Development of a Thermostable Vaccine for Control of Peste des Petits Ruminants (PPR)

Abstract

The current study describes the development of a homologous lineage, live attenuated, thermostable vaccine for control of Peste des Petits ruminants (PPR). The objectives of the study were to isolate and characterize PPR virus (PPRV) from PPR outbreaks in Pakistan, to evaluate the thermostability potential of PPRV local isolates, to attenuate the most thermo-tolerant strain of PPRV and assessing its pathogenicity in goats, to develop a live attenuated thermo-tolerant PPR vaccine and to evaluate the comparative efficacy of different chemical stabilizers on the thermostability of newly developed PPR vaccine. In order to achieve the first objective, a total of 239 clinical samples were collected from 43 suspected outbreaks of PPR reported in different provinces of the country during 2012-2014. The samples were confirmed by Ic-ELISA and RT-PCR. The study revealed an appraisal of 70.17% of positive samples by RT-PCR than Ic ELISA which confirmed 61.58% of samples positive for PPR antigen. It was concluded that RT-PCR is more sensitive assay for confirmation of PPRV from suspected field outbreaks. The results achieved an overall 65.58% of PPR positive samples. The phylogeny and epidemiology of PPRV circulating among small ruminant population were studied by conducting molecular characterization of N gene. The data was analyzed by constructing phylogenetic studies. The analysis of data consorted all the present study isolates into lineage IV. Moreover, Pakistani isolates showed more diversity and were found more closely associated with other isolates from neighboring countries like China, India and Iran. Full genomes of three local PPRV isolates were sequenced using two sequencing methods; random sequencing method or SISPA (Sequence-independent single-primer amplification) and amplification with specific primers. The results showed that amplification with specific primers resulted in high PPRV specific reads (96.7%) as compared to SISPA which did not cover the entire PPRV genome. Considering the cost of amplification and number of reads obtained with the method, sequencing with specific primers is highly suggested. Phylogenetic analysis of full genomes clustered the three isolates within lineage IV clade sharing high sequence identity at nucleotide level with other PPRV strains from same lineage. Moreover, partial sequence of N gene was found informative for a preliminary investigation of origin of a strain. The genome organization of these three strains was also same and was in agreement with the “rule of six” for Paramyxoviridae. Comparative analysis of full genomes of these local strains and one Nigerian strain (75/1) revealed differences at nucleotide and protein level. A comparative analysis of Vero-76 and BTS-34 cells was undertaken by inoculating PPRV isolates on both cell lines. BTS cells showed more pronounced CPEs associated with PPRV along with high virus titer in short time than on Vero cells. The present study also describes the appraisal of thermostability potential using 7 local isolates of PPRV in order to select the most suitable candidate for development PPR vaccine. Results of the thermostability experiments revealed that PPRV was relatively stable at 4°C with no radical loss of infectivity for 3 weeks. PPRV in cell culture medium was impressible to heat and could be inactivated in 7 and 5 minutes at 60°C and 65°C. However, at 56°C, 20 to 25 minutes were required to eliminate PPRV infectivity. Ultraviolet irradiation effectively inactivated PPRV within 2-3 minutes. One isolate (PAK-LRS-13/NARC) appeared more resistant at high temperatures maintaining reasonable required titer after heat treatments. Based on thermostability characteristics, the attenuation of a local thermo-tolerant strain of PPRV was conducted to develop a homologous lineage PPR vaccine by serially passaging the strain upto 60th passage onto Vero cells. The pathogenicity trials were also conducted in goats to monitor the degree of attenuation and progressive loss of pathogenicity after 20th, 40th and 60th passage. It showed that after 20th passage, slight hyperthermia and oculo-nasal discharge were observed in goats. However, after 40th and 60th passage, no clinical manifestations were noticed. It was concluded that 60th passage virus could safely be used for development of PPR vaccine in order to minimize the risks associated with the reversion of attenuated strain to wild type. PPR vaccine was prepared by thermo-stabilization method used for RP vaccine. A comparative study was also conducted to evaluate the effects of three different stabilizers on the thermostability of PPR vaccine. The results were compared by accelerated stability tests. Among the three chemical stabilizers, the loss in viral titer during lyophilization in case of Lactalbumin Hydrolysate-Sucrose (LAH) was only 0.23 TCID50/ml as compared to the Trehalose dehydrate (TD) (0.37 TCID50/ml) and Weybridge medium (WBM) (0.45 TCID50/ml) which suggests LAH as the stabilizer of choice for enhancing thermostability of PPR vaccine. The efficacy of this newly developed PPR vaccine was evaluated by carrying out experimental immunization in goats. Dynamics of humoral immune response was monitored. Challenge protection studies were carried out in animals previously inoculated with PPR vaccine made by Nigerian strain (75/1) and newly developed thermostable vaccine formulation. A virulent field isolate catalogued as PAK-Fjg 14/NARC was used in challenge protection studies. It was found that antibodies developed after 3 days post vaccination. The results were comparable to the Nigerian strain (75/1) vaccine. The animals combated the challenge and did not produce any clinical signs of disease throughout the experimental period. PPRV was not detected in any of nasal, ocular and oral secretions by RT-PCR. Results of humoral immune response revealed that immunization with vaccine prepared from local thermostable strain of PPRV with the new formulation provided equally good protection in goats. The study concluded that the newly developed thermostable vaccine would be beneficial in eliminating cold chain associated problems present in existing vaccine and also help in progressive control of PPR in the country.