Preparation of a standardized, efficacious agricultural H5N3 vaccine by reverse genetics

Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.
Virology (Impact Factor: 3.32). 10/2003; 314(2):580-90. DOI: 10.1016/S0042-6822(03)00458-6
Source: PubMed


Options for the control of emerging and reemerging H5N1 influenza viruses include improvements in biosecurity and the use of inactivated vaccines. Commercially available H5N2 influenza vaccine prevents disease signs and reduces virus load but does not completely prevent virus shedding after challenge with H5N1 virus. By using reverse genetics, we prepared an H5N3 vaccine whose hemagglutinin is 99.6% homologous to that of A/CK/HK/86.3/02 (H5N1). We used the internal genes of A/PR/8/34 and the H5 of A/Goose/HK/437.4/99 (H5N1) after deletion of basic amino acids from its connecting peptide region. The resulting virus was not lethal to chicken embryos and grew to high HA titers in eggs, allowing preparation of HA protein-standardized vaccine in unconcentrated allantoic fluid. The N3 neuraminidase, derived from A/Duck/Germany/1215/73 (H2N3), permitted discrimination between vaccinated and naturally infected birds. The virus construct failed to replicate in quail and chickens. Similar to parental A/PR/8/34 (H1N1), it replicated in mice and ferrets and spread to the brains of mice; therefore, it should not be used as a live-attenuated vaccine. The H5N3 vaccine, at doses of 1.2 microg HA, induced HI antibodies in chickens and prevented death, signs of disease, and markedly reduced virus shedding after challenge with A/CK/HK/86.3/02 (H5N1) but did not provide sterilizing immunity. Thus, reverse genetics allows the inexpensive preparation of standardized, efficacious H5N3 poultry vaccines that may also reduce the reemergence of H5N1 genotypes.

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Available from: Daniel Roberto Perez, Oct 07, 2015
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    • "Virus-containing allantoic fluid was harvested and inactivated with ␤-propiolactone at a ratio of 1:2000 (v/v) for 72 h. Inactivated virus stock was then concentrated using Amicon ultrafiltration and ultracentrifugation through a 25% and 70% sucrose cushion, pelleted at 76,000 × g at 4 • C for an hour, and purified as previously described [18]. The total protein content was determined using the Bradford Assay (Biorad) according to the manufacturer's specifications. "
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    ABSTRACT: The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.
    Vaccine 06/2014; 32(35). DOI:10.1016/j.vaccine.2014.06.016 · 3.62 Impact Factor
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    • "The RNA was extracted with TRIZOL LS Reagent (Life Technologies, Rockville, MD) according to manufacturer's instructions. The HA genes of A/Chicken/Dakahlia/106/ 2008 (106/08), and A/Chicken/Egypt/527/2012 (527/12) isolates were amplified using a one-step RT-PCR kit (Qiagen, Valencia, CA) and two reverse genetic primer sets (primers sequences available on request), which removes multiple basic amino acids at the cleavage site as previously described (Liu et al., 2003). Reassortant viruses containing the modified HA of the H5N1 virus in a genetic background of A/WSN/33 (H1N1) were generated using reverse genetics (Lee et al., 2004b; Neumann et al., 1999). "
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    Veterinary Microbiology 09/2013; 167(3-4). DOI:10.1016/j.vetmic.2013.09.022 · 2.51 Impact Factor
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    • "Besides that, both humoral and cell-mediated immune responses have been shown to induce by DNA vaccines (Liu et al., 2003). Nevertheless, DNA vaccines also have limitations which include low immunogenicity and expression levels, low transfection efficiency and targeting specificity compared to conventional vaccines (Leitner et al., 1999; Liu et al., 2003) which can be addressed by the use of adjuvants. Studies have shown that IL-15 in mammals has a major role in natural killer cell development and proliferation, development of CD4+ T cells and B cells proliferation and differentiation (Huntington et al., 2009; Lauwerys et al., 2000). "
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    ABSTRACT: We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.
    Research in Veterinary Science 08/2013; 95(3). DOI:10.1016/j.rvsc.2013.07.013 · 1.41 Impact Factor
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