Formalin-inactivated Respiratory Syncytial Virus Vaccine Research Articles

A Historical Perspective on Respiratory Syncytial Virus Prevention: A Journey Spanning Over Half a Century From the Setback of an Inactive Vaccine Candidate to the Success of Passive Immunization Strategy.

The efforts to prevent respiratory syncytial virus (RSV) infection in infants span over half a century. RSV vaccine development began in the 1960s, and it confronted a significant disappointment after testing a formalin-inactivated RSV (FI RSV) vaccine candidate. This inactivated RSV vaccine was not protective. A large number of the vaccinated RSV-naive children, when subsequently exposed to natural RSV infection from wild-type virus in the community, developed severe lung inflammation termed enhanced respiratory disease. This resulted in a halt in RSV vaccine development. In the 1990s, attention turned to the potential for passive protection against severe RSV disease with immunoglobulin administration. This led to studies on using standard intravenous immunoglobulins in high-risk infants, followed by high-titer RSV immunoglobulin preparation and, subsequently, the development of RSV monoclonal antibodies. Over the past 25 years, palivizumab has been recognized as a safe and effective monoclonal antibody as a prevention strategy for RSV in high-risk children. Its high cost and need for monthly administration, however, has hindered its use to ~2% of the birth cohort, neglecting the vast majority of newborns, including healthy full-term infants who comprise the largest portion of RSV hospitalizations and the greatest part of the burden of RSV disease. Still these efforts, helped pave the way for the present advances in RSV prevention that hold promise for mitigating severe RSV disease for all infants.

Respiratory Syncytial Virus Vaccines: A Review of the Candidates and the Approved Vaccines.

Respiratory syncytial virus (RSV) is responsible for a significant proportion of global morbidity and mortality affecting young children and older adults. In the aftermath of formalin-inactivated RSV vaccine development, the effort to develop an immunizing agent was carefully guided by epidemiologic and pathophysiological evidence of the virus, including various vaccine technologies. The pipeline of RSV vaccine development includes messenger ribonucleic acid (mRNA), live-attenuated (LAV), subunit, and recombinant vector-based vaccine candidates targeting different virus proteins. The availability of vaccine candidates of various technologies enables adjustment to the individualized needs of each vulnerable age group. Arexvy® (GSK), followed by Abrysvo® (Pfizer), is the first vaccine available for market use as an immunizing agent to prevent lower respiratory tract disease in older adults. Abrysvo is additionally indicated for the passive immunization of infants by maternal administration during pregnancy. This review presents the RSV vaccine pipeline, analyzing the results of clinical trials. The key features of each vaccine technology are also mentioned. Currently, 24 vaccines are in the clinical stage of development, including the 2 licensed vaccines. Research in the field of RSV vaccination, including the pharmacovigilance methods of already approved vaccines, promotes the achievement of successful prevention.

Influenza breakthrough infection in vaccinated mice is characterized by non-pathological lung eosinophilia.

Eosinophils are important mediators of mucosal tissue homeostasis, anti-helminth responses, and allergy. Lung eosinophilia has previously been linked to aberrant Type 2-skewed T cell responses to respiratory viral infection and may also be a consequence of vaccine-associated enhanced respiratory disease (VAERD), particularly in the case of respiratory syncytial virus (RSV) and the formalin-inactivated RSV vaccine. We previously reported a dose-dependent recruitment of eosinophils to the lungs of mice vaccinated with alum-adjuvanted trivalent inactivated influenza vaccine (TIV) following a sublethal, vaccine-matched H1N1 (A/New Caledonia/20/1999; NC99) influenza challenge. Given the differential role of eosinophil subset on immune function, we conducted the investigations herein to phenotype the lung eosinophils observed in our model of influenza breakthrough infection. Here, we demonstrate that eosinophil influx into the lungs of vaccinated mice is adjuvant- and sex-independent, and only present after vaccine-matched sublethal influenza challenge but not in mock-challenged mice. Furthermore, vaccinated and challenged mice had a compositional shift towards more inflammatory eosinophils (iEos) compared to resident eosinophils (rEos), resembling the shift observed in ovalbumin (OVA)-sensitized allergic control mice, however without any evidence of enhanced morbidity or aberrant inflammation in lung cytokine/chemokine signatures. Furthermore, we saw a lung eosinophil influx in the context of a vaccine-mismatched challenge. Additional layers of heterogeneity in the eosinophil compartment were observed via unsupervised clustering analysis of flow cytometry data. Our collective findings are a starting point for more in-depth phenotypic and functional characterization of lung eosinophil subsets in the context of vaccine- and infection-induced immunity.

The third pandemic: The respiratory syncytial virus landscape and specific considerations for the allergist/immunologist.

Background: Since its initial identification in 1956, respiratory syncytial virus (RSV) has been the second most common cause of mortality in infants <6 months of age and a major cause of morbidity and mortality associated with lower respiratory tract infection (LRTI) in older adults (ages >60 years) worldwide. Of particular interest to the allergist/immunologist is a growing body of evidence that suggests an association between LRTI caused by RSV in infants with later-life development of asthma, wheezing, or impaired lung function in adults. Efforts to develop a RSV vaccine have been thwarted for >70 years by the occurrence of enhanced respiratory disease (ERD), an adverse RSV vaccine reaction, in the 1960s, in which more-severe illness occurred on natural infection after vaccination of infants who were RSV naive and with a formalin-inactivated RSV vaccine. Recent advances in knowledge of the structural biology of the RSV surface fusion glycoprotein, however, have revolutionized RSV vaccine development for preventive interventions and have offered, at last, the hope of an effective and safe vaccine for the prevention of RSV disease. Objective: The purpose of this report was to examine the current evidence that supports the epidemiology, disease manifestations, molecular biology, treatments, and new vaccine development of RSV vaccines. Results: The host-immune response to RSV infection is carried out by two distinct but overlapping universes of mucosal and systemic immune systems in which a balanced set of B- and T-cell responses are involved in protective immunity that includes the mucosal immune system in which immunoglobulin A (IgA) prevails and the systemic immune system in which IgG neutralizing antibody predominates. The key to developing an effective vaccine is now thought to be linked to the availability of a stabilized prefusion F protein in the immunizing vaccine, which can perform a dual function of a balanced mucosal and/or systemic immune response as well as an effective antibody specifically directed to critical epitopes on the requisite prefusion F protein. Conclusion: The unfortunate manifestation of RSV ERD that occurred in the 1960s has led to a better understanding of the structural biology of the RSV surface fusion glycoprotein and has provided a basis for the development of more effective and safer RSV vaccines and monoclonal antibody preparations for immunoprophylaxis of the dread effects of RSV disease. There are now a large number of clinical trials in progress that are evaluating these products, which include recombinant vector, subunit, particle-based, live-attenuated, chimeric, and nucleic acid vaccines; and monoclonal antibodies. This article gives an overview of the many aspects of RSV disease and development of virus (RSV) vaccines of particular interest to the allergist/immunologist.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Mycobacterium bovis BCG Given at Birth Followed by Inactivated Respiratory Syncytial Virus Vaccine Prevents Vaccine-Enhanced Disease by Promoting Trained Macrophages and Resident Memory T Cells.

Respiratory syncytial virus (RSV) infects more than 60% of infants in their first year of life. Since an experimental formalin-inactivated (FI) RSV vaccine tested in the 1960s caused enhanced respiratory disease (ERD), few attempts have been made to vaccinate infants. ERD is characterized by Th2-biased responses, lung inflammation, and poor protective immune memory. Innate immune memory displays an increased nonspecific effector function upon restimulation, a process called trained immunity, or a repressed effector function upon restimulation, a process called tolerance, which participates in host defense and inflammatory disease. Mycobacterium bovis bacillus Calmette-Guérin (BCG) given at birth can induce trained immunity as well as heterologous Th1 responses. We speculate that BCG given at birth followed by FI-RSV may alleviate ERD and enhance protection through promoting trained immunity and balanced Th immune memory. Neonatal mice were given BCG at birth and then vaccinated with FI-RSV+Al(OH)3. BCG/FI-RSV+Al(OH)3 induced trained macrophages, tissue-resident memory T cells (TRM), and specific cytotoxic T lymphocytes (CTL) in lungs and inhibited Th2 and Th17 cell immune memory, all of which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented the innate tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. Therefore, BCG given at birth to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants. IMPORTANCE RSV is the leading cause of severe lower respiratory tract infection of infants. ERD, characterized by Th2-biased responses, inflammation, and poor immune memory, has been an obstacle to the development of safe and effective killed RSV vaccines. Innate immune memory participates in host defense and inflammatory disease. BCG given at birth can induce trained immunity as well as heterologous Th1 responses. Our results showed that BCG/FI-RSV+Al(OH)3 induced trained macrophages, TRM, specific CTL, and balanced Th cell immune memory, which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. BCG at birth as an adjuvant to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants.

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.

Roles of the Fc Receptor γ-Chain in Inducing Protective Immune Responses after Heterologous Vaccination against Respiratory Syncytial Virus Infection.

The roles of the Fc receptor (FcR) in protection or inflammatory disease after respiratory syncytial virus (RSV) vaccination and infection remain unknown. Virus-like particles containing RSV fusion proteins (RSV F-VLPs) induce T-helper type 1 antibody responses and protection against RSV. Heterologous RSV F-VLP prime and formalin-inactivated RSV (FI-RSV) boost vaccination has been reported to be effective in providing protection without inflammatory disease. Here, we investigated whether the FcRγ-chain is important for immune protection by the heterologous F-VLP and FI-RSV vaccination using FcRγ-chain knockout (−/−) mice. RSV F-VLP-primed and FI-RSV-boosted FcRγ −/− mice displayed less protective efficacy, as shown by higher lung viral titers upon RSV challenge, compared to RSV F-VLP-primed and FI-RSV-boosted immunized wild-type mice. RSV F-VLP and FI-RSV immunization induced lower levels of neutralizing activity and interferon-γ-producing CD8 T-cells in the bronchoalveolar lavage cells of FcRγ −/− mice than in those of wild-type mice. In addition, FcRγ −/− mice displayed a trend of enhancing lung histopathology after RSV vaccination and infection. This study suggests that the FcRγ-chain plays an important role in inducing antiviral protection and CD8 T-cell responses in RSV F-VLP prime and FI-RSV boost vaccination after RSV infections.

Respiratory Syncytial Virus F Subunit Vaccine With AS02 Adjuvant Elicits Balanced, Robust Humoral and Cellular Immunity in BALB/c Mice.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory illness, particularly in infants, the elderly, and immunocompromised adults. There is no licensed commercial vaccine against RSV. Importantly, formalin-inactivated RSV vaccines mediate enhanced respiratory disease. RSV fusion (F) protein with pre-fusion conformation is a promising candidate subunit vaccine. However, some problems remain to be solved, such as low immunogenicity and humoral immunity bias. Adjuvants can effectively enhance and adjust vaccine immune responses. In this study, we formulated pre-fusion RSV-F protein with the adjuvants, Alhydrogel, MF59, AS03, AS02, and glycol chitosan (GCS). We then conducted head-to-head comparisons of vaccine-induced immune responses in BALB/c mice. All adjuvanted vaccines enhanced antigen-specific and neutralizing antibody titers and viral clearance and gave an order of adjuvant activity: AS02 > AS03, MF59 > GCS, and Alhydrogel. Among them, AS02 elicited the highest antibody expression, which persisted until week 18. Moreover, AS02 significantly enhanced Th1 type immune response in immunized mice. Mice in the AS02 group also showed faster recovery from viral attacks in challenge tests. Further transcriptome analysis revealed that AS02 regulates immune balance by activating TLR-4 and promotes Th1-type immune responses. These results suggest that AS02 may be an excellent candidate adjuvant for RSV-F subunit vaccines. This study also provides valuable information regarding the effect of other adjuvants on immune responses of RSV-F subunit vaccines.

In vitro model for the assessment of human immune responses to subunit RSV vaccines.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.

Natural Killer and CD8 T Cells Contribute to Protection by Formalin Inactivated Respiratory Syncytial Virus Vaccination under a CD4-Deficient Condition.

Respiratory syncytial virus (RSV) causes severe pulmonary disease in infants, young children, and the elderly. Formalin inactivated RSV (FI-RSV) vaccine trials failed due to vaccine enhanced respiratory disease, but the underlying immune mechanisms remain not fully understood. In this study, we have used wild type C57BL/6 and CD4 knockout (CD4KO) mouse models to better understand the roles of the CD4 T cells and cellular mechanisms responsible for enhanced respiratory disease after FI-RSV vaccination and RSV infection. Less eosinophil infiltration and lower pro-inflammatory cytokine production were observed in FI-RSV vaccinated CD4KO mice after RSV infection compared to FI-RSV vaccinated C57BL/6 mice. NK cells and cytokine-producing CD8 T cells were recruited at high levels in the airways of CD4KO mice, correlating with reduced respiratory disease. Depletion studies provided evidence that virus control was primarily mediated by NK cells whereas CD8 T cells contributed to IFN-γ production and less eosinophilic lung inflammation. This study demonstrated the differential roles of effector CD4 and CD8 T cells as well as NK cells, in networking with other inflammatory infiltrates in RSV disease in immune competent and CD4-deficient condition.

Determining Immune and miRNA Biomarkers Related to Respiratory Syncytial Virus (RSV) Vaccine Types.

Respiratory Syncytial Virus (RSV) causes serious respiratory tract illness and substantial morbidity and some mortality in populations at the extremes of age, i.e., infants, young children, and the elderly. To date, RSV vaccine development has been unsuccessful, a feature linked to the lack of biomarkers available to assess the safety and efficacy of RSV vaccine candidates. We examined microRNAs (miR) as potential biomarkers for different types of RSV vaccine candidates. In this study, mice were vaccinated with a live attenuated RSV candidate that lacks the small hydrophobic (SH) and attachment (G) proteins (CP52), an RSV G protein microparticle (GA2-MP) vaccine, a formalin-inactivated RSV (FI-RSV) vaccine or were mock-treated. Several immunological endpoints and miR expression profiles were determined in mouse serum and bronchoalveolar lavage (BAL) following vaccine priming, boost, and RSV challenge. We identified miRs that were linked with immunological parameters of disease and protection. We show that miRs are potential biomarkers providing valuable insights for vaccine development.

Respiratory Syncytial Virus Vaccines: Are We Making Progress?

Respiratory Syncytial Virus Vaccines: Are We Making Progress?

CD4+ T Cells Drive Lung Disease Enhancement Induced by Immunization with Suboptimal Doses of Respiratory Syncytial Virus Fusion Protein in the Mouse Model.

Respiratory syncytial virus (RSV) infection of seronegative children previously immunized with formalin-inactivated (FI) RSV has been associated with serious enhanced respiratory disease (ERD). The phenomenon was reproduced in the cotton rat and the mouse, and both preclinical models have been routinely used to evaluate the safety of new RSV vaccine candidates. More recently, we demonstrated that immunizations with suboptimal doses of the RSV fusion (F) antigen, in its post- or prefusion conformation, and in the presence of a Th1-biasing adjuvant, unexpectedly led to ERD in the cotton rat model. To assess if those observations are specific to the cotton rat and to elucidate the mechanism by which vaccination with low antigen doses can drive ERD post-RSV challenge, we evaluated RSV post-F antigen dose de-escalation in BALB/c mice in the presence of a Th1-biasing adjuvant. While decreasing antigen doses, we observed an increase in lung inflammation associated with an upregulation of proinflammatory cytokines. The amplitude of the lung histopathology was comparable to that of FI-RSV-induced ERD, confirming the observations made in the cotton rat. Importantly, depletion of CD4+ T cells prior to viral challenge completely abrogated ERD, preventing proinflammatory cytokine upregulation and the infiltration of T cells, neutrophils, eosinophils, and macrophages into the lung. Overall, low-antigen-dose-induced ERD resembles FI-RSV-induced ERD, except that the former appears in the absence of detectable levels of viral replication and in the context of a Th1-biased immune response. Taken together, our observations reinforce the recent concept that vaccines developed for RSV-naïve individuals should be systematically tested under suboptimal dosing conditions.IMPORTANCE RSV poses a significant health care burden and is the leading cause of serious lower-respiratory-tract infections in young children. A formalin-inactivated RSV vaccine developed in the 1960s not only showed a complete lack of efficacy against RSV infection but also induced severe lung disease enhancement in vaccinated children. Since then, establishing safety in preclinical models has been one of the major challenges to RSV vaccine development. We recently observed in the cotton rat model that suboptimal immunizations with RSV fusion protein could induce lung disease enhancement. In the present study, we extended suboptimal dosing evaluation to the mouse model. We confirmed the induction of lung disease enhancement by vaccinations with low antigen doses and dissected the associated immune mechanisms. Our results stress the need to evaluate suboptimal dosing for any new RSV vaccine candidate developed for seronegative infants.

Lipopolysaccharide Inhibits FI-RSV Vaccine-enhanced Inflammation Through Regulating Th Responses.

Respiratory syncytial virus (RSV) infection is the primary cause of respiratory disease in infants. The formalin-inactivated RSV (FI-RSV) vaccine resulted in an enhanced respiratory disease (ERD) in infants upon natural RSV infection, which is a major obstacle for development of safe and efficacious vaccines. Excessive and uncontrolled Th immune responses could be involved in the ERD. Agonists of TLRs are used as adjuvants to guide the type of immune response induced by vaccines. We evaluated the impact of lipopolysaccharide (LPS), the agonist of TLR4, on ERD as the adjuvant of FI-RSV. The results showed that LPS remarkably inhibited FI-RSV-enhanced lung inflammation, mucus production, airway inflammatory cell infiltration, and inflammatory cytokines following RSV challenge. Interestingly, LPS inhibited both Th2 and Th17 type cytokines in lungs of FI-RSV-immunized mice following RSV challenge, without an increase in the Th1 type cytokines, suggesting a controlled immune response. In contrast, Pam3Cys and Poly(I:C), the agonist of TLR1/2 or TLR3, partly inhibited FI-RSV-enhanced lung inflammation. Pam3Cys inhibited Th17 type cytokine IL-17, but promoted both Th1 and Th2 type cytokines. Poly(I:C) inhibited Th2 and Th17 type cytokines, but promoted Th1 type cytokines. In addition, LPS promoted IgG and IgG2a antibody production, which might provide protection from RSV challenge. These results suggest that LPS inhibits ERD without impairment in antibody production and protection, and the mechanism appears to be related with regulation of Th responses induced by FI-RSV.

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Vaccination by microneedle patch with inactivated respiratory syncytial virus and monophosphoryl lipid A enhances the protective efficacy and diminishes inflammatory disease after challenge.

Intramuscular (IM) vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) failed in clinical trials due to vaccine-enhanced respiratory disease. To test the efficacy of skin vaccination against respiratory syncytial virus (RSV), we investigated the immunogenicity, efficacy, and inflammatory disease after microneedle (MN) patch delivery of FI-RSV vaccine (FI-RSV MN) to the mouse skin with or without an adjuvant of monophosphoryl lipid A (MPL). Compared to IM vaccination, MN patch delivery of FI-RSV was more effective in clearing lung viral loads and preventing weight loss, and in diminishing inflammation, infiltrating immune cells, and T helper type 2 (Th2) CD4 T cell responses after RSV challenge. With MPL adjuvant, MN patch delivery of FI-RSV significantly increased the immunogenicity and efficacy as well as preventing RSV disease as evidenced by lung viral clearance and avoiding pulmonary histopathology. Improved efficacy and prevention of disease by FI-RSV MN with MPL were correlated with no sign of airway resistance, lower levels of Th2 cytokines and infiltrating innate inflammatory cells, and higher levels of Th1 T cell responses into the lung. This study suggests that MN patch delivery of RSV vaccines to the skin with MPL adjuvant would be a promising vaccination method.

Active prophylaxis for respiratory syncytial virus: current knowledge and future perspectives.

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children but represents also an important cause of morbidity in adults, particularly in the elderly and immunocompromised persons. Despite its global impact on human health, no effective treatment is available except for supportive care and no safe vaccine has been licensed yet. Vaccine development has been hindered by several factors including vaccine enhanced disease associated with formalin-inactivated RSV vaccine, ethical concerns and lack of consensus concerning the most appropriate target antigen. In this review, we analyze history of RSV vaccine and current approaches for preventing RSV including live-attenuated, vector-based, subunit, nucleic acid-based, particle-based vaccines and we debate about concerns on target population, correlates of protection and obstacles that are slowing the progress toward a successful RSV vaccination strategy.

Virus-like particle vaccine primes immune responses preventing inactivated-virus vaccine-enhanced disease against respiratory syncytial virus

Formalin inactivated respiratory syncytial virus (FI-RSV) vaccination caused vaccine-enhanced respiratory disease (ERD) upon exposure to RSV in children. Virus-like particles presenting RSV F fusion protein (F VLP) are known to increase T helper type-1 (Th1) immune responses and avoid ERD in animal models. We hypothesized that F VLP would prime immune responses preventing ERD upon subsequent exposure to ERD-prone FI-RSV. Here, we demonstrated that heterologous F VLP priming and FI-RSV boosting of mice prevented FI-RSV vaccine-enhanced lung inflammation and eosinophilia upon RSV challenge. F VLP priming redirected pulmonary T cells toward effector CD8 T cells producing Th1 cytokines and significantly suppressed pulmonary Th2 cytokines. This study suggests that RSV F VLP priming would modulate and shift immune responses to subsequent exposure to ERD-prone FI-RSV vaccine and RSV infection, suppressing Th2 immune-mediated pulmonary histopathology and eosinophilia.

Development of an intradermal DNA vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus

Respiratory syncytial virus (RSV) is a massive medical burden in infants, children and the elderly worldwide, and an effective, safe RSV vaccine remains an unmet need. Here we assess a novel vaccination strategy based on the intradermal delivery of a SynCon® DNA-based vaccine encoding engineered RSV-F antigen using a surface electroporation device (SEP) to target epidermal cells, in clinically relevant experimental models. We demonstrate the ability of this strategy to elicit robust immune responses. Importantly we demonstrate complete resistance to pulmonary infection at a single low dose of vaccine in the cotton rat RSV/A challenge model. In contrast to the formalin-inactivated RSV (FI-RSV) vaccine, there was no enhanced lung inflammation upon virus challenge after DNA vaccination. In summary the data presented outline the pre-clinical development of a highly efficacious, tolerable and safe non-replicating vaccine delivery strategy.