Development of Respiratory Syncytial Virus Vaccine Candidates for the Elderly.

Respiratory syncytial virus (RSV) is a significant threat to elderly populations and repeated infections that occur throughout life are poorly protective. To assess the role of prior RSV infections as well as elderly immune senescence on vaccine efficacy, we compared immune responses after virus-like particle (VLP) immunization of elderly cotton rats and young cotton rats, both previously RSV infected, in order to mimic the human population. We show that immunization of RSV-experienced young or elderly animals resulted in the same levels of anti-pre-F IgG, anti-G IgG, neutralizing antibody titers, and protection from challenge indicating that the delivery of F and G proteins in a VLP is equally effective in activation of protective responses in both elderly and young populations. Our results suggest that F and G protein-containing VLPs induce anti-RSV memory established in prior RSV infections equally well in both young and elderly animals and thus can be an effective vaccine for the elderly.

Correlative outcomes of maternal immunization against RSV in cotton rats.

Maternal anti-respiratory syncytial virus (RSV) antibodies protect neonates from RSV disease throughout first weeks of life. Previous studies of maternal immunization in cotton rats showed that a single immunization during pregnancy of RSV-primed dams with virus-like particles (VLPs) assembled with pre-fusion F protein and the wild type G protein boosted their RSV serum antibody concentration and protected pups early in life against RSV challenge. We extended these findings by evaluating responses to RSV infection in litters from two consecutive pregnancies of immunized dams. Using an RSV-primed population of VLP-vaccinated and unvaccinated dams, we defined correlations between dams' and litters' RSV neutralizing antibodies (NA); between litters' NA and protection; and between litter's NA and their lung expression of selected cytokines, of a first or of a second pregnancy. Lung pathology was also evaluated. We found positive correlation between the NA titers in the dams at delivery and the NA in their first and second litters and negative correlations between the litters' NA and protection from RSV challenge. Vaccination of dams modulated the mRNA expression for IFNγ and IL-6 and lung pathology in the first and in the second litter at different times after birth, even in the absence of detectable NA. Maternal RSV vaccination enhanced the levels of antibodies transferred to offspring and their protection from challenge. Importantly, maternal vaccination also impacted the immunological and inflammatory response of the offspring's lungs well into maturity, and after the antiviral effect of maternally transferred NA waned or was no longer detectable.

Protection against influenza-induced Acute Lung Injury (ALI) by enhanced induction of M2a macrophages: possible role of PPARγ/RXR ligands in IL-4-induced M2a macrophage differentiation.

Many respiratory viruses cause lung damage that may evolve into acute lung injury (ALI), a cytokine storm, acute respiratory distress syndrome, and ultimately, death. Peroxisome proliferator activated receptor gamma (PPARγ), a member of the nuclear hormone receptor (NHR) family of transcription factors, regulates transcription by forming heterodimers with another NHR family member, Retinoid X Receptor (RXR). Each component of the heterodimer binds specific ligands that modify transcriptional capacity of the entire heterodimer by recruiting different co-activators/co-repressors. However, the role of PPARγ/RXR ligands in the context of influenza infection is not well understood. PPARγ is associated with macrophage differentiation to an anti-inflammatory M2 state. We show that mice lacking the IL-4Rα receptor, required for M2a macrophage differentiation, are more susceptible to mouse-adapted influenza (A/PR/8/34; "PR8")-induced lethality. Mice lacking Ptgs2, that encodes COX-2, a key proinflammatory M1 macrophage mediator, are more resistant. Blocking the receptor for COX-2-induced Prostaglandin E2 (PGE2) was also protective. Treatment with pioglitazone (PGZ), a PPARγ ligand, increased survival from PR8 infection, decreased M1 macrophage gene expression, and increased PPARγ mRNA in lungs. Conversely, conditional knockout mice expressing PPARγ-deficient macrophages were significantly more sensitive to PR8-induced lethality. These findings were extended in cotton rats: PGZ blunted lung inflammation and M1 cytokine gene expression after challenge with non-adapted human influenza. To study mechanisms by which PPARγ/RXR transcription factors induce canonical M2a genes, WT mouse macrophages were treated with IL-4 in the absence or presence of rosiglitazone (RGZ; PPARγ ligand), LG100754 (LG; RXR ligand), or both. IL-4 dose-dependently induced M2a genes Arg1, Mrc1, Chil3, and Retnla. Treatment of macrophages with IL-4 and RGZ and/or LG differentially affected induction of Arg1 and Mrc1 vs. Chil3 and Retnla gene expression. In PPARγ-deficient macrophages, IL-4 alone failed to induce Arg1 and Mrc1 gene expression; however, concurrent treatment with LG or RGZ + LG enhanced IL-4-induced Arg1 and Mrc1 expression, but to a lower level than in WT macrophages, findings confirmed in the murine alveolar macrophage cell line, MH-S. These findings support a model in which PPARγ/RXR heterodimers control IL-4-induced M2a differentiation, and suggest that PPARγ/RXR agonists should be considered as important tools for clinical intervention against influenza-induced ALI.

Alternative Virus-Like Particle-Associated Prefusion F Proteins as Maternal Vaccines for Respiratory Syncytial Virus.

Maternal vaccination may be the most effective and safest approach to the protection of infants from respiratory syncytial virus (RSV) infection, a severe acute lower respiratory tract disease in infants and young children worldwide. We previously compared five different virus-like particle (VLP)-associated, mutation-stabilized prefusion F (pre-F) proteins, including the prototype DS-Cav1 F VLPs. We showed that alternative versions of prefusion F proteins have different conformations and induce different populations of anti-F protein antibodies. Two of these alternative pre-F VLPs, the UC-2 F and UC-3 F VLPs, stimulated in mice higher titers of neutralizing antibodies than DS-Cav1 F VLPs (M. L. Cullen, R. M. Schmidt, M. G. Torres, A. A. Capoferri, et al., Vaccines 7:21-41, 2019, https://doi.org/10.3390/vaccines7010021). Here we describe a comparison of these two pre-F VLPs with DS-Cav1 F VLPs as maternal vaccines in cotton rats and report that UC-3 F VLPs significantly increased the neutralizing antibody (NAb) titers in pregnant dams compared to DS-Cav1 F VLPs. The neutralizing antibody titers in the sera of the offspring of the dams immunized with UC-3 F VLPs were significantly higher than those in the sera of the offspring of dams immunized with DS-Cav1 VLPs. This increase in serum NAb titers translated to a 6- to 40-fold lower virus titer in the lungs of the RSV-challenged offspring of dams immunized with UC-3 F VLPs than in the lungs of the RSV-challenged offspring of dams immunized with DS-Cav1 F VLPs. Importantly, the offspring of UC-3 F VLP-immunized dams showed significant protection from lung pathology and from induction of inflammatory lung cytokine mRNA expression after RSV challenge. Immunization with UC-3 F VLPs also induced durable levels of high-titer neutralizing antibodies in dams.IMPORTANCE Respiratory syncytial virus (RSV) is a significant human pathogen severely impacting neonates and young children, but no vaccine exists to protect this vulnerable population. Furthermore, direct vaccination of neonates is likely ineffective due to the immaturity of their immune system, and neonate immunization is potentially unsafe. Maternal vaccination may be the best and safest approach to the protection of neonates through the passive transfer of maternal neutralizing antibodies in utero to the fetus after maternal immunization. Here we report that immunization of pregnant cotton rats, a surrogate model for human maternal immunization, with novel RSV virus-like particle (VLP) vaccine candidates containing stabilized prefusion RSV F proteins provides significant levels of protection of the offspring of immunized dams from RSV challenge. We also found that antibodies induced by VLPs containing different versions of the prefusion F protein varied by 40-fold in the extent of protection provided to the offspring of vaccinated dams upon RSV challenge.

Efficacy of a respiratory syncytial virus vaccine candidate in a maternal immunization model.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Maternal immunization is an option to increase maternal antibody levels and protect infants from infection. Here we assess the efficacy of virus-like particle (VLP) vaccine candidates containing stabilized pre-fusion (pre-F) or post-fusion (post-F) conformations of the RSV F protein and the attachment RSV G protein in a maternal immunization model using cotton rats. VLP vaccines containing RSV F and G proteins strongly boost pre-existing RSV immunity in dams preventing their perinatal drop in immunity. Boosting is stronger for the pre-F VLP than for the post-F VLP or purified subunit F protein vaccines, giving an advantage on mothers' protection. VLP immunization of dams provides significant protection to pups from RSV challenge and reduced pulmonary inflammation. Collectively, our results show that a VLP vaccine with RSV F and G proteins is safe and effective for maternal and adult vaccination.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.

A recombinant anchorless respiratory syncytial virus (RSV) fusion (F) protein/monophosphoryl lipid A (MPL) vaccine protects against RSV-induced replication and lung pathology.

We previously demonstrated that the severe cytokine storm and pathology associated with RSV infection following intramuscular vaccination of cotton rats with FI-RSV Lot 100 could be completely abolished by formulating the vaccine with the mild TLR4 agonist and adjuvant, monophosphoryl lipid A (MPL). Despite this significant improvement, the vaccine failed to blunt viral replication in the lungs. Since MPL is a weak TLR4 agonist, we hypothesized that its adjuvant activity was mediated by modulating the innate immune response of respiratory tract resident macrophages. Therefore, we developed a new vaccine preparation with purified, baculovirus expressed, partially purified, anchorless RSV F protein formulated with synthetic MPL that was administered to cotton rats intranasally, followed by an intradermal boost. This novel formulation and heterologous "prime/boost" route of administration resulted in decreased viral titers compared to that seen in animals vaccinated with F protein alone. Furthermore, animals vaccinated by this route showed no evidence of enhanced lung pathology upon RSV infection. This indicates that MPL acts as an immune modulator that protects the host from vaccine-enhanced pathology, and reduces RSV replication in the lower respiratory tract when administered by a heterologous prime/boost immunization regimen.

Modeling Human Respiratory Viral Infections in the Cotton Rat (Sigmodon hispidus).

For over three decades, cotton rats have been a preferred model for human Respiratory Syncytial Virus (RSV) infection and pathogenesis, and a reliable model for an impressive list of human respiratory pathogens including adenoviruses, para influenza virus, measles, and human metapneumo virus. The most significant contribution of the cotton rat to biomedical research has been the development of anti-RSV antibodies for prophylactic use in high-risk infants. More recently, however, the cotton rat model has been further explored as a model for infection with other respiratory viral pathogens including influenza and rhinovirus.Together with RSV, these viruses inflict the greatest impact on the human respiratory health.This review will focus on the characteristics of these new models and their potential contribution to the development of new therapies.

The cotton rat Sigmodon hispidus model of respiratory syncytial virus infection.

The cotton rat Sigmodon hispidus is a New World rodent that has become an important model of respiratory syncytial virus (RSV) infection. This small animal is relatively permissive to RSV and can be infected throughout life. It recapitulates the pathology associated with the FI-RSV vaccine-enhanced disease, the phenomenon of maternally transmitted immunity and the ability of passive immunity to suppress efficacy of RSV vaccines. Different highly susceptible human cohort scenarios have been modeled in the cotton rat, including RSV disease in infants, elderly, and immunosuppressed individuals. The cotton rat has accurately predicted efficacy and dose of antibody immunoprophylaxis, and the lack of efficacy of antibody immunotherapy for disease treatment. With the recent development of molecular reagents and tools for the model, the cotton rat is an important model of RSV infection to consider for vaccine and drug testing, and will continue to advance our understanding of RSV disease pathogenesis.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

UNLABELLED: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1ß (IL-1ß) mRNA in WT versus MD-2(-/-) macrophages, where MD-2(-/-) macrophages failed to show IL-1ß expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. IMPORTANCE: This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

New insights for development of a safe and protective RSV vaccine.

Respiratory Syncytial Virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and children <1 year old, resulting in significant morbidity and mortality worldwide. There is currently no RSV vaccine. In the 1960s, a formalin-inactivated RSV (FI-RSV) vaccine trial led to exacerbated disease upon natural infection of vaccinees, including two deaths. The causes involved in the disastrous results of these vaccine trials are still unclear but they remain the engine for searching new avenues to develop a safe vaccine that can provide long-term protection against this important pathogen. This article reviews some of the early history of RSV vaccine development,as well as more recent information on the interaction between RSV and the host innate and adaptive immune responses. A safe and efficacious vaccine for RSV will require "re-education" of the host immune response against RSV to prevent vaccine-enhanced or severe RSV disease.

Methods for monitoring dynamics of pulmonary RSV replication by viral culture and by real-time reverse transcription-PCR in vivo: Detection of abortive viral replication.

Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque-forming unit assay and the real-time reverse-transcription PCR (qRT-PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing lung samples for viral culture and RNA extraction, and finish with the actual methods of viral titration by the qRT-PCR and the plaque-forming unit assays.

Age-related differences in pulmonary cytokine response to respiratory syncytial virus infection: modulation by anti-inflammatory and antiviral treatment.

BACKGROUND: Respiratory syncytial virus (RSV) is the major cause of severe lower respiratory tract infection in infants and young children. Recently, RSV has also been recognized as a serious health risk in elderly individuals, but the pathogenesis of RSV infection in elderly individuals remains unknown. METHODS: Dynamics of pulmonary cytokine response (including interferon- gamma , interleukin [IL]-4, IL-10, IL-6, monocyte chemoattractant protein-1, and growth-regulated oncogene [GRO] mRNA) during acute RSV infection were investigated in young (<2 months old) and aged (>9 months old) cotton rats (Sigmodon hispidus). Therapeutic treatments that diminish viral replication (antiviral antibody) and pulmonary inflammation (anti-inflammatory corticosteroid) in RSV-infected cotton rats were used to evaluate the contribution of virus replication and inflammation to the development of RSV disease with respect to age. RESULTS: The time of the peak expression of the majority of cytokines was shifted with respect to age. Antiviral and anti-inflammatory treatments had a similar effect on cytokine expression in aged and young cotton rats. GRO mRNA transcripts were more abundant in the lungs of aged cotton rats. CONCLUSIONS: The present study reports an age-related delay in the pulmonary cytokine response to RSV and an imbalance in chemokine production with respect to age and underscores different components of RSV pathogenesis with respect to their molecular signature.

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

Prospects of antiviral and anti-inflammatory therapy for respiratory syncytial virus infection.

Respiratory syncytial virus is the leading viral cause of death in children less than 2 years of age, and is an increasing cause of morbidity and mortality in transplant patients and the elderly. Respiratory syncytial virus causes upper and lower respiratory tract infections, which can lead to severe bronchiolitis and pneumonia. High-risk groups for severe respiratory syncytial virus infection include infants with a history of premature birth with or without chronic lung disease, children with congenital heart disease, children with cystic fibrosis or chronic lung diseases, and immunosuppressed patients or patients with immunodeficiency. However, the majority of infants who have severe respiratory syncytial virus disease are born at full term and are otherwise healthy. It is unclear why children, the elderly and the immunosuppressed are at much higher risk for severe disease; however, a respiratory syncytial virus-induced immune pathologic mechanism has long been suspected. Attempts to develop a safe and effective vaccine against respiratory syncytial virus have failed. Antirespiratory syncytial virus immunotherapy, although effective prophylactically, does not provide any beneficial clinical outcome when administered therapeutically, indicating that respiratory syncytial virus-induced pathology is most likely the result of the inflammatory response to infection, rather than a direct viral cytopathic effect. Thus, a combined antiviral and anti-inflammatory therapy may represent the safest and most efficient treatment for acute respiratory syncytial virus infection. In this review, the current knowledge that has set the rationale for the development of such therapy is summarized.