV vaccine development process. One great advantage compared to other difficult viral vaccine targets is that passively administered neutralizing monoclonal antibody is known to protect infants from severe RSV disease. Therefore, the immunological goals for vaccine development are to induce effective neutralizing antibody to prevent infection and to avoid inducing T-cell response patterns associated with enhanced disease. Live-attenuated RSV and replication-competent chimeric viruses are in advanced clinical trials. Gene-based strategies, which can control the specificity and phenotypic properties of RSV-specific T-cell responses utilizing replication-defective vectors and which may improve on immunity from natural infection, are progressing through preclinical testing. Atomic level structural information on RSV envelope glycoproteins in complex with neutralizing antibodies is guiding design of new vaccine antigens that may be able to elicit RSV-specific antibody responses without induction of RSV-specific T-cell responses. These new technologies may allow development of vaccines that can protect against RSV-mediated disease in infants and establish a new immunological paradigm in the host to achieve more durable protection against reinfection.

Pulmonary Eosinophilia Is Attenuated by Early Responding CD8+Memory T Cells in a Murine Model of RSV Vaccine-Enhanced Disease

Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4(+) T-cell response to challenge infection with some features characteristic of Th-2 CD4(+) T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8(+) T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8(+) memory T-cell (CD8(+) Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8(+) T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8(+) Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8(+) T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8(+) Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8(+) T cells regulate this process is not by modulating the differentiation or development of the CD4(+) Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8(+) Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.

The cotton rat model of respiratory viral infections

Development of successful vaccines against human infectious diseases depends on using appropriate animal models for testing vaccine efficacy and safety. For some viral infections the task is further complicated by the frequently changing genetic make-up of the virus, as in the case of influenza, or by the existence of the little-understood phenomenon of vaccine-enhanced disease, as in the case of respiratory syncytial virus (RSV). The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of the RSV vaccine-enhanced disease. Recently, using cotton rats, we have demonstrated that vaccination against another paramyxovirus, human metapneumovirus (hMPV), can also lead to vaccine-enhanced disease. In addition to the study of paramyxoviruses, S. hispidus presents important advantages for the study of orthomyxoviruses such as influenza. The cotton rat is susceptible to infection with unadapted human influenza strains, and heterosubtypic immunity to influenza can be evoked in S. hispidus. The mechanisms of influenza, RSV, and hMPV pathogenesis and immunity can now be investigated in the cotton rat with the development of species-specific reagents for this animal model.

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.

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.

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.