Dead water

How the Russian inactivated vaccine against the new coronavirus was made

Vera Mukhina, N+1

Among the Russian coronavirus vaccines, the Chumakov Scientific Center for Research and Development of Immunobiological Drugs of the Russian Academy of Sciences announced that it had completed animal tests and had already received permission from the Ministry of Health for clinical trials. This is the third Russian development, after the NITSEM named after Gamalei and Vector, approved for human testing. N+1 talked to the staff of the Center about the vaccine and its differences from other Russian developments.

Many Russian developers, in particular Vector, the Gamalei Center and BIOCAD, have put on new technologies and are working on vaccines based on viral vectors, DNA and RNA. In the Chumakov Center, they went the traditional way – they made an inactivated vaccine here.

Inactivated vaccines are more than half a century old – so they have been studied far and wide and tested for various diseases. They were encountered by everyone who was vaccinated against rabies or encephalitis. In addition to excellent knowledge, they can boast a moderate immune response and relative ease of development. The Russian encephalitis vaccine has been produced by the Chumakov Center since the 80s - this is its development. Inactivated polio vaccine is also produced here.

"With our [new] vaccine," says Lyubov Kozlovskaya, head of the department of current and newly emerging infections with pandemic potential, recently established at the Center, "I think everything is pretty clear. It was clear that we would have to develop it on what we have. We have a ready-made platform for inactivated vaccines. When you produce a vaccine on an existing platform, on an existing technology, you can assume what the duration of the immune response will be, how quickly the antibody titers will rise and what approximately they will be, what side reactions will be and how they can be avoided."

The vaccine of the same type, according to the World Health Organization, is made by China (several at once, and one vaccine has already entered the third phase of clinical trials), Kazakhstan and India.

What is the difference

In the case of inactivated vaccines, our immune system learns from the "corpses" of pathogens – they can no longer reproduce or infect cells. In addition to safety, the passivity of this design serves as a guarantee that the immune system will not be overexcited. At the same time, she will be able to get acquainted with the pathogen entirely and independently pick up from the viral "corpses" those details-antigens that she likes – researchers do not need to select them specifically and create synthetic designs (as the creators of new-generation vaccines have to do, which include the development of the Gamalei Center and the Novosibirsk Vector).

However, the disadvantages of inactivated vaccines are also associated with the same characteristics – the integrity of the virion and its passivity. They attract the attention of the immune system – and especially T cells – worse than active vector and attenuated vaccines, and therefore its memory is shorter. Therefore, it is necessary to stimulate it with the help of additional stimuli and to be vaccinated more often. Protein vaccines have the same weakness.

In addition, to get coronavirus "corpses" for the vaccine, researchers first need to multiply the virus itself. Working with pathogenic strains is not always convenient and increases safety requirements.

The production scheme of an inactivated vaccine looks simple. It is necessary to accumulate a sufficient amount of the virus, neutralize it, clean it, add "amplifiers" – and after that it is already possible to inject the resulting drug to patients. Nevertheless, this case is full of nuances. Even with considerable experience behind them, creating an inactivated coronavirus vaccine turned out to be a non-trivial task, the developers admit in an interview with N+1.

"In our case, the main problem was just the new virus itself," says Lyubov Kozlovskaya. – There are no regulatory things: which virus, which strain should be taken. The virus appeared three months before we started developing [the vaccine]. It was unclear whether it would be able to grow on the cells that we use? Won't we have to work out everything [cell line, growth parameters] all over again?"

In addition, she continues, there was no coronavirus strain in the center. Scientists had to isolate it from patient samples, sequence it, find out whether it would be suitable for the role of a vaccine strain, whether it is similar to those that circulate at least in Russia. Fortunately, there were no problems with the cell line and the standard one was quite suitable for the growth of the virus.

How to neutralize the virus

After the scientists received the seed material, the initiative passed to the development department, which took up the technology of vaccine production.

To get enough viruses for a vaccine, they need to be multiplied. On their own, they can't do it, so for this they are hooked to cell cultures. They get infected, synthesize the virus in huge quantities and then die, and the viruses go to the production of the vaccine.

In one form or another, this stage is in the production of any virus-based vaccines. For vector vaccines, it is necessary to multiply the basis, for example, an adenovirus, and in the case of inactivated or attenuated vaccines, developers deal with the pathogen itself. But in different industries, the process is very different depending on the virus, goals and technical equipment.

The danger of the virus plays an important role: a fairly harmless virus is taken as the basis for vectors, and the Chumakov center works with real SARS-CoV-2, which according to Russian metrics belongs to the second class of pathogenicity. In particular, this means that the laboratory should be divided into infectious and non-infectious zones, increased requirements for ventilation and insulation of premises, additional PPE and special rules of conduct for employees.

However, the researchers note that employees are in contact with active virions only at the earliest stages of production, when the primary sowing of the virus occurs.

This is done manually, and then the resulting mixture is seeded with a bioreactor – an aggregate with a large disposable container filled with nutrient medium and micro-carrier balls without direct contact. The nutrient medium is needed for Vero cells, which grow on balls and serve as factories for the reproduction of the virus. Bioreactors with micro-carriers are a fairly new technology, thanks to which it turns out to increase the surface area on which cells grow and provide them with a uniform pH, temperature, oxygen access, and nutrients.

Before infecting cells, they need to multiply themselves. A "rocking" bioreactor is suitable for this: cells grow in a disposable bag in the nutrient medium, the platform under it swings and mixes the contents of the package, and the monitoring system monitors the conditions inside and transmits everything necessary through the tubes. As biomass accumulates, they are transplanted into other, larger reactors. In total, this process takes several days. The images are provided by the Center. Chumakov.

Contrary to the fears of scientists, the virus multiplied well on the standard Vero cell line and nothing new had to be invented here. After the cells grow up, they are infected and wait for another 3-4 days, and then the mixture is neutralized without leaving the bioreactor. This is usually done by heating or formaldehyde, but the Chumakov center uses a more progressive beta-propiolactone. Heating can change the conformation of virus proteins and thereby change them beyond recognition for our immune system, whereas beta-propiolactone simply locks nucleic acids inside the virion forever. Compared with formaldehyde (which also affects mainly nucleic acids), beta-propiolactone is faster and more pleasant to work with: it inactivates the virus in just a day (versus two weeks for formaldehyde) and is then neutralized by simple heating to 37 degrees Celsius.

With the help of such systems, researchers concentrate the virus-containing liquid for further purification. This also happens without direct human contact with the virus.

After that, the virions are sent for filtration and chromatographic purification, and the disposable container is destroyed. Additionally, they check how well beta-propiolactone worked: pour the resulting solution over the cells and see if they were infected with inactivated viruses.

In addition to the virus, a standard adjuvant, aluminum hydroxide, was added to the vaccine. By themselves, "dead" viruses are not able to attract the attention of the immune system, and the adjuvant helps them in this: it collects them on itself and stimulates their eating by immune cells.

Check

The regulations of preclinical and clinical trials are spelled out in detail, but even within it, researchers have a certain freedom of action.

"The most creative approach can be shown just when testing immunogenicity," says Kozlovskaya. – You can take those animals that you think will suit you better. All that is regulated is that there should be two types of them. In our case, they were mice and marmoset monkeys. It's a little easier to work with them than with big [primates]. We have our own colony, and this makes the task easier. Well, primates are much closer to humans than any mouse."

First of all, it was necessary to work out the dose and the vaccination scheme – this is what most of the mice went for. The researchers confirmed the effectiveness of the chosen dose on mice of a different haplotype and only after that they began expensive and complex experiments on marmosets – small non-human monkeys native to the New World.

After selecting the immunogenic dose, the vaccine was tested for safety: the general toxicity, the ability to cause fever and allergies were examined both immediately and after a while. Here everything is according to protocol: allergenicity is usually looked at in rabbits, and chronic toxicity – in guinea pigs.

And how does it work?

In all tests, the researchers compared vaccinated animals with control animals that were injected with a placebo instead of a working drug. According to Anastasia Pinyaeva, head of the department of development and implementation of innovative and semi-industrial technologies, there were no side effects in the test group and control groups, but at the same time, antibodies to the coronavirus S-protein appeared in the test animals in "sufficient" titers from the researchers' point of view.

It is not yet possible to compare this "sufficiency": the Chumakov Center has not published test data, as well as the Vector and the Gamalei Center (although both of them have already switched from animal experiments to humans).

How the immune response is usually viewed The immune response in animal experiments is viewed in almost the same way as in human subjects at the first stages of clinical trials: by antibody titers and T-cell response.

In this case, it is pointless to check the effectiveness of the vaccine by infecting animals with the virus – many of them do not suffer from COVID-19 at all and, in general, this is not included in the standard complex of preclinical studies.
In Russia, lines of mice sensitive to SARS-CoV-2 are being created, and some scientists are conducting tests on Syrian hamsters. But there is no well-developed model for such checks yet.

In addition, the researchers emphasize that due to the very novelty and little knowledge of the infection, it is not entirely clear "how many antibodies are needed, when they will be neutralized, how long they should last."

But in order to get a stable immunity for sure, they – like many other developers – decided to spur the immune system with a repeated, "booster" vaccination. "In view of the fact that this is still a pandemic, and we need to reach the maximum titles that we can achieve as soon as possible... we decided that this [will be] a two-time vaccination with a two-week interval. Again, so that it would just be faster," says Lyubov Kozlovskaya. With this scheme, a month after the first vaccination, the antibodies reach a plateau with titers greater than 1 per hundred.

Inactivated vaccines target an antibody response, and the T-cell response is usually minimal. This is due to the fact that inactivated virions do not infect cells and therefore cannot be properly presented to T-lymphocytes. Nevertheless, preliminary results of experiments on mice suggest that animals still form a pool of T-cells, which, upon repeated encounter with the antigen, begin to divide intensively. This means that the vaccine can include not only an antibody, but also a cellular immune response.

What's next?

Some of the animals will be observed for some time, but in general, preclinical trials have been completed. Scientists are starting tests on humans.

In the very first safety tests, it is planned to vaccinate only 15 people. They will be monitored in the hospital for a couple of weeks and if no serious side effects emerge during this time, the scientists will begin extended trials. In total, two hundred people will take part in them, of which 150 will receive a vaccine, and 50 will receive a placebo. Standard parameters will be evaluated: side effects, antibody titers and T-cell response, and this will be a double–blind study - patients and doctors from field laboratories will not know who got which version of the vaccine.

Based on the results of the tests, it is planned to obtain a temporary registration certificate and begin the third phase of the CI in 2021. When asked whether someone will be vaccinated on the basis of temporary registration outside of clinical trials, the Chumakov center replied that they do not exclude the possibility of producing a vaccine not for clinical trials.

Is there enough for everyone?

Compared to other vaccines under development, inactivated ones require more safety oversight. This may affect the price and availability of the vaccine and become an obstacle to the expansion of production: not every room is suitable for working with SARS-CoV-2 and not every company has the equipment and permission for this.

In the Chumakov center, they plan to cope on their own. They have their own well–established production, where, in particular, they work with viruses of the same class of pathogenicity - with the same tick-borne encephalitis virus. According to the researchers, its capacity should be approximately enough for ten million doses of coronavirus vaccine per year, and if necessary, they can be expanded by installing additional bioreactors.

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