With the COVID-19 pandemic still spreading across the globe, the fight is on to rapidly develop efficient solutions for diagnosis and treatment. In the past, cell-free protein expression has been used to produce antibodies against other strains of coronavirus. As such, could cell-free systems pose a viable option towards finding a solution against SARS-CoV-2 ?
Throughout recent years, there has been promising research using cell-free systems to produce antibodies for either diagnostic tests or neutralizing treatments. For coronaviruses, this has been successfully done before. In particular, previous studies have been able to produce coronavirus antigens and, consequently, antibodies against them . Therefore, there is reason to believe that it can be done again for the current or future outbreaks.
The two major steps in producing an antibody of this type are (a) producing the target antigen and (b) then, generating, selecting and producing a specific antibody against that antigen. Both of which require expression of a highly specific protein in its native form. Hence, it must be correctly folded, without aggregation and expressed at a sufficient yield. Cell-free protein synthesis offers a reliable solution. The technique is known for its ability to produce proteins that are particularly “difficult” to express in their native form using traditional in vivo systems.
In 2016, researchers in Japan successfully developed antibodies to precisely diagnose a strain of coronavirus known as MERS-CoV . MERS-CoV is a virus first identified back in 2012, which causes an illness known as Middle East Respiratory Syndrome. Whilst the antibodies they produced would not attack the virus in COVID-19, their study does show how the cell-free procedure can generate very specific antibodies which can be used in the search for a solution.
Choosing the target
The first step in making antibodies is to produce an antigen. A key part of the breakthrough in the 2016 study was taking the structure of MERS-CoV into account. As with many coronaviruses, MERS-CoV is comprised of four main proteins. Most importantly, the spike (S) proteins, which give coronaviruses their characteristic prickly surface, are heavily targeted by host antibodies. This is thought to drive them to have a high mutation rate, so unlikely to be winners when it comes to making a specific diagnostic test or a vaccine.
On the other hand, the nucleocapsid (N) protein, which is present in high levels in infected cells, is more conserved. Since N-proteins are less likely to change over time, these antigens make better targets for specific diagnostic tests. The team therefore chose this sequence to synthesize as a protein against which they could then raise antibodies.
Producing the antigen
The N-protein that the team had selected is a viral structural protein, which tend to be insoluble. That means traditional systems of protein expression are often unsuccessful in producing the molecule in its native form. This is problematic. If the protein is not correctly folded, the antibodies produced further down the line will not detect the correct sequence.
In cell-free, proteins are produced in a test tube using intra-cellular machinery from cellular extract. The viruses, which are in many cases cytotoxic, cannot kill the host cell so expression rates are much higher than in vivo systems. Therefore, the authors opted for a cell-free protein expression system. In the end, the Japanese team successfully obtained N-protein that was not only properly folded, but also in quantities similar to what can be expected for human cells.
Making the antibody
Once the protein was produced and isolated, it was injected into mice to obtain antibodies against it. The success was from the specificity of the antibody that was produced. As they were required for use in a diagnostic test, the monoclonal antibody binding site needed to be specific to the MERS-CoV virus. Their results showed that it could specifically recognize the desired N-protein, meaning that it could be used to distinguish this coronavirus from other coronaviruses.
Using a cell-free system the team in this study produced a high-quality native protein, specific enough to effectively produce antibodies. It is also worth considering that other studies have shown that, once sequenced, the antibody synthesis can be done using a cell-free system, too [2,3].
There is already research being carried out elsewhere at the moment using cell-free protein synthesis to produce neutralizing antibodies in the hopes of finding a way to efficiently fight against COVID-19 . In this particular case, using a cell-free system also enabled a “game-changing” rapid upscale of production, reducing manufacturing time from nine months to only one . And, during a pandemic, every second counts.
Whilst we hope that the research will be fruitful, the need to raise antibodies for research or clinical purposes goes beyond that of COVID-19. Cell-free systems provide a highly promising option for future study. Hence, the technique could be used to produce coronavirus N-protein for a future a therapeutic solution, diagnostic tests; or even to quickly develop and produce a vaccine.
 Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen
 Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates