Catholic Medical Quarterly Volune 70(4) November 2020
COVID19 Vaccines: Cell lines – Their history and basic information.
Fr Michael Jarmulowicz
This paper, stimulated by ethical concerns relating to use of aborted tissue in production of vaccines, has been produced to help non-scientists understand some of the science surrounding the use of cell lines in medicine but particularly in relation to vaccine production.
Scientists have been trying to grow animal tissue in the laboratory since the late 1800s. Major advances were made in the 1940s and 1950s, with a significant amount of that research been driven by the need to understand viruses. Bacteria will quite happily grow and reproduce outside the body if the environment and nutrients are appropriate. Viruses, however, cannot; they have to invade a living cell. The virus once inside the cell then takes over its metabolic processes to make numerous copies of itself. The cell eventually dies releasing the newly made virus particles.
Scientists were also interested in understanding cancers, and attempting to grow these in the laboratory. Most attempts failed with the cancer cells surviving only a few days in the laboratory. However, in 1951 a surgeon removed part of a cancer from a 31 year old woman named Henrietta Lakes. (She died later that year) Part of it was given to a research scientist and this cancer not only survived but grew prolifically. This was the first successful cell line to be grown in the laboratory; it was labelled using the initials of the donor, Henrietta Lakes, (who did not know the tissue was being used in this way) and so known as HeLa cells. They are immortal and have been widely studied, with two Nobel prizes (2008 & 2009) being awarded, one in virology and the other in genetics, and even sent into space to study effects of zero gravity on cell growth! The HeLa cells were used in the testing and development of the first polio (Salk) vaccine. HeLa cells stick to the walls of the container they are growing in, forming a monolayer of cells. In other cell lines the individual cells stay freely suspended in the culture medium. As a microbiology lab technician in the 1970s I used these cells for isolation of viruses from diagnostic patient samples.
The problem with cancer cell lines (and there are now many) is that they are not ‘normal’ cells. So scientists were looking to grow and study normal cells in the laboratory, starting with animal and subsequently human cells.
Adult Stem Cells
Skin is constantly replacing itself, and if cut we heal. The bone marrow is constantly producing new blood cells. Therefore, we have stem cells within us, which can divide and then differentiate to form the mature skin, kidney or blood cell. Our stem cells are tissue specific. Stem cells in the bone marrow will only produce blood cells; similarly, skin stem cells only skin. A bone marrow transplant essentially harvests the bone marrow, containing stem cells. After injection into the recipient some will lodge in the bone marrow and start producing new blood cells. However, adult stem cells are difficult to find, as they are low in number, and equally do not proliferate well in laboratory conditions. Not all organs have stem cells to replace damaged mature cells, and these include heart muscle and nerve cells.
Fetal Stem Cells / Cell Lines
A fetus also has tissue-specific stem cells, but as the fetus is very actively growing stem cells are more numerous and prolifically dividing. Therefore, scientists started using abortions as sources of fetal stem cells.
Two cell lines relevant to the earlier vaccine debate around the MMR vaccine are WI-38 (Wistar Institute - USA) and MRC-5 (Medical Research Council - UK); both used in production of the MMR vaccine. WI-38 was developed in July 1962 and derived from a 3 month aborted female fetus. MRC-5 was developed from a 14 week aborted male fetus in 1966. (Previously vaccines had been grown on monkey kidney cells, but a significant number are contaminated with a virus (SV-40 – Simian Virus) which lives harmlessly in monkeys.) Both WI-38 and MRC-5 are derived from lung tissue. They can be successfully deep frozen with excellent growth on thawing. The problem with these cell lines is that they are not immortal. After about 50 population doublings, they become senescent. The genetics of this is now understood, and that is why we all die – our cells are genetically programmed not to carry on dividing for ever! The frozen stock of these cell lines is dwindling.
Embryonic cells lines
Within 4-5 days of fertilisation, the single fertilised egg has grown into a hollow ball of cells with a fluid-filled centre, and a small cluster of about 30 cells attached to the inner wall of this cyst (called the blastocyst). The outer layer of cells form the placenta and amniotic membranes, and the small cluster of cells on the inside (the inner cell mass) form the future embryo/fetus/baby.
These cells will develop into every tissue type in the body, and so are called totipotent, or pluripotent. In a suitable culture fluid, these cells will proliferate immortally, still retaining their original characteristics. However, subtle changes in the environment (and sometimes randomly) the cells will start differentiating into one or other tissue type. Creating embryonic cell lines is easier said than done; the majority of attempts fail. All are derived from “left-over” embryos from the IVF industry. At no stage do embryonic cell lines start developing into a proper embryo / fetus.
The reasons scientists want to study embryonic cells, is to understand tissue differentiation, and also understand how/why things go wrong to form disease issues. Furthermore, there is the hope that these cell lines might be used to provide tissue to transplant into adults in organs where there are no stem cells to replace damaged cells – e.g. heart and nerve cells. A good summary of stem cells and their scientific potential can be found on the USA National Institute for Health website.
There are 484 registered stable embryonic cell lines in the US NIH registry, of which 70 carry a disease gene eg muscular dystrophy.
My understanding is that no embryonic cell lines are used in vaccine research/production.
HEK293 and PER.C6
Both are fetal cell lines developed by the same team in the Netherlands and derived from abortions. Both are currently being used in the development of COVID vaccines.
HEK293 was derived from a female fetus aborted in 1972, with the cell line being developed in 1973. HEK stands for ‘Human Embryonic Kidney’ and the 293 is the 293rd experiment to produce this cell line. It is actually a misnomer, as it is not from embryonic kidney, but from fetal kidney, albeit there is some suggestion from the cell characteristics that it is neuronal stem cell of possible adrenal origin. The cell line has been immortalised by inserting a gene from a virus. It was developed specifically for research purposes and is widely used, second only to HeLa cells, in a wide range of biological research.
It is not a ‘normal’ human cell, as it contains 64 chromosomes (hypotriploid), rather than the normal 46 (diploid). It proliferates very rapidly, and its main advantage is that various genes can very easily (with almost 100% efficiency) be inserted into its DNA. It will then produce the relevant protein or even virus that has been inserted into it; hence its use in vaccine work.
PER.C6 was derived from the retinal cells of an 18 week male aborted foetus carried out in 1985, although this cell line was not established until 1995. It was specifically designed for vaccine production, but subsequently found to have characteristics making it suitable for a much wider use. Like HEK293 it has been immortalised through insertion of a viral gene.
Before a vaccine is approved for use it has to go through a rigorous three phase clinical trial. The candidate vaccine is initially given to animals to test its safety and also its efficacy. If it is deemed safe and efficacious in animals then human trials can start. In phase 1 it is given to a small number of volunteers, initially in a tiny dose, but gradually increasing the dose, with close monitoring of any adverse effects. This is to demonstrate safety. At the same time tests are seen to check whether it produces an appropriate immune response. Phase 2 is given to a larger number of healthy adults, again checking for any adverse effects and also demonstration that it produces an appropriate antibody response. Some of the volunteers are given a placebo, rather than the vaccine. This is a standard “double blind” approach to clinical trials, where neither the volunteer or administering doctor knows what has been given, and so it minimises any bias in interpreting any findings. On completion of satisfactory phase 2 trials, it is then given to a much larger group of volunteers (thousands) in a double-blind fashion. Half will receive the vaccine, the other half a placebo. They are then closely monitored to see how many develop the infection. Hopefully in the vaccinated group, none will get the disease, and in the unvaccinated group the number of infections should be that of the background population. Obviously to get valid results, the virus does need to be present in the population. The phase 3 trial is the one that proves its efficacy. As scientists keep stressing, the production of antibodies is not proof that the person is immune from infection (though it is hoped that they are).
Russia has recently approved its vaccine for use, but it has only passed phases 1 and II of the trial, so in reality its issue to the general population will be phase 3 – but presumably without the close monitoring normally expected in a properly conducted double blind trial. In 1976 President Gerald Ford authorised fast tracking of an H1N1 flu vaccine, which sadly resulted in 450 cases of Gullaine-Barrie syndrome, which led to the abandonment of the mass vaccination program.
Oxford Covid Vaccine
The Oxford group vaccine is using a chimpanzee common cold virus, which has been genetically modified so it cannot replicate in humans or cause human disease. Into this modified chimpanzee virus, they further modified the virus by inserting the gene from the COVID-19 virus, which produces the ‘spike protein’ present on the outer wall of the coronavirus. This spike protein binds to a receptor on human cells which then allows the coronavirus to be taken up into the cell and start proliferating. It is only this single gene from the coronavirus that is used in this vaccine.
This modified virus is then grown in the HEK293 (and not sure if PER.C6) cell line to produce the actual vaccine. This vaccine was rapidly developed, and has completed phase I and II trials, with results from 1077 volunteers, published in the Lancet on 20th July 2020. The Phase III trial is just starting, with recruitment of 10,000 UK volunteers. In addition, the trial will be also be done with volunteers in South Africa, Brazil and USA. At the time of writing the trial has been paused while a case of possible adverse reaction to the vaccine is investigated. Such pauses are normal while potential reactions are investigated. This is the second pause in the trial. The first pause after a suspected reaction was diagnosed as multiple sclerosis. The media are reporting that the second case has transverse myelitis, but this has been declared as incorrect by Astra Zeneca as the diagnostic investigations are not complete.
Imperial College Covid Vaccine
The Imperial group are using a completely novel approach to this vaccine. This method has been extensively tested in animals, but is the first time it is being tried on humans. The gene encoding the spike protein of the coronavirus is incorporated into tiny fat particles. This is then injected into a muscle, and the fat particles fuse with the cell membranes of the skeletal muscle, and the gene taken into the cell. There it self amplifies itself (this is not the whole virus or its genetic code) and also produces the spike protein which the muscle cell then exhibits on its surface. The body’s immune system recognises this as foreign and produces the immune response. No fetal cells are used, and so there is no association with any abortions. The proposed benefit of this technique is the large scale of production which can be achieved, so millions of doses of vaccine (if efficacious) can be very quickly produced, and so useful in pandemics.
In June the first 15 volunteers were injected with the new vaccine. In mid-July a further 105 volunteers started being given one of three different doses of the vaccine, to be followed by a booster four weeks later. So this vaccine is much less advanced in its clinical trials than the Oxford vaccine.
Fr Michael Jarmulowicz. BSc, MBBS, BDiv. KSG
consultant histopathologist, now priest.
10th September 2020
- Significant Research Advances Enabled by HeLa Cells. https://osp.od.nih.gov/scientific-sharing/hela-cells-timeline/
- Characteristics of this cell line and origin described in https://www.lgcstandards-atcc.org/products/all/ccl-75.aspx?geo_country=gb#generalinformation
- Stem cell basics: https://stemcells.nih.gov/info/basics.htm
- NIH Human Embryonic Stem Cell Registry eligible for use in NIH-funded research. https://grants.nih.gov/stem_cells/registry/current.htm
- See https://www.hek293.com/ for details of this cell line. See also https://en.wikipedia.org/wiki/HEK_293_cells
- See https://www.nature.com/articles/ncomms5767
- See https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(02)00045-X
- Details of the vaccine given to trial volunteers https://covid19vaccinetrial.co.uk/files/uclhlocalisedcov002pisages70yearsandoverv1005aug2020pdf
- Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31604-4/fulltext
All internet addresses accessed and verified as correct 3/11/20