Bench Press

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)

As Ben mentioned on Monday, we at Bench Press were disappointed that we were unable to attend the 2009 Science Online London Conference, but we were glad to see the amazing coverage within the blogosphere. One of the panels I was interested in was the first breakout session titled “What is a scientific paper?”. A discussion on the essence of scientific papers could be incredibly enlightening about steps needed to modernize scientific communication and publishing.

Having read through various notes and posts about the panel I have to agree with Cameron Neylon that while the panel’s discussion on methods to modernize papers themselves had some interesting ideas, a key issue with papers was glossed over; their continued publication in antiquated vessels known as journals. As Cameron Neylon writes:

The journal used to play an important role in publication. The publisher still has an important role but we need to step outside the notion of the journal and present different types of content and objects in the best way for that set of objects. The journal as brand may still have a role to play although I think that is increasingly going to be important only at the very top of the market. The idea of the journal is both constraining our thinking about how best to publish different types of research object and distorting the way we do and communicate science. Data publication should be optimized for access to and discoverability of data, software publication should make the software available and useable. Neither are particularly helped by putting “papers” in “journals”. They are helped by creating stable, appropriate publication mechanisms, with appropriate review mechanisms, making them citeable and making them valued. The point at which our response to needing to publish things stops being “well we’d better create a journal for that” then we might just have made it into the 21st century.

Cameron argues effectively that the journal, as used today, does little if anything to optimize access and discovery of data thereby constraining scientific communication and handicapping scientific progress.

While the panel may not have addressed this key issue thoroughly enough, I was happy to see that PLoS has taken a first step to address the limitations of the traditional journal with their new project PLoS Currents: Influenza. As described in their FAQ PLoS Currents: Influenza is

a website for immediate, open communication and discussion of new scientific data, analyses, and ideas in a critical research area. Submissions are screened by a group of leading researchers in the field, and those deemed appropriate are posted immediately and publicly archived at the National Center for Biotechnology Information (NCBI). All content is open access, available under the terms of the Creative Commons Attribution License.

The explicit goal of this project is to provide easy and efficient access to data on Influenza, in hopes that scientific discussion and breakthroughs can be made in a more timely manner. While PLoS still differentiates Currents from Journals, their step out of the typical scientific publishing space with this project is commendable and looks like a great first step at expanding scientific publication. Hopefully this experiment works out and other publishers will begin to experiment as well.

For more coverage on PLoS Currents: Influenza, Bora Zivkovic has a great introductory post.