Formalin-inactivated Respiratory Syncytial Virus Research Articles

Mucosal immunization with a low-energy electron inactivated respiratory syncytial virus vaccine protects mice without Th2 immune bias.

The respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections associated with numerous hospitalizations. Recently, intramuscular (i.m.) vaccines against RSV have been approved for elderly and pregnant women. Noninvasive mucosal vaccination, e.g., by inhalation, offers an alternative against respiratory pathogens like RSV. Effective mucosal vaccines induce local immune responses, potentially resulting in the efficient and fast elimination of respiratory viruses after natural infection. To investigate this immune response to an RSV challenge, low-energy electron inactivated RSV (LEEI-RSV) was formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) or 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DD-LEEI-RSV) for vaccination of mice intranasally. As controls, LEEI-RSV and formalin-inactivated-RSV (FI-RSV) were used via i.m. vaccination. The RSV-specific immunogenicity of the different vaccines and their protective efficacy were analyzed. RSV-specific IgA antibodies and a statistically significant reduction in viral load upon challenge were detected in mucosal DD-LEEI-RSV-vaccinated animals. Alhydrogel-adjuvanted LEEI-RSV i.m. showed a Th2-bias with enhanced IgE, eosinophils, and lung histopathology comparable to FI-RSV. These effects were absent when applying the mucosal vaccines highlighting the potential of DD-LEEI-RSV as an RSV vaccine candidate and the improved performance of this mucosal vaccine candidate.

A Historical Perspective on RSV 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 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 naïve 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.

Ally, adversary, or arbitrator? The context-dependent role of eosinophils in vaccination for respiratory viruses and subsequent breakthrough infections.

Eosinophils are a critical type of immune cell and central players in Type 2 immunity. Existing literature suggests that eosinophils also can play a role in host antiviral responses, typically Type 1 immune events, against multiple respiratory viruses, both directly through release of antiviral mediators and indirectly through activation of other effector cell types. One way to prime host immune responses towards effective antiviral responses is through vaccination, where typically a Type 1-skewed immunity is desirable in the context of intracellular pathogens like respiratory viruses. In the realm of breakthrough respiratory viral infection in vaccinated hosts, an event in which virus can still establish productive infection despite pre-existing immunity, eosinophils are most prominently known for their link to vaccine-associated enhanced respiratory disease (VAERD) upon natural respiratory syncytial virus (RSV) infection. This was observed in a pediatric cohort during the 1960s following vaccination with formalin-inactivated RSV (FI-RSV). More recent research has unveiled additional roles of the eosinophil in respiratory viral infection and breakthrough infection. The specific contribution of eosinophils to the quality of vaccine responses, vaccine efficacy, and antiviral responses to infection in vaccinated hosts remains largely unexplored, especially regarding their potential roles in protection. Based on current findings, we will speculate upon the suggested function of eosinophils and consider the many potential ways by which eosinophils may exert protective and pathological effects in breakthrough infections. We will also discuss how to balance vaccine efficacy with eosinophil-related risks, as well as the use of eosinophils and their products as potential biomarkers of vaccine efficacy or adverse events.

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.

Laser-assisted intradermal delivery of a microparticle vaccine for respiratory syncytial virus induces a robust immune response.

Respiratory syncytial virus (RSV) is an infectious disease that poses a significant public health risk in young children. Vaccine studies conducted in the 1960s using an intramuscular injection of formalin-inactivated respiratory syncytial virus (Fi-RSV) resulted in an enhanced respiratory disease and led to the failure of the vaccine. Thus, the virus-like particles (VLP) of the RSV fusion (F) protein was used as the vaccine antigen in this study. The F-VLP was encapsulated in a microparticle (MP) matrix composed of cross-linked bovine serum albumin (BSA) to enhance the antigen presentation and uptake. Moreover, a painless vaccination method would be desirable for an infectious disease that mainly affects young children. Thus, an ablative laser device, Precise Laser Epidermal System (P.L.E.A.S.E), was utilized to create micropores on the skin for vaccine delivery. We observed enhanced antigen presentation of the vaccine microparticles (F-VLP MP) with and without the adjuvant monophosphoryl lipid A (MPL-A) MP in dendritic cells. Consequently, Swiss Webster mice were immunized with the adjuvanted vaccine microparticles using the P.L.E.A.S.E laser to study the in vivo immunogenicity. The immunized mice had high serum immunoglobulin (IgG, IgG2a) levels, indicating a Th1 response. Subsequent analysis of lung homogenates post- RSV challenge revealed high IgA, indicating generation of a mucosal immune response upon intradermal immunization. Flowcytometry analysis showed high CD8+, and CD4+ expression in the lymph node and spleen of the adjuvanted vaccine microparticle immunized mice. Increased expression of interferon gamma (IFN-γ) in the spleen cells further proved Th1 polarized immune response. Finally, an immune plaque assay indicated significantly low lung viral titer in the mice immunized with intradermal adjuvanted vaccine microparticles. Thus, ablative laser-assisted immunization with the F-VLP based adjuvanted vaccine microparticles could be a promising vaccine candidate for RSV.

Microparticle RSV Vaccines Presenting the G Protein CX3C Chemokine Motif in the Context of TLR Signaling Induce Protective Th1 Immune Responses and Prevent Pulmonary Eosinophilia Post-Challenge.

Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.

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.

Development of a respiratory syncytial virus vaccine using human hepatitis B core-based virus-like particles to induce mucosal immunity

BackgroundRespiratory syncytial virus (RSV) is an important viral pathogen responsible for severe infection of the lower respiratory tract in children under the age of 5 years. No vaccines against RSV are currently in clinical use. Vaccine-associated enhanced respiratory disease (ERD) caused by excess Th2 type responses was observed in a clinical trial of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. Thus, inducing a balanced immune response is a crucial issue in the development of an RSV vaccine. MethodsIn this study, we constructed, expressed, and purified a recombinant RSV vaccine candidate (i.e., HRØ24) containing the two heptad repeat regions and the antigenic sites Ø, II, and IV of the RSV F protein. The RSV vaccine candidate was intranasally administrated to BALB/c and C57BL/6 mice in combination with virus-like particles (VLPs) derived from the core protein of the hepatitis B virus (HBc). Mucosal immunity to HRØ24 was then assessed. ResultsIntranasal administration of HBc VLPs in combination with HRØ24 induced serum IgGs against HRØ24 as well as lung HRØ24-specific sIgAs in both C57BL/6 and BALB/c mouse models. The secretion of IFN-γ from splenocyte re-stimulation and an elevated ratio of serum IgG2a to IgG1 indicated that the immune response induced by the HBc VLPs/HRØ24 mixture was Th1-biased. Weight loss of <5% and no to low eosinophil infiltration was observed in histological analysis of the lung following a challenge with the RSV A2 strain. These results suggest that the HBc VLPs/HRØ24 combination conferred substantial partial protection against RSV-induced illness in mice. ConclusionsLong-term immunity to RSV-induced illness was achieved via intranasal vaccination using a mixture of HBc VLPs and HRØ24 in mouse models.

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.

Neutralizing epitope of the Fusion Protein of Respiratory Syncytial Virus Embedded in the HA Molecule of LAIV Virus is not Sufficient to Prevent RS Virus Pulmonary Replication but Ameliorates Lung Pathology following RSV Infection in Mice

Aims:To develop experimental bivalent vaccines against influenza and RSV using a cold-adapted LAIV backbone.Background:Respiratory syncytial virus (RSV) is a causative agent of bronchiolitis and pneumonia in young children, elderly and immunocompromised adults. No vaccine against RSV has been licensed to date for various reasons. One of the promising platforms for designing RSV vaccine is the use of live attenuated influenza vaccine (LAIV) viruses to deliver RSV epitopes to the respiratory mucosa.Objective:To generate recombinant LAIV viruses encoding a neutralizing epitope of the RSV fusion protein and assess their protective potential against both influenza and RSV infections in a mouse model.Methods:Reverse genetics methods were used to rescue recombinant LAIV+HA/RSV viruses expressing chimeric hemagglutinins encoding the RSV-F epitope at its N-terminus using two different flexible linkers. BALB/c mice were intranasally immunized with two doses of the recombinant viruses and then challenged with the influenza virus or RSV. The LAIV viral vector and formalin-inactivated RSV (FI-RSV) were included as control vaccines. Protection was assessed by the reduction of virus pulmonary titers. In addition, RSV-induced lung pathology was evaluated by histopathology studies.Results:Two rescued chimeric LAIV+HA/RSV viruses were identical to the LAIV vector in terms of replication capacityin vitroandin vivo. The RSV-F neutralizing epitope was successfully expressed only if inserted into the HA molecule via G-linker, but not A-linker. Both chimeric viruses induced high influenza-specific antibody levels and fully protected mice against a lethal influenza challenge virus. However, they induced weak anti-RSV antibody responses which did not prevent RS virus replication upon challenge, and only LAIV-HA+G-RSV variant protected mice against RSV-induced lung pathology.Conclusion:Although the designed LAIV-RSV chimeric viruses were unable to neutralize the RS virus pulmonary replication, the LAIV-HA+G-RSV reduced RSV-induced lung pathology and can be considered a promising bivalent vaccine against influenza and RSV infections and warrants its further development.

WITHDRAWN: Nonglycosylated RSV F protein elicited protective immunity in mice

Abstract Human respiratory syncytial virus (RSV) is the primary cause of severe respiratory tract disease in infants and young children, but no vaccine is yet on the market. The fusion (F) protein of RSV, especially the prefusion F protein (pre-F), is the best antigen for eliciting a protective immunity and neutralizing antibody response in the host. In this study, we constructed and purified the nonglycosylated pre-F protein using Escherichia coli BL21, called pre-RBF. We found that pre-RBF could be produced at high levels with stable trimer conformations. Importantly, pre-RBF was shown to bind 5C4, a monoclonal antibody specific for the pre-F. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with different doses of pre-RBF all showed superior protective immunity, which was confirmed by serum-neutralizing activity and viral clearance after challenge. Pre-RBF induced a balanced Th1-biased immunity response, whereas FI-RSV induced Th2-biased immune responses. So pre-RBF represents a potential vaccine candidate for the prevention of human infection with RSV.

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.

Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice.

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.

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.

2592. Lung Function as an Indicator of Vaccine Enhanced RSV Disease in Cotton Rats

BackgroundRespiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract infections in infants and young children. Seronegative children previously vaccinated with formalin-inactivated live RSV formulated with aluminum (FIRSV) developed vaccine enhanced RSV disease (VERD), which is characterized by fever, wheezing, bronchopneumonia, and airway hyperresponsiveness (AHR). We investigated whether impaired lung function can serve as a marker for VERD in an animal model of RSV infection.MethodsUninfected and RSV-infected cotton rats intranasally challenged with 106 pfu of RSV A2 were anesthetized with pentobarbital and tracheostomized. A cannula was placed in the trachea and animals were connected to flexiVent™ (Scireq), which is a computer-controlled piston ventilator that analyzes pressure and volume signals in response to an oscillatory waveform applied at the animal’s airways. Vecuronium bromide was administered to ventilated animals to prevent independent breathing. To measure AHR, animals were exposed to increasing doses of inhaled methacholine, and methacholine-induced bronchoconstriction was measured.ResultsTwo independent studies showed that RSV-infected cotton rats (n = 4) exhibited increased total respiratory system resistance (Rrs) and airway resistance (Rn) following methacholine challenge on days 4 and 6 post-infection compared with uninfected cotton rats (n = 4).ConclusionRSV-induced impairment in lung function can be exploited for the development of a more robust and objective method for assessing vaccine safety in a cotton rat model of respiratory disease compared with traditional histopathological analysis.Disclosures All authors: No reported disclosures.