development of a multivalent vaccine against respiratory syncytial virus

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.