Development of a multivalent vaccine against Respiratory Syncytial Virus

1. Development of a multivalent vaccine against Respiratory Syncytial Virus By Lucious Vaughn Alabama State University Department of Biological Sciences

2. Introduction Human Respiratory Syncytial Virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants and children, with almost everyone having contracted RSV at least once by the age of 2.

4. The science of RSV • Family: Paramyxoviridae Genus: Pneumovirus • Negative sense single strand RNA virus translated into 11 proteins

5. The Goal To design a multivalent vaccine against Respiratory Syncytial Virus • Multivalent- having several sites of attachment for an antibody or antigen. In this case F, M2, & G proteins.

6. The Virus

7. Genomics of RSV

8. The F (fusion) protein • Viral penetration • Syncytium formation • High titers of neutralizing antibodies Syncytia

9. The G (attachment) protein • Aides in the attachment of the virus to the host. • Has epitopes recognized by the host antibody response.

10. The M2 (matrix) protein • M2-1:Transcription elongation factor • M2-2:Regulates viral transcription • Induces CD8 T-cells

11. The F gene Entire length of F gene = omitted on purpose

12. The M2 gene Entire length of M2 gene = omitted on purpose

13. The G gene Entire length of G gene = omitted on purpose

14. Newly designed multivalent gene FM2G Sal I- R.E. digestion end Nco I- R.E. digestion end

15. pET-32a(+) Vector

16. Linear view of pET-32a(+) Vector showing Restriction Enzyme sites Nco I Sal I

17. Insertion and Cloning of multivalent gene with vector

18. Test ligation 1 2 3 4 5 Lane-1: 1kb ladder Lane-2: RFM2G cut with Nco I Lane-3: pET-32 cut with Nco I and Sal I Lane-4: RFM2G cut with Nco I and Sal I Lane-5: 100kb ladder 550 bp Restriction enzyme analysis of multivalent gene on agarose gel

19. The Methods CLONING PROTEIN EXPRESSION PROTEIN PURIFICATION IMMUNIZATION

20. Expression of protein E. coli BL21 cells pET-32 with FM2G E. coli Protein expression

21. SDS-PAGE analysis of multivalent protein 1 2 3 4 5 Lane-1: SDS-Marker Lane-2: Cytoplasmic Extract Lane-3: Soluble Fraction Lane-4: Pellet Lane-5: Purified FM2G protein 38KDa

22. Western blot analysis of the multivalent protein 1 2 3 4 38KDa Lane-1: Pellet Lane-2: Soluble Fraction Lane-3: Cytoplasmic Extract Lane-4: Magic Marker 38KDa

23. Conclusion • Multivalent gene cloned into pET-32a vector • Successful transformation • Successful protein expression • Confirmation analyses identified the created protein • Immunization testing in various specimens is presently ongoing

24. REFERENCES • 1. Collins, P.L., et al., Nucleotide sequences for the gene junctions of human respiratory syncytial virus reveal distinctive features of intergenic structure and gene order. PNAS, 1986. 83: p. 4594-4598. • 2. Domachowske, J.B. and H.F. Rosenberg, Respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment. Clin. Micro. Rev., 1999. 12(2): p. 298-309. • 3. Hacking, D. and J. Hull., Respiratory syncytial virus- Viral biology and the host response. Journal of infection., 2002. 45: p. 18-24.