The highly conserved stem domain of hemagglutinin (HA) protein presents an attractive target for vaccine designers. Unlike the head domain, which varies from year to year, the stem domain remains relatively constant. If it could be targeted by a flu vaccine, it might remain vulnerable over multiple seasons, removing the need for annual vaccinations.

A vaccine that effectively targets the HA stem, however, has proven very difficult to produce. Several researchers developed constructs consisting of portions of the HA stem domain, but these investigators were unable to produce properly folded stem trimer. Another investigation, initiated at Stanford, has shown more promise. Scientists representing Stanford’s departments of chemical engineering and bioengineering have reported developing a simple procedure that results in high yields of HA stem trimer. This trimer, the scientists assert, is recognized identically by a panel of neutralizing antibodies as compared with recognition of the full-length HA ectodomain.

The procedure is described in the Proceedings of the National Academy of Sciences, in a paper entitled “Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.” The researchers made use of a section of DNA that contained the instructions for making the protein structure for one important strain of flu, the H1N1 virus that caused the pandemic of 1918 and recurred in milder form in 2009. After beginning with the DNA sequence that defines the entire HA protein, both head and stem, the researchers subtracted the DNA coding for the head. Thus, they isolated a DNA strand that contained only the instructions for making the protein stem. At this point, the Stanford team used a relatively new and experimental process to manufacture the viral stem. This process is called cell-free protein synthesis (CFPS).

In CFPS, scientists bust open bacterial cells to create a molecular goop that contains multiple ribosomes to which scientists may transmit their DNA instructions. The advantage of CFPS is that it can produce proteins in a few hours versus a couple of weeks or even a couple of months, which is how long it takes to make proteins for flu vaccines using the practices that are approved for medical use today.

The Stanford researchers used this CFPS process to create and refine a viral protein stem that would be useful as an experimental vaccine antigen. But first the scientists needed to overcome a number of challenges, including the need to weave monomers into a trimer, as well as finding a way to solubilize their bioengineered antigen, which was initially insoluble.

To produce the correctly folded trimeric HA stem domain protein, the authors developed a refolding method. In their paper, the authors wrote, “We suggest this stepwise procedure may provide a valuable paradigm for producing properly folded multimeric domains of membrane-associated cell and viral surface proteins. To reduce the aggregation and the formation of incorrect intermolecular disulfide bonds, several newly exposed hydrophobic residues were replaced, the isoelectric points of newly exposed protein fragments were decreased, and the number of cysteines in the HA stem domain was reduced from eight to four. The HA stem trimer was further stabilized by introducing intermolecular disulfide bonds between foldon monomers and between stem domain monomers.”

“This has been a tough process,” said James R. Swartz, D.Sc., study leader and James H. Clark Professor in Stanford’s school of engineering. “Many labs have been trying to develop an HA stem vaccine, and we’re glad to have made these contributions.”

Many steps remain before the research community knows whether this viral stem approach yields a better flu vaccine. Next, Dr. Swartz and his team will attach their stem protein to a virus-like particle. The idea will be to create a bigger, better target with which to elicit an immune system response.

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