Alphavirus as Vaccine-vectors
The use of alphavirus as a vector vaccine has become an attractive technology for the generation of novel and improved vaccines. Expression vectors have now been engineered from at least three alphaviruses in which the structural protein gene region has been replaced by a heterologous gene and have been shown to express high levels of heterologous proteins in a broad-spectrum host of many mammalian and insect cells. Due to its good properties, alphavirus replicon vectors are being developed as platform vaccine technologies for many viruses, bacteria, protozoa, and tumor antigens.
Introduction of Alphavirus
Alphaviruses are enveloped viruses carrying a single-stranded RNA genome of positive polarity, belonging to the family of Togaviridae. The alphavirus targets a wide range of host animal cells via common surface receptors such as heparin and laminin and then fuses to the cell membrane to enter the cell. Genomic RNA is replicated in the cytoplasm and subgenomic RNA is used as a template for direct expression of viral capsids and membrane proteins. The next full-length RNA molecule is incorporated into the capsid protein to produce a nucleocapsid that is transported to the cell surface. At the same time, the membrane proteins travel through Golgi and endoplasmatic reticulum before embedding nucleocapsids and resulting in budding of mature viral particles.
Alphavirus as Vaccine-vectors
Alphaviruses contain a single strand RNA genome that can be easily modified to express heterologous genes at very high levels in a broad variety of cells, including tumor cells. Alphavirus vectors can also be used as viral particles containing a packaged vector RNA, or directly as nucleic acids in the form of RNA or DNA. In the latter case alphavirus RNA is cloned within a DNA vector downstream of a eukaryotic promoter. The alphaviruses Semliki Forest virus (SFV), Venezuelan equine encephalitis (VEE) and Sindbis virus (SIN) have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles.
The Design for Alphavirus as Vaccine-vectors
Several types of vector systems have been engineered. Alphavirus vectors have been used for vaccine development as naked RNA, recombinant viral particles, and layered DNA plasmids (Fig 2). The application of naked RNA vectors involves the use of in vitro transcribed RNA from an expression vector which consists of viral non-structural genes and the exogenous gene of interest downstream of a strong subgenomic promoter. Generation of recombinant particles requires co-transfection of in vitro transcribed RNA from an expression vector (as described above) and a helper vector that provides viral structural genes into mammalian cell lines (eg, baby hamster kidney (BHK) cells). The resulting particles are capable of performing a round of infection on a range of host cells. The layered DNA vector system includes a DNA delivery vector that providing foreign gene expression under a CMV promoter. In addition, engineering of vectors with additional subgenomic promoters for full-length genomes allows for the production of replication-proficient particles that can provide improved delivery and extended gene expression.
In addition to the standard vectors designed for each expression system, Creative Biolabs has also developed some improved vectors and expression systems. For example, point mutations in non-structural genes of SFV and SIN expression vectors reduce the cytotoxicity of host cells and enhance the expression levels of recombinant proteins. A single point mutation in the SIN nsP2 gene results in persistent infection of the transduced host cells. In addition, the use of engineered expression vectors based on non-structural genes from avirulent SFV A7 (74) strains resulted in reduced cytotoxicity in mammalian cell lines and primary neurons compared to conventional SFV vectors. Moreover, 5-10 fold enhanced expression has been achieved from SFV vectors containing translational enhancement signals from capsid proteins.
Fig 2 Alphavirus vector systems for vaccine delivery. (A) Naked RNA vector in vitro transcribed from plasmid DNA. (B) Replication-deficient alphavirus particles are produced in baby hamster kidney (BHK) cells after co-transfection of in vitro transcribed RNA from an expression vector and a helper vector. (C) A layered DNA vector for plasmid immunization. SP6, polymerase promoter; 26S, subgenomic alphavirus promoter; CMV, cytomegalovirus promoter; polyA tail, polyadenylation signal. (Kenneth. 2014)
Advantages of Alphavirus as Vaccine-vectors
- Easy to operate and mass production
- High-level expression of recombinant protein
- Can transduce multiple mammalian cells
- Reasonable production costs and process stability
With years of experience and advanced vaccine development platform, Creative Biolabs provides different types of alphavirus vectors according to different customers’ needs. If you are interested in any of the alphavirus vectors, please feel free to contact us.
Reference
- Kenneth Lundstrom. (2014). Alphavirus-Based Vaccines. Viruses, 6(6): 2392-2415.
All of our products can only be used for research purposes. These vaccine ingredients CANNOT be used directly on humans or animals.
