Rhodomentosones A and B: Two Pairs of Enantiomeric Phloroglucinol Trimers from Rhodomyrtus tomentosa and Their Asymmetric Biomimetic Synthesis.

Rhodomentosones A and B (1 and 2), two pairs of novel enantiomeric phloroglucinol trimers featuring a unique 6/5/5/6/5/5/6-fused ring system were isolated from Rhodomyrtus tomentosa. Their structures with absolute configurations were elucidated by NMR spectroscopy, X-ray crystallography, and ECD calculation. The bioinspired syntheses of 1 and 2 were achieved in six steps featuring an organocatalytic asymmetric dehydroxylation/Michael addition/Kornblum-DeLaMare rearrangement/ketalization cascade reaction. Compounds 1 and 2 exhibited promising antiviral activities against respiratory syncytial virus (RSV).

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors.

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus Infectivity with a Single Agent.

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.

Structure basis of neutralization by a novel site II/IV antibody against respiratory syncytial virus fusion protein.

Globally, human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in newborns, young children, and the elderly for which there is no vaccine. The RSV fusion (F) glycoprotein is a major target for vaccine development. Here, we describe a novel monoclonal antibody (designated as R4.C6) that recognizes both pre-fusion and post-fusion RSV F, and binds with nanomole affinity to a unique neutralizing site comprised of antigenic sites II and IV on the globular head. A 3.9 Å-resolution structure of RSV F-R4.C6 Fab complex was obtained by single particle cryo-electron microscopy and 3D reconstruction. The structure unraveled detailed interactions of R4.C6 with antigenic site II on one protomer and site IV on a neighboring protomer of post-fusion RSV F protein. These findings significantly further our understanding of the antigenic complexity of the F protein and provide new insights into RSV vaccine design.

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein.

Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity.

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein.IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545-1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577-587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749-1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110-128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion.

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F1 and F2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic α-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.

Induction of an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications for Vaccine Design.

RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I IFN response, SAM vaccines were evaluated in IFN receptor knockout mice. Our data indicate that minimizing the early type I IFN responses may be a useful strategy to increase primary SAM expression and the resulting vaccine potency. RNA sequence modification, delivery optimization, or concurrent use of appropriate compounds might be some of the strategies to finalize this aim.

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells.

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research, and in human serum, these antibodies contribute to >90% of the neutralization response; however, detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV, CD27+ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells, 0.35% recognized RSV-A2-infected cells, of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells, 44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein, while, unexpectedly, 41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However, these processes did not seem to depend on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCE Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly, and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries, RSV lower respiratory tract disease has a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease.

Formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity.

Respiratory syncytial virus (RSV) causes serious upper and lower respiratory tract infections in newborns and infants. Presently, there is no licensed vaccine against RSV. We previously reported the safety and efficacy of a novel vaccine candidate (ΔF/TriAdj) in rodent and lamb models following intranasal immunization. However, the effects of the vaccine on the innate immune system in the upper and lower respiratory tracts, when delivered intranasally, have not been characterized. In the present study, we found that ΔF/TriAdj triggered transient production of chemokines, cytokines and interferons in the nasal tissues and lungs of BALB/c mice. The types of chemokines produced were consistent with the populations of immune cells recruited, i.e. dendritic cells, macrophages and neutrophils, in the nose-associated lymphoid tissue (NALT), lung and their draining lymph nodes of the ΔF/TriAdj-immunized group. In addition, ΔF/TriAdj stimulated cellular activation with generation of mucosal and systemic antibody responses, and conferred complete protection from viral infection in the lungs upon RSV challenge. The effect of ΔF/TriAdj was short-lived in the nasal tissues and more prolonged in the lungs. In addition, both innate and adaptive immune responses were lower when mice were immunized with ΔF alone. These results suggest that ΔF/TriAdj modulates the innate mucosal environment in both upper and lower respiratory tracts, which contributes to robust adaptive immune responses and long-term protective efficacy of this novel vaccine formulation.

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.

Influence of Respiratory Syncytial Virus F Glycoprotein Conformation on Induction of Protective Immune Responses.

UNLABELLED: Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE: Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.

Facile and rapid detection of respiratory syncytial virus using metallic nanoparticles.

BACKGROUND: Respiratory syncytial virus (RSV) causes severe respiratory infection in infants, children and elderly. Currently, there is no effective vaccine or RSV specific drug for the treatment. However, an antiviral drug ribavirin and palivizumab is prescribed along with symptomatic treatment. RSV detection is important to ensure appropriate treatment of children. Most commonly used detection methods for RSV are DFA, ELISA and Real-time PCR which are expensive and time consuming. Newer approach of plasmonic detection techniques like localized surface plasmon resonance (LSPR) spectroscopy using metallic nanomaterials has gained interest recently. The LSPR spectroscopy is simple and easy than the current biophysical detection techniques like surface-enhanced Raman scattering (SERS) and mass-spectroscopy. RESULTS: In this study, we utilized LSPR shifting as an RSV detection method by using an anti-RSV polyclonal antibody conjugated to metallic nanoparticles (Cu, Ag and Au). Nanoparticles were synthesized using alginate as a reducing and stabilizing agent. RSV dose and time dependent LSPR shifting was measured for all three metallic nanoparticles (non-functionalized and functionalized). Specificity of the functionalized nanoparticles for RSV was evaluated in the presence Pseudomonas aeruginosa and adenovirus. We found that functionalized copper nanoparticles were efficient in RSV detection. Functionalized copper and silver nanoparticles were specific for RSV, when tested in the presence of adenovirus and P. aeruginosa, respectively. Limit of detection and limit of quantification values reveal that functionalized copper nanoparticles are superior in comparison with silver and gold nanoparticles. CONCLUSIONS: The study demonstrates successful application of LSPR for RSV detection, and it provides an easy and inexpensive alternative method for the potential development of LSPR-based detection devices.

Imaging viral RNA using multiply labeled tetravalent RNA imaging probes in live cells.

Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from ∼80nm to ∼10µm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes, their loading into the virion represents a critical step in the creation of infectious particles. RNA imaging tools represent a powerful approach to gain insight into fundamental viral processes, including virus entry, replication, and virion assembly. Imaging viral processes in live cells is critical due to both the heterogeneity of these processes on a per cell basis, and the inherent dynamics of these processes. There are a number of methods for labeling RNA in live cells; we'll introduce the myriad of methods and then focus on one approach for labeling viral RNA, using multiply-labeled tetravalent RNA imaging probes (MTRIPs), which do not require engineering of the target RNAs. We feel this approach is advantageous given many viral genomes may not tolerate large nucleotide insertions into their sequences.

Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera.

Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø-specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F-specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø.

Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.

The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss.

Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract.

Globally, Respiratory Syncytial Virus (RSV) is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs) composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications.

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.

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.