Development of a process for upscaling and production of thermotolerant Peste-des-petits ruminants vaccine.

Vaccination is the most effective means of preventing Peste-des-petits-ruminants (PPR), an important disease of small ruminant population. The thermolabile nature of PPR vaccine poses a major constraint in shipping, storage and its successful application. In view of limited thermotolerance of PPR virus and ongoing global PPR control and eradication program, development of a thermotolerant PPR vaccine was tried using a novel lyophilization protocol and improved thermostabilization. A lyophilization cycle of 16 h (h) using 200 µl of PPR vaccine virus (stock titre 5.8 log10 TCID50/vial in 200 µl) was developed. For this, five stabilizer formulations were selected out of ten formulations based on the stability of liquid vaccine at 37 °C and three freeze-thaw cycles. Improved thermostabilization of PPR vaccines was obtained by inclusion of 5% trehalose and 0.5% gelatine to Lactalbumin hydrolysate-sucrose (LS) formulations which significantly improved the stability of lyophilized vaccines with a shelf-life of at least 1305.3 days at 2-8 °C, 23.68 days at 25 °C, 20.88 days at 37 °C, 5.01 days at 40 °C and 3.22 days at 45 °C which qualifies the standards of a thermotolerant PPR vaccine as defined by the FAO and OIE. In reconstituted vaccines, the combination of LS, trehalose and gelatin (LSTG) provided a shelf-life of 1.77 days at 37 °C, 22.41 h at 40 °C and 10.05 h at 45 °C. The study suggested that use of the short lyophilization protocol standardized with 200 µl of lyophilized PPR vaccine stabilized with LSTG formulation, can be used to develop and upscale thermotolerant PPR vaccines during national and global PPR control and eradication as targeted by the FAO and OIE by 2030.

Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

Molecular characterization of lapinized classical Swine Fever vaccine strain by full-length genome sequencing and analysis.

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6-98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5'-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3'-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3'-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3'-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3'-UTR indicated that Indian CSFV strain requires further passage to obtain a 3'-UTR structure similar to most of the attenuated strains.

Induction of immune responses and protection in mice against rabies using a self-replicating RNA vaccine encoding rabies virus glycoprotein.

A self-replicating RNA vaccine encoding rabies virus glycoprotein gene was developed utilizing sindbis virus RNA replicon. The in vitro transcribed RNA (Sin-Rab-G RNA) was transfected in mammalian cells and analysed for self-replication and expression of rabies glycoprotein. To generate immune responses against rabies, mice were immunized with 10microg of Sin-Rab-G RNA and immune responses developed were compared with mice immunized with rabies DNA vaccine and commercial cell culture vaccine (Rabipur). The self-replicating rabies RNA vaccine generated cellular and humoral IgG responses similar to rabies DNA vaccine. On challenge with rabies virus CVS strain, rabies RNA vaccine conferred protection similar to rabies DNA vaccine. These results demonstrated that replicon-based self-replicating rabies RNA vaccine with 10microg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine.

A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies.