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The goal of this study is to prepare and evaluate influenza virus-like particles (VLPs) as a candidate trivalent vaccine to protect poultry against avian influenza (AI). VLPs will be configured to express HA proteins derived from avian H5N1, H7N2, and H9N2 strains. <P> The objectives will include (i) determination that the HA proteins can be efficiently incorporated into a VLP envelope in the context of M1 and NA proteins; (ii) preparation of VLPs in Sf9 cells; (iii) examination of the genetic stability of recombinant baculovirus, determination of morphological, functional, antigenic, and stability characteristics of VLPs, as well as (iv) determination of immunogenicity and protective characteristics of the trivalent VLPs (H5-H7-H9) in experimental animal challenge model (BALB/c mice). We expect that trivalent AI VLPs will elicit immune responses including induction of hemagglutination-inhibition (HAI) antibody following intranasal vaccination of mice. Morbidity and mortality in animals vaccinated with trivalent VLP vaccine will be determined after challenges with three corresponding virulent AI viruses. <P> We expect that if found feasible, trivalent VLP vaccine may represent a promising candidate for further testing in poultry to protect agricultural poultry against multiple AI viruses.
NON-TECHNICAL SUMMARY: In this Phase I SBIR application, we propose the trivalent influenza virus-like particle (VLP) vaccine that is not dependent on egg production and allows protection of poultry against multiple AI viruses. Our hypothesis is that expression of H5, H7, and H9 HA proteins will result in a trivalent vaccine that will contain HA proteins derived from three distinct AI viruses. We hypothesize that trivalent VLPs will elicit broad protection against multiple AI strains, especially if vaccine is administered through the respiratory route. We propose preparation and feasibility testing in mice of the prototype trivalent VLPs as a broadly protective AI vaccine. VLPs will be prepared in Sf9 insect cells using baculovirus expression system. Characteristics of VLPs will be determined including expression levels in Sf9 cell culture and genetic stability. Immunogenicity and efficacy of trivalent vaccine will be evaluated in BALB/c mice in collaboration with the Centers for Disease Control and Prevention (CDC, Atlanta, GA). If successful, this technology may allow rapid preparation of inexpensive and highly effective poultry vaccines against multiple AI strains.
<P>APPROACH: Influenza genes of H5, H7 and H9 subtypes will be cloned within a pFastBac1-based baculovirus transfer vector. Recombinant baculoviruses (rBV) expressing H9, H7, and H5 genes will be generated using a Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA). Spodoptera frugiperda Sf9 cells (ATCC, Manassas, VA) will be maintained as suspension cultures in HyQ-SFX insect serum free medium (HyClone, Logan, UT) at 27 C. For production of VLPs, Sf9 cells at 2 x 10'6 cells/ml will be infected at a multiplicity of infection of 3.0 for 72 hr with respective rBV. VLPs will be harvested from the growth medium supernatant, concentrated and partially purified by using a 20% (w/v) sucrose step gradient in phosphate buffered saline (PBS). VLPs will be stored at 2-8 C in a PBS buffer. For testing by electron microscopy, VLP samples will be adsorbed onto a freshly discharged 400 mesh carbon parlodion-coated copper grids (Poly- Sciences, Warrington, PA). The grids will be rinsed with buffer containing 20mM Tris, pH 7.4, and 120mM KCl and negatively stained with 1% phosphotungstic acid, then dried by aspiration. Influenza VLPs will be visualized on a Hitachi H-7600 transmission electron microscope (Hitachi High Technologies America, Schaumburg, IL) operating at 80 kV and digitally captured. Trivalent VLP vaccine will be prepared, formulated in PBS and used for vaccinations. BALB/c mice, 30 animals per group, will be vaccinated intranasally (i.n.) with VLPs containing 5 ug of HA on days 0 and 21. Thirty control animals will receive PBS as placebo. Following vaccinations on days 0 and 21, serum will be collected on days 21 and 35, respectively. Sera will be tested by HAI for the presence of influenza-specific humoral responses. HAI assay will be performed using 0.5% turkey RBC with 4 HA units of viruses using standard method For virus challenge, vaccinated and control mice will be transferred into a BSL3+ containment facility (CDC, Atlanta, GA), divided into groups of ten and challenged intranasally (i.n.) on day 35 with 10'6 EID50 of influenza H5N1, H7N2, or H9N2 viruses. Morbididty and mortality will be recorded daily. Nasal wash samples will be taken from mice on days 2, 4, 6, and 8 postchallenge, and influenza viruses will be titrated to determine virus shedding in the upper respiratory tract. Statistical significance of virus titers in nasal washes will be determined.