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Vaccine Detail

Defective adenovirus expressing VEEV E2 glycoprotein
Vaccine Information
  • Vaccine Name: Defective adenovirus expressing VEEV E2 glycoprotein
  • Target Pathogen: VEE Virus
  • Target Disease: Venezuelan equine encephalitis
  • Vaccine Ontology ID: VO_0004116
  • Type: Recombinant vector vaccine
  • Antigen: VEEV E2 glycoprotein
  • POLS_EEVVT Structural polyprotein (p130) gene engineering:
    • Type: Recombinant protein preparation
    • Description: Recombinant defective type 5 adenoviruses expressing the E3E26K structural genes of VEEV were prepared and examined (Phillpotts et al., 2005).
    • Detailed Gene Information: Click Here.
  • Preparation: To make recombinant viruses, the core gene, the first three nucleotides at the 5′-end of the VEE E3 gene, the 3′-end of the 6K gene and the E1 gene were removed by restriction digestions. An initiation codon, the three nucleotides deleted from E3, the nucleotides removed from the 3′-end of the 6K gene and a termination codon were all added by ligation of oligonucleotide linkers. The E3–E2–6K fragment was then cloned into plasmid pMV101, derived from pMV100 by the addition of a unique Eco R1 restriction site at an Xba I site located downstream of the promoter sequence, to generate plasmid pVEEV. Plasmid pMV100, which contains the CMV major immediate early promoter and a polyadenylation signal. Three sites within the VEEV E2 glycoprotein were mutated by site-directed mutagenesis from the sequence found in the attenuated TC-83 strain to the sequence found in the virulent TrD strain. Each of the E3–E2–6K sequences from plasmids pVEEV, pVEEV#2 and pVEEV#3 were inserted into AHuman adenovirus type 5 (Ad5) dl309 to make recombinant adenoviruses. Ad5 dl309 has a deleted E1a gene (Jones et al., 1979) and is replication defective, requiring E1a complementation for replication. The E1a gene product may be supplied in trans by stably transfected 293 cells. A second deletion is found in the E3 region of this virus (Bett et al., 1995). The Ad5 vector is designed to enter mammalian cells and express proteins but it is defective for production of infectious progeny virus. All of the recombinant adenoviruses were purified by three rounds of terminal dilution in 293 cells cultured in 96-well plates. The VEEV inserts in the recombinant adenoviruses were characterised by sequencing (Phillpotts et al., 2005).

    To prepare virulent virus stocks, suckling mice were infected intracerebrally of each supplied virus. Infected brains were harvested, prepared as tissue suspensions and clarified by centrifugation. The titre of each VEEV strain was determined by plaque formation under a carboxymethyl cellulose overlay in Vero cells (Phillpotts et al., 2005).
  • Description: Recombinant defective type 5 adenoviruses, expressing the E3E26K structural genes of VEEV were examined for their ability to protect mice against airborne challenge with virulent virus. After intranasal administration, good protection was achieved against the homologous serogroup 1A/B challenge virus (strain Trinidad donkey). There was less protection against enzootic serogroup II and III viruses, indicating that inclusion of more than one E3E26K sequence in a putative vaccine may be necessary. These studies confirm the potential of recombinant adenoviruses as vaccine vectors for VEEV and will inform the development of a live replicating adenovirus-based VEEV vaccine, deliverable by a mucosal route and suitable for use in humans (Phillpotts et al., 2005).
Host Response

Mouse Response

  • Host Strain: Balb/c
  • Vaccination Protocol: Balb/c mice, 6–8 weeks old (Charles River Laboratories, UK) were immunised intranasally under halothane anaesthesia on days 0, 7 and 21 with 107.0 pfu of each recombinant adenovirus in 50 μl PBS.
  • Immune Response: The ability of serum to neutralise VEEV was examined in standard plaque reduction assays. Briefly, dilutions of serum and VEEV suspended in L15MM were mixed and incubated at 4 °C overnight. Residual infectious virus was estimated by plaque assay in Vero cells. A reduction in plaque numbers of equal to or greater than 50% was considered indicative of virus neutralisation.

    Immunisation with RAd/VEEV#3 provides cross-protection against other epizootic and enzootic strains
  • Challenge Protocol: Seven days after the final immunisation, the animals were challenged via the airborne route by exposure for 20 min to a polydisperse aerosol generated by a Collison nebuliser (Phillpotts et al., 1997). Mice were contained loose within a closed box during airborne challenge. The virus dose was calculated by sampling the air in the box and assuming a respiratory minute volume for mice of 1.25 ml/g (Guyton, A.C., 1947). After challenge, mice were observed twice daily for clinical signs of infection (piloerection, hunching, inactivity, excitability and paralysis) by an observer who quantified these and was unaware of the treatment allocations. In accordance with UK Home Office requirements and as previously described, humane endpoints were used (Wright et al., 1998). These experiments therefore record the occurrence of severe disease rather than mortality. Even though it is rare for animals infected with virulent VEEV and showing signs of severe illness to survive, our use of humane endpoints should be considered when interpreting any virus dose expressed here as 50% lethal doses (LD50).
  • Efficacy: Optimal protection within the VEEV IA/B serogroup depends upon sequence homology
  • Description: Balb/c mice, 6-8 weeks old
References
Bett et al., 1995: Bett AJ, Krougliak V, Graham FL. DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309. Virus research. 1995 Nov; 39(1); 75-82. [PubMed: 8607286].
Guyton, A.C., 1947: Guyton, A.C.. Measurement of the respiratory volume of laboratory animals. Am. J. Physiol.. 1947; 150; 10-11.
Jones et al., 1979: Jones N, Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul; 17(3); 683-9. [PubMed: 476833].
Phillpotts et al., 1997: Phillpotts RJ, Brooks TJ, Cox CS. A simple device for the exposure of animals to infectious microorganisms by the airborne route. Epidemiology and infection. 1997 Feb; 118(1); 71-5. [PubMed: 9042037 ].
Phillpotts et al., 2005: Phillpotts RJ, O'brien L, Appleton RE, Carr S, Bennett A. Intranasal immunisation with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus. Vaccine. 2005 Feb 18; 23(13); 1615-23. [PubMed: 15694514].
Wright et al., 1998: Wright AJ, Phillpotts RJ. Humane endpoints are an objective measure of morbidity in Venezuelan encephalomyelitis virus infection of mice. Archives of virology. 1998; 143(6); 1155-62. [PubMed: 9687872 ].