E. coli expression and immunological assessment of expressed recombinant Newcastle disease virus hemagglutinin-neuraminidase protein in chickens.

Every year, the poultry industry experiences significant economic losses due to epidemics of Newcastle disease virus (NDV). Developing new vaccines by identifying and using the immunogenic hemagglutinin-neuraminidase (HN) protein can protect the poultry industry. In the present study, the full-length HN protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells, purified via affinity chromatography and detected via western blot analysis using His-specific antibodies. The purified HN protein was further evaluated in chickens to study the immune response against NDV. The successful production of HN-specific IgY proved the activity of the purified HN protein. IgY was present in the serum of immunized chickens. However, the immune response was higher in chickens immunized with purified HN protein along with complete and incomplete adjuvants than in chickens immunized with only the HN protein. Keywords: protein; Newcastle disease virus; poultry; infectious diseases; vaccines.

Molecular evolution of the hemagglutinin-neuraminidase (HN) gene in human respirovirus 3.

Human respirovirus 3 (HRV3) is a major causative agent of acute respiratory infections in humans. HRV3 can manifest as a recurrent infection, although exactly how is not known. In the present study, we conducted detailed molecular evolutionary analyses of the major antigen-coding hemagglutinin-neuraminidase (HN) gene of this virus detected/isolated in various countries. We performed analyses of time-scaled evolution, similarity, selective pressure, phylodynamics, and conformational epitope prediction by mapping to HN protein models. In this way, we estimated that a common ancestor of the HN gene of HRV3 and bovine respirovirus 3 diverged around 1815 and formed many lineages in the phylogenetic tree. The evolutionary rates of the HN gene were 1.1 × 10-3 substitutions/site/year, although the majority of these substitutions were synonymous. Some positive and many negative selection sites were predicted in the HN protein. Phylodynamic fluctuations of the gene were observed, and these were different in each lineage. Furthermore, most of the predicted B cell epitopes did not correspond to the neutralization-related mouse monoclonal antibody binding sites. The lack of a link between the conformational epitopes and neutralization sites may explain the naturally occurring HRV3 reinfection.

Assessment of one-dose mumps-containing vaccine effectiveness on wild-type genotype F mumps viruses circulating in mainland China.

To clarify the protective effect of one-dose mumps-containing vaccines (MuCV) in mainland China, the antigenic variations of HN gene and cross-neutralization capacities between MuCV and wild type genotype F MuVs were studied. In total, 70 HN gene sequences of genotype F MuV representative strains obtained from 2001 to 2015, two types of MuCV strains, 139 pairs of pre- and post-vaccination serum samples from infants receiving one dose of MuCV vaccination were analyzed. Genotype-specific amino acid variations were observed in the potential antigenic epitopes between MuCV and wild-type genotype F MuVs circulating in mainland China. The mumps neutralization antibody titers induced by one-dose MuCV were found to be generally low. Moreover, significant differences in neutralization titers were observed between vaccine and wild-type strains. It could be concluded that one-dose MuCV had a cross-protective effect against the wild-type genotype F MuVs, but its effectiveness was limited, which might be caused by insufficient doses of MuCV vaccination and the genotype-specific antigenic differences between vaccine and wild-type MuVs as well. In addition, a poor linear correlation between mumps-specific IgG concentrations and neutralization titers was observed in this study, indicating the concentration of MuV-specific IgG could not fully reflect the neutralizing antibody titer in serum. Therefore, it is highly recommended to provide a second dose of MuCV to preschool children to increase MuV neutralizing antibody titers and use MuV cross-neutralization test as preferred tool for assessment of mumps-containing vaccine effectiveness on wild-type MuVs. This is the first report to assess the effectiveness of one-dose Chinese MuCV against wild-type genotype F MuVs, which would be benefit for the development of mumps vaccination strategy.

Assessment of selected media supplements to improve F/HN lentiviral vector production yields.

The development of lentiviral-based therapeutics is challenged by the high cost of current Good Manufacturing Practices (cGMP) production. Lentiviruses are enveloped viruses that capture a portion of the host cell membrane during budding, which then constitutes part of the virus particle. This process might lead to lipid and protein depletion in the cell membrane and affect cell viability. Furthermore, growth in suspension also causes stresses that can affect virus production yields. To assess the impact of these issues, selected supplements (Cholesterol Lipid Concentrate, Chemically Defined Lipid Concentrate, Lipid Mixture 1, Gelatin Peptone N3, N-Acetyl-L-Cysteine and Pluronic F-68) were assayed in order to improve production yields in a transient transfection production of a Sendai virus F/HN-pseudotyped HIV-1-based third generation lentiviral vector in FreeStyle 293 (serum-free media) in suspension. None of the supplements tested had a significant positive impact on lentiviral vector yields, but small non-significant improvements could be combined to increase vector production in a cell line where other conditions have been optimised.

Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine.