Transcript Development of a recombinant multivalent vaccine against

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.
Viruses That Cause Respiratory Illness
Influenza A or B, 12.5%
12.50%
Parainfluenza, 9.7%
9.70%
44.30%
RSV, 8.90%
8.90%
Metapneumovirus, 8.9%
8.90%
4.30%
11.40%
Other, 11.4%
Adenovirus, 4.3%
Entero/Rhinovirus, 44.3%
• Family: Paramyxoviridae
Genus: Pneumovirus
• Negative sense single strand RNA virus
translated into 11 proteins
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.
• Viral penetration
• Syncytium formation
• High titers of neutralizing antibodies
Syncytia
• Aides in the attachment of the virus to the
host.
• Has epitopes recognized by the host
antibody response.
• M2-1:Transcription elongation factor
• M2-2:Regulates viral transcription
• Induces CD8 T-cells
Entire length of F gene = omitted on purpose
Entire length of M2 gene = omitted on purpose
Entire length of G gene = omitted on purpose
Sal I- R.E. digestion end
Nco I- R.E. digestion end
Sal I
Nco I
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
Restriction enzyme analysis of multivalent
gene on agarose gel
CLONING
PROTEIN EXPRESSION
PROTEIN PURIFICATION
IMMUNIZATION
E. coli BL21 cells
pET-32 with FM2G
Protein expression
1
Lane-1: SDS-Marker
Lane-2: Cytoplasmic Extract
Lane-3: Soluble Fraction
Lane-4: Pellet
Lane-5: Purified FM2G protein
38KDa
2
3
4
5
1
2
38KDa
Lane-1: Pellet
Lane-2: Soluble Fraction
Lane-3: Cytoplasmic Extract
Lane-4: Magic Marker
3
4
• 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
• 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 virusViral biology and the host response. Journal of
infection., 2002. 45: p. 18-24.