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Influenza viruses are negative-stranded, enveloped orthomyxoviruses that contain eight gene segments encoding various viral proteins. During the viral life cycle point mutations drive genetic drift responsible for seasonal influenza epidemics. Novel influenza viruses can also be produced through genetic reassortment between viruses. These viruses can result in devastating pandemics as they expose their host populations to novel antigenicity. These alterations in the influenza viruses’ genomes require that vaccine strains be updated annually to account for changes in virus populations.

Currently two types of vaccines are utilized to combat seasonal flu, each with their own limitations:

1. Chemically inactivated virus delivered via injection – mainly acts by inducing antibody response rather than cellular immunity and has suboptimal efficacy in elderly patients.
2. Live attenuated influenza virus vaccine of cold-adapted virus delivered via nasal spray – induces both humoral and cellular immunity, but is restricted to healthy children, adolescents, and adults, performing better in immunologically naive young children than adults.

So despite the relative effectiveness of the current offerings there is definitely room for improvement. That’s where research being done by Dr. Steffen Mueller and colleagues at Stony Brook University comes in. Building on prior research on producing synthetically attenuated polio viruses, Dr. Mueller’s group utilized a technique dubbed Synthetic Attenuated Virus Engineering (SAVE) to generate synthetic influenza virus vaccine candidates. They detail their work in a paper in Nature Biotechnology titled Live attenuated influenza virus vaccines by computer-aided rational design.

What’s truly unique about SAVE is that it only uses silent mutations within the viral genome to produce live attenuated virus. As described in the paper:

The central idea of SAVE is to recode and synthesize a viral genome in a way that perfectly preserves the WT amino acid sequence, while rearranging existing synonymous codons to create a suboptimal arrangement of pairs of codons. For reasons that are not understood, some pairs of codons occur more frequently, and others less frequently, than expected. … Although the mechanism of attenuation is unclear, preliminary evidence suggests that translation is affected. Attenuation can be ‘titrated’ by adjusting the extent of codon-pair deoptimization. Because codon-pair deoptimization results from miniscule effects at each of hundreds or thousands of nucleotide mutations (without changing amino acid sequences), reversion to virulence is extremely unlikely. Aided by computer algorithms, codon pair–deoptimized viral genomes can be rapidly designed and synthesized, and live virus can be generated by reverse genetics.

Figure 1

Utilizing SAVE Dr. Meuller’s group generated influenza strains with the following proteins synthetically deoptimized, polymerase subunit B1 (PB1), nucleoprotein (NP), and hemagglutinin (HA). They tested strains with a single deoptimized gene as well as one strain with all three altered genes. First Dr. Meuller’s group analyzed each of their strains in vitro to assess their growth characteristics. They found that all the mutant viruses produced plaques similar to wild type and produced reasonable titers although slightly lower (about tenfold lower). What was most interesting about their in vitro characterization however was their western blot analysis of the synthetically designed proteins. In each mutant it is clear that the gene that underwent SAVE produces significantly less protein in comparison to wild type (PR8) (Figure 1). The effect is specific and lends support to the idea that codon-pair deoptimization results in an effect on translation.

After checking their in vitro characteristics, Dr. Meuller’s group tested for attenuation by infected mice. They found that despite the reasonably robust viral growth each mutant strain demonstrated an attenuation effect. A strain combining all three modified genes (PR8^3F) resulted in an increase in median LD₅₀ of about 13,000 fold. Further tests on PR8^3F showed effective attenuation of symptoms and viral load in comparison to wild type (Figure 2).

Figure 2

In addition to assessing attenuation of the virus, Dr. Meuller’s group assessed the immune response and protective immunity in detail by immunizing mice and then challenging them 28 days after inoculation. As seen in Figure 3 below (a, b) strain PR8^3F had a much larger range of safe doses in comparison to wild type. Viral load is effectively limited (c) in PR8^3F inoculated animals and antibody response is robust (d) even in very low doses in comparison to LD₅₀ of PR8^3F.

Figure 3

Ultimately, this paper illustrates a really interesting new technology that seems capable of revolutionizing vaccine development. It provides a novel vaccine design strategy that appears to produce robust immune protection. It remains to be seen if SAVE vaccine candidates can address the limitations of current flu vaccines as well as other issues, but this paper is a strong first step for this technology.

(Source – Nature Biotechnology)