human respiratory syncytial virus (rsv) is the most common cause of bronchiolitis and pneumonia...
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Development of a multivalent vaccine against Respiratory Syncytial Virus
By Lucious Vaughn
Alabama State UniversityDepartment of Biological Sciences
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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.
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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%
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The science of RSV
• Family: Paramyxoviridae
Genus: Pneumovirus
• Negative sense single strand RNA virus translated into 11 proteins
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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.
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The Virus
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Genomics of RSV
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The F (fusion) protein
• Viral penetration • Syncytium formation• High titers of neutralizing antibodies
Syncytia
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The G (attachment) protein
• Aides in the attachment of the virus to the host.
• Has epitopes recognized by the host antibody response.
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The M2 (matrix) protein
• M2-1:Transcription elongation factor
• M2-2:Regulates viral transcription
• Induces CD8 T-cells
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The F gene Entire length of F gene = omitted on purpose
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The M2 geneEntire length of M2 gene = omitted on purpose
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The G geneEntire length of G gene = omitted on purpose
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Newly designed multivalent gene FM2GSal I- R.E. digestion end Nco I- R.E. digestion end
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pET-32a(+) Vector
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Linear view of pET-32a(+) Vector showing Restriction Enzyme sites
Sal I Nco I
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Insertion and Cloning of multivalent gene with vector
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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
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The Methods
CLONING
PROTEIN EXPRESSION
PROTEIN PURIFICATION
IMMUNIZATION
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Expression of protein
E. coli BL21 cells pET-32 with FM2G
E. c
oli
Protein expression
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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
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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
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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
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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.