Anti-Viral Vaccines

Live Vaccines

• 3. Live recombinant vaccines
• It is possible, using genetic engineering, to introduce a gene coding for an immunogenic protein from one organism into the genome of another (such as vaccinia virus). The organism expressing a foreign gene is called a recombinant. Following injection into the subject, the recombinant organism will replicate and express sufficient amounts of the foreign protein to induce a specific immune response to the protein.
• Attributes
• Good immune response
• Both Cell Mediated Immunity and antibody responses.
• Immunity is long lived
• Single dose
• Safety
• Danger of reversion to virulence, or
• Severe disease in immunocomprised
• Stability
• Organisms in the vaccine must remain viable in order to infect and replicate in the host
• Vaccine preparations are therefore very sensitive to adverse storage conditions
• Maintenance of the cold chain is very important.
• Expense
• Cheap to prepare

Vaccinia Virus

• The most unusual, and perhaps technologically the most useful, feature of poxviruses is their ability to replicate in the infected cell's cytoplasm, and not nucleus. Infectious virions have a lipoprotein envelope surrounding a complex core of linear duplex DNA connected at each end by hairpin loops. Virus encoded enzymes, a multi-subunit DNA-dependent RNA polymerase, a transcription factor, capping and methylating enzymes, and a poly(A) polymerase are all contained within the core. Vaccinia is therefore well equipped to synthesize translatable mRNA.

Vaccinia Virus

• Vaccinia undergoes homologous recombination during replication in infected cells. When used as an expression vector, this innate ability to recombine is used to introduce foreign DNA coupled to a vaccinia promoter. The steps below outline the construction of the vaccinia expression vector.
• (1) The gene (YFG) is flanked with vaccinia DNA sequences, especially the vaccinia promoters and multicloning sites for cleavage and ligation.
• The following are often included:
• The promoters are necessary DNA sequences because the endogenous viral RNA polymerase binds here to initiate transcription. The promoter also determines the direction of translation for the insert, and more importantly the ability to express proteins (depending on how tightly regulated the promoter is).
• In addition, DNA sequences, such as the lacO/lacIq repressor system, that act in conjunction with promoters and also bind repressor molecules can regulate the induction of transcription. Hence, by adding or removing a particular substrate, expression of YFG can be turned on and off as necessary.
• Stabilizing elements such as transcription terminators can also be incorporated downstream of the multicloning site. These anti-termination elements signal the RNA polymerase to release the DNA template and stop transcription, and prevent pausing, pre-mature termination, and overreading which adversely affect plasmid replication.
• Finally, small open reading frames, known as ribosome binding sites, upstream of YFG, can be included to encourage binding and translation of the target sequence.