Oral Vaccination of Baculovirus-Expressed VP28 Displays Enhanced Protection against White Spot Syndrome Virus in Penaeus monodon

White Spot Syndrome Virus (WSSV) is an infectious pathogen of shrimp and other crustaceans, and neither effective vaccines nor adequate treatments are currently available. WSSV is an enveloped dsDNA virus, and one of its major envelope proteins, VP28, plays a pivotal role in WSSV infection. In an attempt to develop a vaccine against WSSV, we inserted the VP28 gene into a baculovirus vector tailored to express VP28 on the baculovirus surface under the WSSV ie1 promoter (Bac-VP28). The Bac-VP28 incorporated abundant quantity (65.3 µg/ml) of VP28. Shrimp were treated by oral and immersion vaccination with either Bac-VP28 or wild-type baculovirus (Bac-wt). The treatment was followed by challenge with WSSV after 3 and 15 days. Bac-VP28 vaccinated shrimp showed significantly higher survival rates (oral: 81.7% and 76.7%; immersion: 75% and 68.4%) than Bac-wt or non-treated shrimp (100% mortality). To verify the protective effects of Bac-VP28, we examined in vivo expression of VP28 by immunohistochemistry and quantified the WSSV copy number by qPCR. In addition to that, we quantified the expression levels shrimp genes LGBP and STAT by real-time RT-PCR from the samples obtained from Bac-VP28 vaccinated shrimp at different duration of vaccine regime. Our findings indicate that oral vaccination of shrimp with Bac-VP28 is an attractive preventative measure against WSSV infection that can be used in the field

A Novel Peptide ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

BACKGROUND: Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met. METHODOLOGY/PRINCIPAL FINDINGS: Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA. CONCLUSION/SIGNIFICANCE: The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas

Time-mortality relationship of (a) oral and (b) immersion vaccinations and WSSV challenge experiment.

<p>Shrimp were vaccinated with Bac-VP28, Bac-wt or PBS (positive control) and challenged with WSSV or PBS buffer (negative control) on 3 and 15 days post vaccination (dpv).</p

Analysis of the protection generated by recombinant baculovirus by (a) quantification of WSSV viral copies by real-time PCR (b) immunohistochemistry analysis of <i>in vivo</i> expression of VP28 from Bac-VP28 and Bac-wt vaccinated shrimp tissues.

<p>In Figure a, each column represents the mean of triplicate assay with standard deviation. Asterisks denote significant differences (P<0.01) between samples. All shrimp in the 10 dpi and 15 dpi samples of Bac-wt, Positive control group were died and data is Not Detectable. In Figure b, the arrow marks indicates the expression of VP28 in shrimp tissues.</p

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

Virology3912315-32

Schematic structure of recombinant baculovirus construct, analysis the expression of VP28 on baculovirus system using anti-mouse VP28 polyclonal antibodies and Quantification of VP28 expressed from Bac-VP28.

<p>(a) The VP28 gene was cloned downstream of WSSV ie1 promoter and upstream of Tsv40 of pFasBacHT vector and named as Bac-VP28 and above construct with out VP28 gene named as Bac-wt. (b) Immunofluorescence assay of expression of VP28 in Sf9 cells. The cells were infected with Bac-VP28 and Bac-wt at MOI of 0.5 and at 48 hrs post infection the cells fixed and analysed. (c) Western blot analysis of baculovirus expressed VP28 compared with WSSV virions. Lane 1-supernatant of baculovirus expressed VP28; Lane 2-complete Bac-VP28 virions; Lane 3-complete Bac-wt virions; Lane 4-purified wild type WSSV virions from WSSV infected shrimp tissue. (d) (i) -quantitative western blot analysis of VP28 expressed in baculovirus system and compared with different concentration of PrVP28 expressed in bacterial system as a standard. Lane 1 to Lane 6- purified PrVP28 protein from 0.01 µg, 0.05 µg, 0.1 µg, 0.25 µg, 0.5 µg and 1 µg; Lane Bac-VP28-1 µg total protein; Lane Bac-wt-1 µg of total protein. The nitrocellulose membrane was scanned and analysed by Odyssey Infrared Imager. (ii)-rVP28 protein standard curve; (iii)-the amount of VP28 protein presents in Bac-VP28 culture.</p

Set up for vaccination experiments.

<p>Set up for vaccination experiments.</p

Temporal expression analysis LGBP and STAT genes in healthy or WSSV infected shrimp and compared with Bac-VP28 vaccinated shrimp at 2, 5, 7, 10 and 15 dpi.

<p>The relative expression level was expressed by ΔCt (Ct value of LGBP/STAT gene-Ct value of EF-1α gene) and determined for each samples. The average ΔCt for WSSV infected or vaccinated samples from each days post infections was then used to plot the graph.</p

Antimicrobial Analysis of Biosynthesized Lectin-Conjugated Gold Nanoparticles

To enhance the bioactivity of molecules through nanoparticles is being tested which has potential use in sustained-release drug delivery systems and to enhance the therapeutic effectiveness of drugs. Our current investigation s is to conjugate lectin to that of a gold nanoparticle (GNP) surface without disturbing the bioactive properties and enhances the antibacterial activity of lectin. Au-lectin nanoparticles were checked for their hemagglutination activity, characterized by transmission electron microscopy (TEM) and UV-visible spectrophotometer. The antibacterial effect of nanoparticle lectin, Au salt nanoparticle, and conjugated Au-lectin was estimated by Kirby-Bauer disc method; MICs were determined by microbroth dilution and compared with ciprofloxacin. These tests were done using known species of bacterial strain of multidrug resistant. The hemagglutination activity of lectin was improved to fourfold after purification. Lectin and Au nanoparticles combined had a significant effect on the inhibition of bacterial growth. No significant differences were observed in the inhibition zone diameters from killed bacteria and its supernatant towards any of the tested organisms. Lectin-conjugated gold particles showed good efficacy as antimicrobial agents and the nanoparticle-killed bacteria to work against the viable population of the same bacterium and/or other bacterial species too