Clever —

Virus engineered to rely on artificial amino acids, used as vaccine

You make the virus in cells with a weird amino acid, then use it as a vaccine.

Flu viruses showing the protein coat that surrounds their genetic material.
Flu viruses showing the protein coat that surrounds their genetic material.

Synthetic biology has become a catch-all term for attempts to engineer organisms to do things they normally wouldn't. Efforts so far have ranged from assembling logic circuits inside bacteria to replacing an entire bacterial genome with one synthesized from scratch. So far, however, the field has largely produced some extremely impressive proofs-of-concept. There haven't been a lot of advances with obvious practical uses.

That may be about to change. Researchers have taken a technique that's been used a number of times before—engineering cells to use an artificial amino acid—and applied it to make a flu virus that acts as a vaccine. The vaccine is highly effective and, because it depends on an amino acid our cells don't use, it can't cause infections in us. Best yet, if the vaccine gets into cells with a normal flu virus, it interferes with its ability to generate an infection.

All of our proteins are made with different combinations of the same 20 amino acids. While many additional amino acids exist, those 20 appear to be the standard toolkit that all life shares. There are a few exceptional organisms that use a 21st, but these oddball amino acids are usually close chemical relatives of existing ones.

Revising the genetic code

In recent years, researchers have figured out how to get cells to use some of those additional amino acids, which can be chemically distinct from the existing 20. These artificial amino acids open up the possibility of proteins with some dramatically different chemistry, ones that can catalyze different reactions or interact with chemicals that life normally doesn't deal with. So far, the results have been mostly potential.

A team from Peking University, however, has been working on a different use for artificial amino acids: creating viruses that don't work in normal cells. The idea is to turn these semi-artificial viruses into a vaccine.

The logic here is extremely clever. Many vaccines just contain one or a few proteins from an infectious agent. But these aren't always effective, since they lack the complexity and context of an intact virus or bacteria. The same can be true for a vaccine made from a deactivated virus. The alternative, to use a weakened virus, runs the risk that people with a weak immune system may experience a full infection.

How can artificial amino acids help with this? Well, if you make a virus that depends on them, then it can only reproduce in cells that will provide that amino acid. Since that would exclude all of our cells, using this virus as a vaccine poses no risk of creating an infection. To the immune system, it should look a lot like a normal virus, so it should be an effective vaccine.

Rather than focusing on an artificial amino acid that has a radically different chemistry, the people behind the new work decided to use one that's a close chemical relative to an existing one. And to make things convenient, they picked one that's already used by a microbe. This meant that all the genes needed to put the amino acid into proteins existed; they just had to be pulled out of the microbe and put into cells that a flu virus could infect.

(The artificial amino acid in question is Nϵ-2-azidoethyloxycarbonyl-L-lysine. It's closely related to the normal amino acid lysine, and chemically look similar to another normal amino acid. Technically, it's not artificial since it's naturally used by some microbes. But it's artificial in human cells, so we'll keep using that term.)

The system is also effective at making sure any genes that use the artificial amino acid won't work without it. The three-base code for the artificial one is UAG. Humans and most other organisms interpret that code as telling the cell to stop making the protein. Thus, any gene with UAG in the middle will be made into a protein normally by cells with the microbial genes, but will be stopped early and produce a severely truncated version in normal cells.

The genes from the microbe were placed into a human kidney cell line. Tests with a fluorescent protein indicated that, as long as it was supplied in the media used to feed these cells, the artificial amino acid would be incorporated into proteins.

Engineering the virus

The system was tested next using a flu virus. A single amino acid code in one of its genes was altered to UAG. When this version of the virus was placed in normal cells, they didn't produce any functional virus, because translation of that gene was terminated early. But when placed in the kidney cells that carried the microbial system, virus was produced normally. The resulting virus could infect other cells, but if those didn't have the microbial system too, the infection stopped there.

That is, for the most part. Mutations occur at a steady rate, and some of these changed the UAG so that it coded for some other amino acid. If that change didn't inactivate the virus, then it could infect normal cells again. The researchers saw precisely this happening: at a low frequency, normally infective viruses emerged during their tests.

The authors went back and tested 21 other different sites that they changed to UAG, targeting any amino acid that looked chemically similar to the artificial one. Some of these disabled the virus entirely; the artificial replacement was close, but not close enough. But seven of the changes produced a viable virus. And several of these could be combined, making the gene very resistant to this sort of evolutionary change. Gradually, they engineered UAGs into genes on every one of the eight different RNA segments that make up the flu virus.

This virus would grow just fine in the kidney cells engineered to carry the microbial system, and the resulting virus could infect other cells. But unless the cells that it infected also carried the microbial system, then the virus stalled there. No mature viruses were ever produced. And because so many different mutations would be needed to return the virus to its original state, the virus remained dependent upon the microbial genes to reproduce.

This was true when they tested it in animals. While a specific amount of normal virus would kill half the mice it was injected into, they found they could inject 100,000 times as much of the engineered virus and there wouldn't be any indication of any health issues. The mice, however, mounted a robust immune response against the virus, one that was broader than the one generated against a normal flu vaccine. The virus also worked as a vaccine in ferrets and guinea pigs.

The authors also tested what happened if cells were infected both with the engineered virus and a normal flu virus. It turned out the engineered version suppressed the infection of the normal one. Remember where we mentioned that the flu virus has a genome made from eight distinct RNA molecules? In cells infected with both viruses, the progeny were a mixture of segments taken from both sources at random. Thus, the vast majority of viruses produced contained at least one engineered segment and couldn't go on to successfully infect normal cells.

A lot of the promise of synthetic biology seems a bit hand-wavy—we can probably do something useful with this at some point. This has included the use of artificial amino acids. Yes, they could potentially expand life's chemistry, but it wasn't clear that doing so would allow us to do things that the normal amino acids couldn't. Still, this is clearly a case where the artificial ones are central to the biotechnology, and the applications are obvious.

Science, 2016. DOI: 10.1126/science.aah5869  (About DOIs).

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Channel Ars Technica