Unlike drugs, virtually all vaccines need to be transported at cold temperatures (usually between 2 and 8 degrees Celsius) prior to use. If they are exposed to higher temperatures, many vaccines lose potency. Re-cooling does not help. Thus we need what is called the cold chain of handling before use. It would therefore be extremely useful if we can make and transport a vaccine at room temperature, with no cold chain needed.
Warm vaccine
It is towards such a “warm vaccine” that an Indian group, led by Raghavan Varadarajan of the Indian Institute of Science, Bengaluru (IISc) and other collaborators at IISER Trivandrum, THSTI Faridabad, and the IISc-incubated startup Mynvax have worked. Their preprint, entitled, “Design of a thermo-tolerant, immunogenic SARS-CoV-2 Spike fragment,” is publicly available in Biorxiv at https://doi.org/10.1101/2020.08.18.252437.
Active domain
The COVID virus has, on its surface, a protein called the spike which is about 1,300-amino-acids long. Within this, a sequence that is about 250 amino acids long, called the receptor binding domain (RBD), attaches itself to the “host” cell surface and starts the process of infection. The group decided to synthesise in the laboratory, not the full length spike protein, but a 200-amino-acid fragment of the RBD, and study its structure, (three-dimensional architecture, or the shape which allows it to fit snugly to the host cell surface — a “lock and key” fit), thermal stability (can it work at temperatures higher than the normal lab conditions), and so forth. Happily, the authors found that this fragment, when freeze dried (or “lyophilized”), is highly stable, can withstand transient exposure to temperatures as high as 100 degrees Celsius and can be stored for over a month at 37 degrees Celsius, suggesting a cold-chain should not be required for this molecule.
Clues from structure
On an aside, for the past 70 years, India has been a pioneer in the field of how the structure or architecture of a protein offers clues to its function. It still is. For example how the triple helical structure of collagen, experimentally discovered by the late G. N. Ramachandran in 1954, explains why it is found in the skin and tendons, offering them rope-like strength. Prof. Ramachandran also suggested, given a protein sequence, how we may predict the ways it may fold into a 3-dimensional architecture. This, in turn, has led biochemists to synthesise proteins with changes in their amino acid sequence that function the way they want it to.
The authors have done exactly this by carefully choosing the sequence of the RBD fragment to be expressed, and demonstrating that the resulting protein is thermo-tolerant. This exemplifies the power of protein structural analysis and “genetic engineering”.
The RBD protein was produced in large amounts in mammalian cells as well as in a yeast called Pichia pastoris, which is a highly cost-effective, inexpensive, host. However, when they compared the two proteins, they found that the yeast-expressed protein was more heterogeneous and did not yield the desired antibodies when tested in animals. They also tried expressing the identical RBD fragment in the usual model bacterial system, E.coli, but the protein was non-functional.
Adjuvants and trials
Now that we have a thermo-tolerant RBD, can it be tried as a vaccine candidate, generating antibodies that will block the receptor-binding motif of the spike protein and prevent infection? Generally, immunologists add what is called an adjuvant which, when co-injected along with the vaccine material (cells or molecules), stimulates the immune system and enhances the ability of the vaccine to work. Aluminium salts are often used.
The authors chose to use guinea pigs in their initial immunisations, since these are thought to be better models than mice for respiratory illnesses. As adjuvant, they used a generic version of the MF59 adjuvant which has an excellent safety record in humans, and they injected the RBD formulation in guinea pigs. After two doses, tests showed substantial levels of the desired receptor-blocking antibodies in the animals. So, it works.
They point out that numerous other groups have used the entire full length spike protein, or new RNA-based approaches to express antigens, including the RBD. However, unlike all the COVID-19 vaccine formulations currently in clinical trials which require a cold chain, this particular thermo-tolerant RBD fragment, (and possibly other RBD formulations), can be stored at room temperature for extended periods.
The researchers are now testing the ability of the formulation to protect animals from challenge with infectious virus and will simultaneously carry out safety and toxicity assessments prior to testing in human clinical trials. We wish the group all success.
dbala@lvpei.org
