Development and Evaluation of Virosome Based Vaccine(s) Against New Castle Disease

Abstract

Poultry is one of the fast growing and economically viable industries of Pakistan. Unfortunately, its growth has always been confronted with number of infectious and non infectious diseases. Among infectious diseases, Newcastle disease (ND) commonly known as Ranikhet is on top of the list. The strict biosecurity measures and immunoprophylaxis are two main ways to control this deadly infection. In spite of the use of killed and live and vaccines in commercial poultry, the continuous outbreaks of NDV is still recorded causing morbidity and mortality in Pakistan. Keeping the above scenario in view, present study was aimed for the development of virosome vaccine using local very virulent strain of NDV and evaluation of its immunogenicity in chicken. In Phase-I, NDV was isolated and identified from field outbreaks using standard techniques. Virus was purified using sucrose velocity density gradient ultracentrifugation and pathogenicity was tested through embryo lethal dose fifty (ELD50), intracerebral pathogenicity index (ICPI) and mean death time (MDT). Molecular characterization was done by amplifying fusion gene through RT-PCR and sequencing whereas viral proteins were analyzed through SDS-PAGE. The band of fusion protein was utilized and further characterized using in-gel digestion and mass spectrometry. In Phase-II, virosome was prepared using characterized strain of local NDV. Briefly, Triton X-100 was used as detergent and SM2 Bio-Beads were employed to eliminate detergent and reconstruct the viral membrane into virosome. Confirmation was carryout by transmission electron microscopy (TEM) and SDS-PAGE was employed for protein analysis. In-vitro cell attachment property of prepared virosomes was examined by incorporation of virosome into plasmid having green fluorescent protein (GFP). Microscopic examination was done through fluorescent microscope and images were captured. In Phase-III, after in-vitro fusion and characterization studies of ND virosome in cell culture were appraise as vaccine. Stability, Sterility and safety of virosome based vaccine were examined before in-vivo valuation of immunogenicity and challenge protection study in experimental broiler. Virosome vaccine was given (30µg/bird) at day seven and fourteen through intranasal route whereas commercially available live and killed ND vaccines were used for comparison along with negative control. Humoral immune response was monitored at different days post-vaccination using HI test. A xv challenge protection test was done to check the protective efficacy of vaccines using very virulent NDV. The results revealed that local NDV belonged to very virulent strain. Phylogenic analysis showed that it was more related to UVAS-2015 strain (Accession No. MF437287, Lahore Pakistan origin) followed by UVAS-2016 strain (Accession No. KX91187, Lahore Pakistan origin) and Tehran strain (Accession No. MG871466, Tehran Iran origin). The images of electron micrographs denote oval to circular shaped virosomes with size of approximately 75 to 200 nm whereas, SDS-PAGE revealed only 2 proteins i.e. haemagglutinin-neuraminidase (HN) and fusion (F) in prepared virosomes. The positive results of fusion study in vero cells showed the existence of intact HN and Fusion viral envelop proteins on ND virosomes and hence confirmed their ability of fusion to animal cells in-vitro. Virosome based vaccine was found to be sterile showing no bacterial or fungal growth on any culture media, stable at wide range of temperatures, safe as no lesions were examined in chicken embryos up to seven days post-inoculation. The experimental trials in broiler chicken showed highly significant (p<0.05) and clinically protective HI antibody titers at seven, fourteen, twenty-one and twenty-eight days post-immunization with virosome vaccine as compared to negative control group. The GMTs were equal to both live and killed vaccines with non-significant (p>0.05) difference throughout the experiment. The antibody levels raised in all vaccinated groups gradually from 7th day and were maximum at 28th day post-vaccination. In group (G1) vaccinated with virosome based vaccine, GMTs was 83.18 and 77.62 at 21st day and 28th day post vaccination, respectively. Challenge with very virulent NDV post-immunization revealed 80%, 90% and 100% protection in virosome, live and killed vaccinated groups, respectively. It was concluded that virosome ND vaccine prepared from local very virulent strain of NDV for the first time in the country was found to be completely safe and revealed good clinical protection along with comparable immunogenicity to already available live and inactivated vaccines in broiler chicken.