After reading Part 1 and Part 2A, you should now understand why we use medium, FBS, and antibiotics to grow cells and that it’s important to passage cells if you want to continue or expand your cell line. We also discussed fibroblast cells and how they are typically used in the lab because they are easy to grow and you can expand your cell line into multiple flasks rapidly. These concepts are absolutely vital in understanding how vaccines are made.
Let us move on to the next big concept in vaccine manufacturing: attenuation of the virus. You may have already heard the term attenuate. For example, the measles vaccine is a live, attenuated vaccine. What does this mean and how does this fit in with our ingredients list?
To attenuate means to weaken; to attenuate a virus means to weaken it. Remember that for an efficacious vaccine, we want to expose people to the germ and activate the immune system without causing illness. In the lab, scientists have figured out how to change the measles virus so it will still activate your immune response, but not actually make you sick. Here’s how:
- Virus attenuation via adaptation into a new host cell. 1. Sample is taken from host. 2. Passaged through chick embryos multiple times. 3. Passaged through chicken embryo fibroblasts multiple times. 4. Attenuated virus is isolated from cells and sequenced.
Wild measles virus is well suited for human cells: once you breathe it in, measles will invade your lung (epithelial) cells, take over its machinery, and reproduce itself. A scientist name Enders first discovered that if you add measles virus to non-human host cells, like chicken egg (embryo) cells, the virus will adapt (aka mutate) to its new host. This is survival of the fittest at its best! Please note that the measles vaccine virus used in MMR is passaged in chicken embryo fibroblast cells and not in eggs.
- Diagram of how measles virus was attenuated. Key: Names within the colored boxes are the names of the measles strains. 1. Measles virus was isolated from a patient with last name Edmonston. 2. The Edmonston strain was serially passaged 24 times in human kidney cells, 28 times in amnion cells, 6 times in chick embryos, and then in chicken embryo fibroblasts to create the Edmonston B strain. The current measles vaccine virus in the U.S. (Attenuvax, Merk) was made by further attenuating the Edmonston B strain 40 times in chicken embryo fibroblasts. We call this live, further attenuated virus the Moraten or Edmonston-Enders strain. The Schwarz strain, made by attenuating the Edmonston A strain, is widely used in vaccines in Europe and other countries around the world.
By repeating the process of adding virus to cells, letting it grow, then isolating the virus and adding it to a new plate of cells, over time you can create a virus that is so well adapted to surviving in its chicken host that it can no longer efficiently reproduce in the human host. We call this process passaging the virus in cells or tissues. Please read our previous article for a nice summary of how we passage cells in the lab. The measles vaccine virus is passaged in chicken embryo fibroblasts while other vaccine viruses, like rubella, is passaged in other host cells like monkey kidneys or human diploid cells (more on this in a bit). Theoretically any cell that is not the typical cell for measles invasion could work–though some viruses will not grow at all in certain hosts. It’s important to know that we passage the virus at least 40 times (or even more depending on the specific vaccine) in chicken embryo cells. This is necessary (and great) because when you give the attenuated (weakened) virus back to the human host, there is only an extremely small chance of it reverting back to the wild virus—each round of passaging created a new batch of mutations that helped the virus survive in the chicken. It is almost impossible for the virus to jump back to the human-disease causing form.
- The rubella virus vaccine strain currently used in the U.S. in the MMR vaccine (Meruvax II) was created by serially passaging rubella virus in WI-38 cells at a low temperature (for viruses). Growing the virus in the diploid cells at low temperature resulted in the production of the avirulent (attenuated) strain RA 27/3. Two other strains were previously used in vaccines in the 1960’s and 70’s (HPV-77 & HPV-77/DEC but were continued due to side effects and because they didn’t provide as much protection as the RA 27/3 strain did.
So now when you see non-human cells listed in the vaccine package insert, you know why: wild virus is passaged in non-human cells many times, forcing it to adapt to the new host and no longer causing disease in the human host.
Common concern: Use of chicken embryo fibroblasts to passage virus Some people have legitimate concerns about vaccines that are prepared in chicken embryos. After isolating the virus from the egg, there still could be a small amount of chicken proteins that some people have an allergic reaction to (i.e. when you get redness and other symptoms from the flu shot). Viruses that are grown in chicken fibroblast cells (not in the egg, just in cells) do not experience an allergic reaction because the virus was never in the whole egg. So even though the measles vaccine is grown in chicken fibroblast cells, there is no reported evidence of having an egg allergy to this. Another concern is the use of chicken embryos in general. Often when people hear the word embryo, they do not realize that we mean a chicken egg. So if you don’t have an issue with eating chicken or egg products, there should also be no issue with this process. However, some people are ethically against the use and/or consumption of animals, period, and thus this is a legit concern to have. Unfortunately, we don’t have a better way to mass produce the amount of virus needed for vaccines and other research purposes. Some researchers are currently working on genetically engineering attenuated viruses and others are working on alternatives to animal testing. But these standard practices are currently the most reliable tools we have.
Common concern: Use of human fetal diploid cell lines to passage virus Let’s get some definitions out of the way. You may see the term ‘human diploid cells’. Diploid refers to the number of copies of chromosomes found in a cell (di = two). If you remember from science class, most cells of the body, besides your sex cells, are diploid. So nothing out of the ordinary there. Second, I’ve observed online that some people are confused about the term “cell line.” A cell line is a cell culture developed from a single cell and therefore consisting of cells with more or less identical genetic makeup. You can propagate some cell lines indefinitely. This is important to understand because we can isolate cells from a tissue once and then culture them for decades–we don’t isolate cells from new tissues over and over again. There are two cell lines that were first created in the 1960’s that are used to make some vaccine viruses and were originally derived from two human fetuses called Wi-38 and MRC-5. Both the Wi-38 and MRC-5 cell lines are composed of fibroblast cells from lung tissue. I’ve observed some confusion online regarding the synthesis of these cell lines: some people think fetuses are sacrificed every time this cell line is used. It’s important to understand that this cell line was made in the 1960’s from two fetuses that were already destined for abortion. The parents agreed to donate the fetuses for research at the time of termination. Others are concerned about the religious implications of using fetus cells in vaccines. First, some people are under the impression that you can find fetus cells in the final vaccine product. This is not true: the virus is isolated from the cells and washed several times. Second, the Vatican has issued a statement several years ago that is it acceptable to use vaccines that were prepared with these cells as it serves for the greater good. They also ask for better methods of vaccine manufacturing that will avoid using these cell lines. Asking for better methods is definitely a great suggestion, just remember that you need to support science funding in order for that to happen! Please read this article for more information about the cell lines used and the religious implications.
Common concern: vaccines contain cells (whether they be human, animal, other)
Please note that viruses are removed and purified from cells before vaccine manufacturing. You will see on some package inserts that residual amounts of cellular products MAY be found in the final product. I am personally not concerned about this after reading Plotkin’s article here and knowing that we consume plant and animal cells at a much larger rate than the miniscule amount found in vaccines. The vaccine product is also extensively tested and scanned for any dangerous contaminants before release.
If you read the previous articles, I hope you can see that production of the vaccine virus is separate from production of the final vaccine product that is injected into you. This means that the ingredients we have discussed so far, including animal and human cell lines, are not found in the final product (except possibly in amounts less than 1 ppm). In the next article, we will get into how the vaccine virus is packaged into the vaccine product and what ingredients are actually found in the injectable form.
Figure 1. Virus Attenuation. Taken from: http://www.nature.com/nm/journal/v14/n2/full/nm1726.html
“Attenuation of measles strain” diagram modified from Plotkin et al. “Vaccines.” (textbook). Philadelphia: Saunders; 2012.
Plotkin et al. “Vaccines.” (textbook). Philadelphia: Saunders; 2012.
Plotkin. “The history of Rubella and Rubella vaccination leading to elimination.” Clin Infect Dis. (2006) 43 (Supplement 3): S164-S168.
Hilleman et al. “Live, Attenuated Rubella-Virus Vaccine.” N Engl J Med 1968; 279:300-303
Please let me know if you spot any inaccuracies in this text at PHUpodcast@gmail.com