Development of effective vaccine against local isolates of foot and mouth disease virus in Bangladesh

Abstract

Foot and mouth disease (FMD) is a highly contagious picornaviral disease of domestic and wild cloven-hoofed animals throughout the world including Bangladesh. The endemicity of this disease is causing notifiable economic loss and an impediment to the full potential surge of livestock industry in Bangladesh. Vaccination using imported or locally produced vaccines is the existing practice of controlling the disease in Bangladesh, although vaccine failure is common upon vaccination. The causes of these failures are multifold including selection of inappropriate vaccine virus strain(s), inadequate antigenic payload and improper vaccine production process. It was presumed that a quality assured FMD vaccine tailored to the circulating indigenous FMDV strains might efficiently protect the susceptible animals in Bangladesh. Hence, the present investigation was envisaged to carry out an extensive epidemiological study and molecular characterization of the circulatory FMDVs in Bangladesh, selection of appropriate vaccine candidates, optimization of vaccine production processes and effective vaccine development. A total of 319 FMDV infected tissue samples from 47 FMD outbreaks were collected and analyzed over a period of seven years from 2012 to 2018. Polymerase Chain Reaction (PCR) based analysis of the samples revealed that FMDV serotypes O, A and Asia1 are prevalent in Bangladesh with predominance of serotype O (81%) followed by serotype A (13%) and Asia1 (6%). Intra-serotype genetic divergence specifically in serotype O was evident by VP1 protein coding gene based phylogeny studies. Serotype O isolates of Bangladesh clustered in Ind2001d, Ind2001BD1 and Ind2001BD2 sublineages of Ind2001 lineage, although most of the recent isolates belonged to novel and dominating sublineage Ind2001BD1. Phylogeny of FMDV serotype A isolates of Bangladesh clustered all of them in Genotype VII of Asia Topotype. Depending on the prevalence and dominance, FMDV serotype O isolate BAN/TA/Dh-301/2016 from Ind2001BD1 sublineage and serotype A isolate BAN/CH/Sa-304/2016 from Genotype VII were selected as vaccine candidates. Comparative genetic distance analysis confirmed that selected vaccine candidates are significantly (p=0.001-0.002) genetically closer to the respective isolates of Bangladesh than the current Indian vaccine strains. Z-test for selection predicted that the vaccines prepared by selected candidates will protect animals against all the circulatory FMDV serotype O and A isolates of Bangladesh. The selected vaccine candidates were adapted and propagated in BHK-21 cell line and subjected to complete genome sequencing. The complete genome of vaccine candidates O/BAN/TA/Dh-301/2016 and A/BAN/CH/SaPage | II 304/2016 were found to be 8211 nt and 8221 nt in length respectively with an ORF of 6999 nt for each. Deduced amino acid (aa) sequences comparison deciphered that the VP1, VP2 and VP3 regions of O/BAN/TA/Dh-301/2016 had 5%, 3% and 5% structural variability with respective regions of current Indian vaccine strain O/India/R2/75. Three aa substitutions in critical antigenic site 1 in G-H loop of VP1 and 2 aa substitutions in antigenic site 2 of VP2 regions were evident. Similar regions of A/BAN/CH/Sa-304/2016 showed 8%, 6% and 5% variability compared to respective regions of serotype A vaccine strain A/IND40/00 with four aa substitutions in critical antigenic sites of VP1. For the purpose of vaccine production, bulk amount of both the vaccine candidates were successfully inactivated by Binary Ethylenimine within 9-10 hours after the onset of inactivation. Extrapolation of inactivation kinetics of both the vaccine candidates confirmed that there will be less than 1 infectious particle in a 104 liter batch after 24 hours inactivation cycle. Complete inactivation of virus was assured by the absence of CPE even at third serial passage of inactivated virus in BHK-21 cell monolayers. Inactivated virus particles in the fluids were significantly (p=0.002-0.003) concentrated by ultrafiltration system using 100 kDa hollow fiber cartridge. After concentration, the final concentrations of inactivated virus particles in the fluids were found to be 16.33±1.15 µg/ml and 14.33±0.57 µg/ml for serotype O and A respectively. Vaccines were formulated with Montanide ISA 201 oil adjuvant and the final concentrations of inactivated FMD viral antigens per 2 ml of vaccine were 14.5 µg and 12.41 µg for serotype O and A respectively. Absence of adverse reactions in the recipient guinea pigs during in-vivo vaccine efficacy testing confirmed the safety of developed vaccines. The minimum inactivated virus particle contents that protected 100% animals from virus challenge and elicited SN50 titer significantly higher than the protective level was 3.53 µg and 3.10 µg for FMDV serotype O and A vaccine candidates respectively. In 2D-MNT tests, the antigenic relationship (r) values of the circulating FMD serotype O and A field viruses with respective vaccine strains were found to be >0.3. Strong antigenic relationship between field virus and selected vaccine strains confirmed that vaccine produced using these candidate viruses will protect all genetically divergent FMDV strains of respective serotypes circulating in Bangladesh.