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Significance Hepatitis C virus chronically infects approximately 1% of the world’s population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and 1H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and (1)H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of ‘membrane-anchored’ RSV F protein to elevated temperature (45–55 °C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

3-O-(3′,3′-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

The complete nucleotide sequences of the attachment glycoprotein (G) genes of three strains of avian metapneumovirus subgroup C (AMPV-C) were determined from the viral genomic and mRNAs. The G gene of AMPV-C was 1798 nt (1015 nt longer than previously reported) and the derived polypeptide had 585 aa. The deduced amino acid sequence of the predicted G protein of AMPV-C strain Colorado (AMPV-CO) showed 21-25 % amino acid identity to the G proteins of human metapneumoviruses, but only 14-16 % amino acid identity to those of other AMPV subgroups. The predicted G protein of AMPV-CO showed 98 and 81 % amino acid identity to those of AMPV-C strains Mn-1a and Mn-2a, respectively, indicating considerable sequence variation in the G proteins of AMPV-C isolates. Comparison of the G protein sequences of AMPV-CO and Mn-2a identified a highly divergent domain (48 % amino acid identity) at aa 300-450.

Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3′-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves. BRSV infection is associated with epithelial cell death and inflammation. Over the past few years, a growing number of viruses have been found to induce apoptosis. In order to determine the ability of BRSV to induce apoptosis, we studied the effect of BRSV infection in cultured MDBK cells. We used ligation-mediated PCR assay to detect specific blunt-end cellular DNA fragments produced by cellular endonucleases cleaving the genomic DNA between the nucleosomes during apoptosis. We found that BRSV infection resulted in apoptosis in MDBK cells. This data demonstrates for the first time that BRSV can induce apoptosis. This data also may contribute to delineate the mechanisms that regulate tissue injury and potential lung repair following BRSV infection.

Infectious bovine respiratory syncytial virus (BRSV) was produced by intracellular co-expression of five plasmid borne cDNAs, each under the control of a T7 RNA polymerase promoter. These separately encoded a full-length, genetically-marked copy of BRSV antigenome along with either BRSV or human respiratory syncytial virus (HRSV) support plasmids, which express N, P, L and M2-1 proteins. HEp2 cells were used in transfection and recombinant vaccinia virus (MVA-T7) provided T7 RNA polymerase to drive the transcription. The recovery of recombinant BRSV (rBRSV) was confirmed by immunological staining of plaques, restriction enzyme digestion and nucleotide sequencing of PCR fragments carrying the genetic markers from the rescued virus. The rBRSV was indistinguishable from its parental wild-type virus in its growth characteristics in cell culture. The present work has completed the entire genome sequence of BRSV strain A51908 (15,140 nt) and has also identified changes in sequence and growth characteristics in cell culture from the original BRSV strain A51908 laboratory isolate.

International audience; Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves resulting in a substantial economic loss for the cattle industry worldwide. In order to determine the presence of BRSV in Uruguay, an immunoenzymatic test was set up, using a recombinant BRSV nucleocapsid (N) protein as the antigen. The N protein was produced in sf9 insect cells by a recombinant baculovirus expressing the N protein. Serum samples collected from one hundred cattle from four different geographic regions of Uruguay were analyzed. Antibodies against the N protein of BRSV were detected in 95% of the serum samples analyzed. These results show for the first time the presence of BRSV antibodies and suggest a widespread BRSV infection in the cattle population of Uruguay.; Résumé - Première mise en évidence sérologique du virus syncytial respiratoire bovin en Uruguay. Le virus syncytial respiratoire bovin (VSRB) est une cause majeure de maladie respiratoire chez le veau, provoquant des pertes économiques importantes pour l'industrie bovine dans le monde entier. Afin de mettre en évidence la présence du VSRB en Uruguay, un test immunoenzymatique a été élaboré, qui utilise comme antigène une protéine recombinante de nucléocapside (N) du VSRB. La protéine N a été produite dans des cellules d'insecte sf9 par un baculovirus recombinant exprimant la protéine N. Des échantillons de sérum prélevés sur100 bovins, provenant de quatre régions différentes d'Uruguay, ont été analysés. Des anticorps dirigés contre la protéine N du VSRB ont été détectés dans 95% des échantillons analysés. Ces résultats montrent, pour la première fois, la présence d'anticorps anti-VSRB et suggèrent que l'infection par le VSRB est largement répandue dans la population bovine en Uruguay.

The complete nucleotide sequence of a functional clone of the large polymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomic and mRNAs. The BRSV L gene is 6573 nt in length and the derived polypeptide has 2162 aa. Alignment of the sequences of the BRSV L gene, and its encoded protein, with sequences of the L gene and protein of human respiratory syncytial virus strain A2 showed 77% identity at the nucleotide level and 84% identity at the amino acid level. By comparison, the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level. A minigenome was constructed to encode a BRSV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative BRSV transcription motifs and flanked by the BRSV genomic termini. Transfection of plasmids encoding the BRSV minigenome, nucleocapsid protein (N), phosphoprotein (P) and L protein, each under the control of T7 promoter, into cells infected with a vaccinia virus recombinant expressing the T7 RNA polymerase gave rise to CAT activity and progeny with the minigenome. This result indicates that the N, P and L proteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional. Further, inclusion of small amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.

We report here the nucleotide and deduced amino acid (aa) sequences of the small hydrophobic (SH) gene of the avian pneumovirus strain Colorado (APV/CO). The SH gene of APV/CO is 628 nucleotides in length from gene-start to gene-end. The longest ORF of the SH gene encoded a protein of 177 aas in length. Comparison of the deduced aa sequence of the SH protein of APV/CO with the corresponding published sequences of other members of genera metapneumovirus showed 28% identity with the newly discovered human metapneumovirus (hMPV), but no discernable identity with the APV subgroup A or B. Collectively, this data supports the hypothesis that: (i) APV/CO is distinct from European APV subgroups and belongs to the novel subgroup APV/C (APV/US); (ii) APV/CO is more closely related to hMPV, a mammalian metapneumovirus, than to either APV subgroup A or B. The SH gene of APV/CO was cloned using a genomic walk strategy which initiated cDNA synthesis from genomic RNA that traversed the genes in the order 3'-M-F-M2-SH-G-5', thus confirming that gene-order of APV/CO conforms in the genus Metapneumovirus. We also provide the sequences of transcription-signals and the M-F, F-M2, M2-SH and SH-G intergenic regions of APV/CO.

The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of the viral genomic RNA. To investigate the domains and specific residues involved in different activities of the P protein, we generated a total of 22 deletion and 17 point mutants of the P protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome transcription, (2) direct minigenome replication, and (3) form complexes with nucleocapsid protein (N) and large polymerase protein (L). These studies revealed that all the regions of P protein except amino acids 41–80 are essential for minigenome transcription and replication. Interestingly, amino acids 41–60 appeared to contain sequences that negatively regulate transcription and replication. Analysis of the N- or C-terminal ends indicated that deletion of up to 3 amino acids from the N- or C-terminus completely ablated the replication, while leaving substantial residual transcription. Single amino acid substitutions within the N-terminal 4 or C-terminal 13 amino acids showed that substitution at position 2, 4, 234, 236, 238, 240, or 241 was highly inhibitory to both transcription and replication, whereas substitution at position 3 was highly inhibitory to replication while leaving substantial residual transcription. Substitution of serine residues at the C-terminus indicated that loss of phosphorylation sites did not appear to have any effect on transcription and replication. Coimmunoprecipitation of P–N and P–L complexes with P-specific antiserum revealed that substitution mutations at the N- or C-terminus did not affect binding to N and L proteins, except that substitution mutation at C-terminus position 234, 236, 238, 240, or 241 affected binding to N protein by 10-fold.

The interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV–CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161–180 amino acids and a C-terminal region of 221–241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121–160. The data suggest that N and L protein binding domains on the P protein do not overlap.

The nucleotide sequences of the 3' leader and 5' trailer regions were determined for genomic RNA of bovine respiratory syncytial virus (BRSV) strain A-51908. The leader and trailer sequences are '45' and '161' nucleotides in length, respectively. The functionality of BRSV leader and trailer sequences and their recognition by HRSV and ovine respiratory syncytial virus (ORSV) proteins were examined with a in vitro transcribed BRSV genomic RNA analog carrying the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of BRSV transcription signals. Upon transfection into BRSV, HRSV or ORSV infected cells, the BRSV minireplicons were 'rescued' such that the reporter gene was expressed, the minigenome was replicated and packaged into micrococcal nuclease resistant-infectious minireplicons. The passage of infectious minireplicons could be blocked by a polyclonal BRSV neutralizing antiserum. Bovine parainfluenza virus-3, a heterologous paramyxovirus was inactive in rescuing BRSV genomic RNA analog. Mutational substitution of the G residue at position 4 of leader sequence in the BRSV genomic RNA analog, with an A or U residue inhibited its transcription and replication, while replacement with a C residue had no significant effect on rescue. These results show that the cis-acting elements of BRSV are functional and are also recognized by the proteins of HRSV and ORSV. The helper virus complemented rescue system developed here will be useful for characterizing the cis-acting elements of BRSV.

The G and P genes of bovine, ovine and caprine respiratory syncytial (RS) viruses were analyzed by RNase A one-dimensional fingerprinting, using A 51908 as the reference strain. Antisense G or P RNA probes of bovine RS virus strain A 51908 were hybridized to total RNA extracted from bovine turbinate cells infected with bovine, ovine or caprine RS virus strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products were analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed heterogeneity among bovine, ovine and caprine RS virus isolates. Ovine RS virus strains generated RNA cleavage patterns more distantly related to the bovine or caprine RS virus strains, particularly in the G gene. Statistical analysis of the results obtained indicated that genetic differences between bovine and ovine viruses were larger, compared with the ones among bovine strains themselves. The same analysis also revealed a close genetic relation among bovine and caprine strains. These results are discussed in terms of ungulate RS virus genetic variation and vaccine development.

The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA. To investigate the domains and specific residues involved in different N activities, we generated a total of 27 deletion and 12 point mutants of the N protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsidation, and (3) form a complex with the phosphoprotein (P). The mutations tested were defective in synthesis of RNA from the BRSV-CAT minigenome template with the exception of the following: a deletion involving the first N-terminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues. Micrococcal nuclease enzyme protection assays showed that mutations involving amino acids 1–364 of the 391-amino-acid N protein prevented minigenome encapsidation. Thus the BRSV N protein has a C-terminal, 27-amino-acid tail that is not required for encapsidation. Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving deletion of the N-terminal amino acid and the mutant involving a substitution at position 2. This finding indicates that the formation of a nucleocapsid sufficient to protect the RNA from nuclease is not required for template function. Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association with the P protein: a central portion of 244–290 amino acids and a C-terminal portion of 338–364 amino acids.