Development of a multivalent vaccine against Respiratory Syncytial Virus
By Lucious Vaughn
Alabama State UniversityDepartment of Biological Sciences
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.
12.50%9.70%
8.90%
8.90%11.40%
4.30%
44.30%
Viruses That Cause Respiratory Illness
Influenza A or B, 12.5%
Parainfluenza, 9.7%
RSV, 8.90%
Metapneumovirus, 8.9%
Other, 11.4%
Adenovirus, 4.3%
Entero/Rhinovirus, 44.3%
The science of RSV
• Family: Paramyxoviridae Genus: Pneumovirus
• Negative sense single strand RNA virus translated into 11 proteins
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.
The Virus
Genomics of RSV
The F (fusion) protein
• Viral penetration • Syncytium formation• High titers of neutralizing antibodies
Syncytia
The G (attachment) protein
• Aides in the attachment of the virus to the host.
• Has epitopes recognized by the host antibody response.
The M2 (matrix) protein
• M2-1:Transcription elongation factor
• M2-2:Regulates viral transcription
• Induces CD8 T-cells
The F gene Entire length of F gene = omitted on purpose
The M2 geneEntire length of M2 gene = omitted on purpose
The G geneEntire length of G gene = omitted on purpose
Newly designed multivalent gene FM2GSal I- R.E. digestion end Nco I- R.E. digestion end
pET-32a(+) Vector
Linear view of pET-32a(+) Vector showing Restriction Enzyme sites
Sal I Nco I
Insertion and Cloning of multivalent gene with vector
Test ligation
550 bp
Restriction enzyme analysis of multivalent gene on agarose gel
Lane-1: 1kb ladderLane-2: RFM2G cut with Nco ILane-3: pET-32 cut with Nco I and Sal ILane-4: RFM2G cut with Nco I and Sal ILane-5: 100kb ladder
1 2 3 4 5
The Methods
CLONING
PROTEIN EXPRESSION
PROTEIN PURIFICATION
IMMUNIZATION
Expression of protein
E. coli BL21 cells pET-32 with FM2G
E. c
oli
Protein expression
SDS-PAGE analysis of multivalent protein
Lane-1: SDS-MarkerLane-2: Cytoplasmic ExtractLane-3: Soluble FractionLane-4: PelletLane-5: Purified FM2G protein
38KDa
1 2 3 4 5
Western blot analysis of the multivalent protein
Lane-1: PelletLane-2: Soluble FractionLane-3: Cytoplasmic Extract Lane-4: Magic Marker
1 2 3 4
38KDa
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
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.