Antibodies and passive immunization

Vaccines and their origins

We all know vaccines, and the vast majority of people have been vaccinated several times throughout their lives, starting with the childhood vaccination program. In such programs, many receive their first vaccine when they are 3 months old.

The world’s first vaccine was developed by Edward Jenner in 1796 against smallpox. Today, smallpox is the only disease that has been eradicated worldwide due to vaccine programs. Since the first smallpox vaccine, vaccines have been developed against many different types of diseases. One way to make a vaccine, is using a less virulent (infectious) type of the bacteria/virus you want to vaccinate against, as is the case with the yellow fever vaccine. You can also inactivate the virus/bacteria you want to vaccinate against and then use it directly as the vaccine. This is the case for Hepatitis A vaccination. In the past few years, new types of vaccines have also emerged on the market. Johnson & Johnson’s COVID-19 vaccine is based on DNA that encodes protein from the COVID-19 virus. After vaccination, your cells absorb the DNA and then produce the coronavirus proteins themselves. The same principle, but using mRNA instead of DNA, is used in Moderna and Pfizer vaccines. You can learn more about vaccines here and specifically about mRNA vaccines here.

The 1796 smallpox vaccine consisted of live viruses that were similar enough to smallpox to lead to immunity, but without the same virulence (infectiousness). Edward Jenner’s idea for the vaccine came after he observed that milkmaids were not contracting smallpox. He hypothesized that milk maids close association with cows gave them immunity to the disease, because the cows had a similar disease, cowpox He therefore took inflammed tissue from cows with cowpox and placed it in scratches on humans and found that this protected the humans from contracting smallpox.

Regardless of the type of vaccine used, they all work by activating the immune system of the receiver. When the inside of the body is exposed to something non-human (a virus, a bacterium), it is recognizes as foreign, which activates the immune system. The body’s immune system starts producing antibodies that can specifically bind and neutralize the foreign proteins/viruses/bacteria they encounter. You can learn more about the body’s immune system here.

Snake bite antivenom – a type of vaccination

Antivenoms are the only specific treatment for snakebites today, and although many people don’t realize it, antivenoms have a surprising similarity with vaccines. Antivenoms were first invented by Albert Calmette in 1895. The principle of the treatment is similar to the principle behind vaccines, in which you immunize using a less dangerous version of a virus/bacteria. However, there are also major differences between traditional vaccines and snakebite treatment. People who are at high risk of being bitten by snakes are not vaccinated. Instead, a production animal (usually a horse) is ‘vaccinated’ and the animal’s antibodies are then used to treat humans (Figure 16) who have been bitten.

There are many reasons why people are not vaccinated against snake bites. First of all, there are hundreds of venomous snake species, all of which have unique venom compositions. This would make it very difficult to develop a vaccine that covered enough snake species. In addition, it is hard for the immune system to make antibodies against snake venom toxins (snake toxins generally have low immunogenicity). Snake venom is also, as the word implies, venomous, which means that you can only inject very limited amounts of venom to prevent the toxins in the venom from exerting their toxic effects. In total, it is simply too complicated to vaccinate all people who are at risk of being bitten by snakes against all the snake venoms they can potentially encounter. It would be a year-long process with many injections. Despite all of the above, there are still a few people (try searching for Steve Ludwin or Tim Friede on Google) who inject venom on a weekly basis to build up tolerance to certain snake venoms.

The current treatment for snakebites is still somewhat similar to vaccines – just not as a vaccination of the people who have been bitten or are at risk of being bitten. Instead, larger production animals such as horses and sheep are vaccinated (Figure 16). In fact, this process is called immunization instead of vaccination. In practice, this works by ‘milking’ venom from the snakes you want to create an antivenom against and then injecting low doses of these venoms into the production animal. You start with very low doses, which are gradually increased over a period of up to 1.5 years. The reason you can increase the dose through the process is that animal’s immune system is “learning” to fight the toxins, during the process. By increasing the dose slowly, you may actually end up being able to inject doses so high that they would have killed the animal, if you had started at that dose. The way the immune system develops this tolerance is that the B-cells of the white blood cells, start producing antibodies that can bind to the snake toxins thereby neutralizing the toxic effects. Once immunization is complete, blood is drawn from the production animal, isolating the white blood cells while returning the red blood cells back into the animal. From the white blood cells, the antibodies can be isolated relatively easily. It is these antibodies that are the active ingridient in the treatment of snake bites; antivenom. Despite the fact that antivenom have saved thousands of lives since their invention, there are many ways in which antivenom can be improved. More of these will be discussed later.

Antibodies

Antibodies is an intriguing class of molecules. The term ‘antibodies’ covers a number of different molecules. IgG (immunoglobulin G) is the most known molecule in this class. Antibodies are part of the human immune system, more specifically, the adaptive immune system. The adaptive immune system can fight previously unseen threats (such as infections) throughout our lives, and constantly adapts to new pathogens. As mentioned earlier, the antibodies are produced by a immune cells termed B cells. Antibodies are highly specific towards the target molecule (e.g. a toxin) they are made for. In other words, an antibody only works against one specific threat. If you are infected with a virus/bacteria for a second time, antibodies are very helpful. They are the reason why you usually only get sick the first time you are infected with a specific disease, such as the flu or coronavirus. In the vast majority of cases, you only get sick the second time you are infected with a specific virus/bacteria, in cases where the bacteria or virus that infects you has changed so radically that the B-cell antibodies can no longer recognize it.

Antibodies are protein-based molecules that can recognize and bind other specific molecules. They are Y-shaped, binding to the target molecule (e.g. a toxin) with the “end of the two arms of the Y” (Figure 17).

How do antibodies work?

Antibodies have several different effects in the body and different ways to fight foreign intruders. The simplest method is that antibodies can bind molecules or organisms in the body, preventing them from exerting their effects. For example, in the case of an infectious disease, an antibody can specifically bind to a protein on the surface of a virus that prevents the virus from infecting the host. Another example comes from snake bites, where the antibodies in the antivenom work by binding the toxins in the snake venom, preventing them from exerting their effects. For enzymatic toxins, the antibody binds to the toxin in a way that disturbs the active site, preventing the toxin from binding to its substrate and thereby carrying out the enzymatic process that the enzyme catalyzes. For toxins without enzymatic activity, such as neurotoxins, antibodies can neutralize the toxin’s effect by binding the toxin on the site where the toxin interacts with the receptor where it exerts its toxic effects. It also means that not all antibodies that can bind to a toxin necessarily neutralize the effect of the toxin. It’s easy to imagine antibodies that bind to toxins without preventing the toxin from carrying out its toxic effect. These types of antibodies are called non-neutralizing antibodies, whereas antibodies that prevent toxins from exerting their effects are called neutralizing antibodies (Figure 18).

Antibodies can also work in ways other than preventing molecules from exerting their effects. Antibodies are a natural part of the human immune system and therefore there is also an interaction between the cells of the immune system and the antibodies. For example, antibodies can be used to ‘recruit’ immune system cells. When many antibodies bind to a bacterium, for example, it signals the cells of the immune system to come and ‘eat’ the bacterium. In this way, the antibodies guide the immune system towards threats in the body.

Antibody discovery – Hybridoma technology and phage display

As described, antibodies are an important group of proteins produced by the immune system. In addition to the antibodies we naturally develop in the body, antibodies can also be used as medication to treat a number of diseases such as cancer and autoimmune diseases. The total worldwide market for antibody-based medicine in 2020 was $125,000,000,000,000 USD – that’s a LOT of money! The development of such treatments requires standardized methods for the discovery of these therapeutic antibodies. Therapeutic antibodies are developed as monoclonal antibodies, meaning they originate from the same B-cell and thus recognize only one specific antigen or a defined group of antigens. In the following section, we will describe two methods used for antibody discovery; hybridoma technology and phage display. These two technologies are very different and have their own advantages and disadvantages.

Hybridoma technology

Hybridoma technology (Figure 19) was invented in 1975 and was the first method for the discovery of monoclonal antibodies. The method consists of immunizing an animal (usually a mouse) with the molecule/organism you want to find antibodies against (also called an antigen). After immunizing the mouse with your antigen (similar to immunizing horses and sheep to produce an antivenom), you isolate the B cells from the mouse. Each B-cell produces a unique antibody. Out of all these B cells, each producing a unique antibody, a proportion will recognize the antigen you have immunized the mouse with, while the remaining will recognize other antigens that the mouse has developed antibodies against throughout its life. Once you have isolated these antibody-producing B cells, you can grow them in a lab to see which antibodies best recognize your antigen. However, these B cells have a limited lifespan, like most other cells in humans and mice. Therefore, in order to work with and analyze the antibodies produced by the B cells for an unlimited period of time, the B cells are fused with special cancer cells (also known as myeloma cells) that can live forever. The process of merging is called cell fusion. When a myeloma cell (the cancer cell) and a B-cell are fused, the fused cells are called hybridoma cells and they have characteristics of both the B-cell and the myeloma cell. Hybridoma cells thereby retain the antibody-producing function of B cells and the immortality of myeloma cells. Using these cells, you can examine the different antibodies produced by the different cells without time constraints and eventually select an antibody with the exact properties you want for your therapeutic antibody.

Phage display

The phage display technology was invented after the hybridoma technology in 1985 and the inventors received the 2018 Nobel Prize in Chemistry for this discovery. The technology is very different from hybridoma technology, for example, it does not require the use of animals. The technology is based on a coupling between antibodies and bacteriophages (also known as phages). Bacteriophages are a type of virus that can only infect bacteria.

Antibodies and phages are linked inserting a gene encoding an antibody into the phage’s DNA. The Antibody-DNA is inserted as an extension of a gene encoding the phage’s surface proteins, such that the antibody protrudes from the surface of the phage (Figure 20). This method of combining DNA from two different organisms is called recombinant DNA technology. This creates a phage that carries the specific antibody gene and at the same time displays this antibody on its outer surface. Instead of doing this with just one antibody, you create what we call a library, a collection of billions of phages, each carrying a unique antibody on their surface. The antibody genes used to create these libraries often come from people who donate a blood sample from which the antibody-coding genes can be isolated from the B-cells.

Once you have created one of these subject display libraries, you can start finding relevant antibodies for a specific target. The process is called phage display selection and is used to identify the antibodies that recognize the antigen you want to find antibodies against (Figure 21). This can be done by attaching the antigen to the bottom of a specially designed plastic tube and then adding your phage display library. Once the antibodies have had time to bind to the antigen, the antibodies that didn’t recognize the antigen are washed away. This process is repeated 2-3 times until you have accumulated a pool of antibodies, all of which can bind to your antigen. The combination of the antibody on the surface of the phage (phenotype) and the gene that codes for antibodies in the phage’s DNA (genotype) makes it easy to find the ‘recipe’ for the best antibodies afterwards (using DNA sequencing). This ‘recipe’ can then be cloned into cells (bacteria, yeast or mammalian cells), which can then produce the antibodies.

Working questions

1. Why are horses and sheep most commonly used to produce antivenom? Why not larger animals, elephants or giraffes? Why not similar animals like pigs or cows?

2. Explain why blood is separated into red blood cells and plasma after centrifugation

3. Which cells are fused in hybridoma technology and for what purpose?

4. Which antibody discovery method would you choose if you were tasked with discovering an antibody against a toxin that was incredibly dangerous to mice even at very low concentrations. Reason.