3. 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.

5. Family: Paramyxoviridae Genus: Pneumovirus Negative sense single strand RNA virus translated into 11 proteins

6. 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.

9. Viral penetration Syncytium formation High titers of neutralizing antibodies Syncytia

10. Aides in the attachment of the virus to the host. Has epitopes recognized by the host antibody response.

11. M2-1:Transcription elongation factor M2-2:Regulates viral transcription Induces CD8 T-cells

12. Entire length of F gene = omitted on purpose

13. Entire length of M2 gene = omitted on purpose

14. Entire length of G gene = omitted on purpose

15. Sal I- R.E. digestion endNco I- R.E. digestion end

17. Sal I Nco I

19. Restriction enzyme analysis of multivalent gene on agarose gel 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 1 2 3 4 5

20. CLONING PROTEIN EXPRESSION PROTEIN PURIFICATION IMMUNIZATION

21. E. coli BL21 cells pET-32 with FM2G Protein expression

22. Lane-1: SDS-Marker Lane-2: Cytoplasmic Extract Lane-3: Soluble Fraction Lane-4: Pellet Lane-5: Purified FM2G protein 38KDa 1 2 3 4 5

23. Lane-1: Pellet Lane-2: Soluble Fraction Lane-3: Cytoplasmic Extract Lane-4: Magic Marker 1 2 3 4 38KDa

24. 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

25. 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.