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.

Protective antigenic sites identified in respiratory syncytial virus fusion protein reveals importance of p27 domain.

Respiratory syncytial virus (RSV) vaccines primarily focused on surface fusion (F) protein are under development. Therefore, to identify RSV-F protective epitopes, we evaluated 14 antigenic sites recognized following primary human RSV infection. BALB/c mice were vaccinated with F peptides, F proteins, or RSV-A2, followed by rA2-Line19F challenge. F peptides generated binding antibodies with minimal in vitro neutralization titers. However, several F peptides (including Site II) reduced lung viral loads and lung pathology scores in animals, suggesting partial protection from RSV disease. Interestingly, animals vaccinated with peptides (aa 101-121 and 110-136) spanning the F-p27 sequence, which is only present in unprocessed F0 protein, showed control of viral loads with significantly reduced pathology compared with mock-vaccinated controls. Furthermore, we observed F-p27 expression on the surface of RSV-infected cells as well as lungs from RSV-infected mice. The anti-p27 antibodies demonstrated antibody-dependent cellular cytotoxicity (ADCC) of RSV-infected A549 cells. These findings suggest that p27-mediated immune response may play a role in control of RSV disease in vivo, and F-p27 should be considered for inclusion in an effective RSV vaccine.

Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.

Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies.

Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.

A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

A proof of concept for structure-based vaccine design targeting RSV in humans.

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.

Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

Protective antigenic sites in respiratory syncytial virus G attachment protein outside the central conserved and cysteine noose domains.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants. Previously, we elucidated the antibody repertoire following primary RSV infection in infants. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes from RSV bound 100-fold more phages within attachment protein (G) following primary RSV infection. The G-reactive epitopes spanned the N- and C-termini of G ectodomain, in addition to the central conserved domain (CCD). In the current study, we examined the contribution of antigenic regions of G outside of the CCD to RSV-specific immunity. We evaluated the immunogenicity, neutralization and protective efficacy of all RSV-G antigenic sites identified following primary RSV infection using recombinant E. coli expressed G ectodomain (REG), CCD-deleted G ectodomain (REG ΔCCD), N- and C-terminal G subdomains, and antigenic site peptides. The REG ΔCCD, N- and C-terminal subdomains and peptides generated antibody titers in rabbits and mice that bound fully glycosylated Recombinant Mammalian expressed G ectodomain (RMG) and intact RSV virion particles but minimal in vitro neutralization titers compared with the intact G ectodomain. Vaccinated mice were challenged intranasally with RSV-A2 Line 19F. Viral replication in nasal cavity and lungs was significantly reduced in vaccinated animals compared to unimmunized controls. Control of viral loads post-RSV challenge correlated with serum antibody binding to the virus particles. In addition, very low Th2/Th1 cytokine ratios were found in the lungs of REG ΔCCD vaccinated mice after challenge. These data demonstrate the presence of multiple protective sites in RSV G protein outside of the CCD that could contribute to the development of a bacterially produced unglycosylated G protein as safe and protective vaccine against RSV disease.

The respiratory syncytial virus fusion protein formulated with a polymer-based adjuvant induces multiple signaling pathways in macrophages.

Respiratory syncytial virus (RSV) causes acute respiratory tract infections in infants, the elderly and immunocompromised individuals. No licensed vaccine is available against RSV. We previously reported that intranasal immunization of rodents and lambs with a RSV vaccine candidate (ΔF/TriAdj) induces protective immunity with a good safety profile. ΔF/TriAdj promoted innate immune responses in respiratory mucosal tissues in vivo, by local chemokine and cytokine production, as well as infiltration and activation of immune cells including macrophages. The macrophage is an important cell type in context of both innate and adaptive immune responses against RSV. Therefore, we characterized the effects of ΔF/TriAdj on a murine macrophage cell line, RAW264.7, and bone marrow-derived macrophages (BMMs). A gene expression study of pattern recognition receptors (PRRs) revealed induction of endosomal and cytosolic receptors in RAW264.7 cells and BMMs by ΔF/TriAdj, but no up-regulation by ΔF in PBS. As a secondary response to the PRR gene expression, induction of several chemokines and pro-inflammatory cytokines, as well as up-regulation of MHC-II and co-stimulatory immune markers, was observed. To further investigate the mechanisms involved in ΔF/TriAdj-mediated secondary responses, we used relevant signal transduction pathway inhibitors. Based on inhibition studies at both transcript and protein levels, JNK, ERK1/2, CaMKII, PI3K and JAK pathways were clearly responsible for ΔF/TriAdj-mediated chemokine and pro-inflammatory cytokine responses, while the p38 and NF-κB pathways appeared to be not or minimally involved. ΔF/TriAdj induced IFN-ß, which may participate in the JAK-STAT pathway to further amplify CXCL-10 production, which was strongly up-regulated. Blocking this pathway by a JAK inhibitor almost completely abrogated CXCL-10 production and caused a significant reduction in the cell surface expression of MHC-II and co-stimulatory immune markers. These data demonstrate that ΔF/TriAdj induces multiple signaling pathways in macrophages.

Dose Selection for an Adjuvanted Respiratory Syncytial Virus F Protein Vaccine for Older Adults Based on Humoral and Cellular Immune Responses.

This is the second phase 1 study of a respiratory syncytial virus (RSV) vaccine containing RSV fusion protein (sF) adjuvanted with glucopyranosyl lipid A (GLA) in a squalene-based 2% stable emulsion (GLA-SE). In this randomized, double-blind study, 261 subjects aged ≥60 years received inactivated influenza vaccine (IIV), a vaccine containing 120 µg sF with escalating doses of GLA (1, 2.5, or 5 µg) in SE, or a vaccine containing 80 µg sF with 2.5 µg GLA in SE. Subjects receiving 120 µg sF with 2.5 or 5 µg GLA were also randomized to receive IIV or placebo. Immunity to RSV was assessed by detection of microneutralizing, anti-F immunoglobulin G, and palivizumab-competitive antibodies and F-specific gamma interferon enzyme-linked immunosorbent spot assay T-cell responses. Higher adjuvant doses increased injection site discomfort, but at the highest dose, the reactogenicity was similar to that of IIV. Significant humoral and cellular immune responses were observed. The 120 µg sF plus 5.0 µg GLA formulation resulted in the highest responses in all subjects and in older subjects. These results confirm previous observations of vaccine tolerability, safety, and immunogenicity and were used to select the 120 µg sF plus 5.0 µg GLA formulation for phase 2 evaluation. (This study has been registered at ClinicalTrials.gov under registration no. NCT02289820.).

A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome replication-deficient Sendai vector that has many favorable vaccine characteristics. The specific vaccine design guarantees genetic stability of the transgene; furthermore, it supports a favorable presentation of the antigen, activating the adaptive response, features that other vectored vaccine approaches have often had difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies, and it could also be applied to other infectious diseases.

Immunopathogenesis associated with formaldehyde-inactivated RSV vaccine in preclinical and clinical studies.

INTRODUCTION: Respiratory syncytial virus (RSV) infection is responsible for one-third of deaths of acute lower respiratory infection in children less than one-year-old. The formaldehyde-inactivated RSV vaccine trial conducted in the 1960s predisposed the vaccinees to more serious RSV infection instead of protection. Better understanding of the underlying mechanism is of critical importance for better designing of safe and effective RSV vaccines. Areas covered: PubMed was searched to review immunopathology induced by RSV vaccines. We intend to dissect the differences in clinical and pathological manifestations of enhanced respiratory disease (ERD) in different animal models in comparison with humans. Formaldehyde-inactivated RSV vaccine causes ERD in both humans and animals, while RSV vaccine without formaldehyde treatment could also induce similar disease in animals, suggesting multiple pathways may be involved. Expert commentary: Identification of biomarkers pertinent to clinical evaluation should be further explored for safety assessment of RSV vaccines in human trials.

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein.

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation.

Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE: hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.

Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

Impact of formulation and particle size on stability and immunogenicity of oil-in-water emulsion adjuvants.

Oil-in-water emulsions have gained consideration as vaccine adjuvants in recent years due to their ability to elicit a differentiated immunogenic response compared to traditional aluminum salt adjuvants. Squalene, a cholesterol precursor, is a natural product with immunostimulatory properties, making it an ideal candidate for such oil-in-water emulsions. Particle size is a key parameter of these emulsions and its relationship to stability and adjuvanticity has not been extensively studied. This study evaluates the effect of particle size on the stability and immunogenicity of squalene emulsions. We investigated the effect of formulation parameters such as surfactant concentration on particle size, resulting in particles with average diameter of 80 nm, 100 nm, 150 nm, 200 nm, or 250 nm. Emulsions were exposed to shear and temperature stresses, and stability parameters such as pH, osmolarity, size, and in-depth visual appearance were monitored over time. In addition, adjuvanticity of different particle size was assessed in a mouse model using Respiratory Syncytial Virus Fusion protein (RSV-F) as a model antigen. Temperature dependent phase separation appeared to be the most common route of degradation occurring in the higher particle sizes emulsions. The emulsions below 150 nm size maintained stability at either 5 °C or 25 °C, and the 80 nm diameter ones showed no measurable changes in size even after one month at 40 °C. In vivo studies using the emulsions as an adjuvant with RSV F antigen revealed that superior immunogenicity could be achieved with the 80 nm particle size emulsion.

Recombinant influenza virus carrying the conserved domain of respiratory syncytial virus (RSV) G protein confers protection against RSV without inflammatory disease.

Respiratory syncytial virus (RSV) is one of the most important causes for viral lower respiratory tract disease in humans. There is no licensed RSV vaccine. Here, we generated recombinant influenza viruses (PR8/RSV.HA-G) carrying the chimeric constructs of hemagglutinin (HA) and central conserved-domains of the RSV G protein. PR8/RSV.HA-G virus showed lower pathogenicity without compromising immunogenicity in mice. Single intranasal inoculation of mice with PR8/RSV.HA-G induced IgG2a isotype dominant antibodies and RSV neutralizing activity. Mice with single intranasal inoculation of PR8/RSV.HA-G were protected against RSV infection as evidenced by significant reduction of lung viral loads to a detection limit upon RSV challenge. PR8/RSV.HA-G inoculation of mice did not induce pulmonary eosinophilia and inflammation upon RSV infection. These findings support a concept that recombinant influenza viruses carrying the RSV G conserved-domain can be developed as a promising RSV vaccine candidate without pulmonary disease.

Virus-like nanoparticle and DNA vaccination confers protection against respiratory syncytial virus by modulating innate and adaptive immune cells.

Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses.