How do tiny antibodies take on the responsibility of protecting us?

Author: Zhang Yuyi (Shanghai Pasteur Institute, Chinese Academy of Sciences)

The article comes from the Science Academy official account (ID: kexuedayuan)

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You must have heard a lot about "antibodies produced in patients who have recovered from COVID-19", but do you really know what antibodies are? How do they protect us?

Image source: veer gallery

Where do antibodies come from?

Before introducing antibodies, let’s first take a look at the human immune system.

Schematic diagram of the three major lines of defense of the human body

(Image source: http://tushuo.jk51.com/)

The human immune system is mainly composed of three lines of defense. The first line of defense is the skin and mucous membranes, such as our epidermis, nasal hairs in the nostrils, and mucous membranes in the mouth, which can block and remove most foreign matter. The second line of defense is the bactericidal substances and phagocytes in body fluids. Bactericidal substances can destroy the cell walls of bacteria and kill them, and phagocytes can engulf and eliminate various pathogens that invade the human body.

The human body's first and second lines of defense are innate. They are a natural, broad protective force that is not targeted at a specific pathogen and is therefore called nonspecific immunity or innate immunity (if our body is likened to a city, then the first and second lines of defense are the city walls and moats).

The third line of defense of the immune system is the acquired defense function that is gradually established after birth under the stimulation of specific antigens and works only against the antigen. It is mainly composed of various immune organs (such as tonsils, lymph nodes, thymus, bone marrow and spleen, etc.) and immune cells (lymphocytes, monocytes/macrophages, granulocytes, mast cells) with the help of blood circulation and lymphatic circulation. It is also called specific immunity or adaptive immunity (police in the city). Antibodies are produced by specific immune cells.

The criminals in the body that do bad things are antigens (Ag), which refer to any substance that can induce the body's immune response, such as bacteria, viruses, pollen, etc. When some criminals crossed the moat and climbed over the wall, they complacently thought they had successfully sneaked into the city, but they didn't expect that they had already been targeted by several different mysterious organizations.

The members of these mysterious organizations are well-trained. They know that they are weak and cannot deal with the vicious criminals alone, so they quietly track and block them, and promptly contact the city police to eliminate the criminals. These mysterious organizations are antigen-presenting cells (APCs).

Antigen presenting cells process antigens and present them to specific immune cells (Image source: Wikipedia)

Antigen presenting cells are also called accessory cells. They can absorb, process and transmit antigen information. After a series of signal transmission processes, they stimulate B cells to differentiate into effector B cells and secrete antibodies. Therefore, antibodies are the corresponding protective proteins produced by the body after experiencing these antigen stimulations. They are a high-performance, multi-purpose and powerful weapon "invented" by activated B cells to eliminate specific antigens.

How do antibodies protect us?

An antibody is a protein with a "Y"-shaped structure, and its function is closely related to its structure.

Schematic diagram of antibody structure: The two branches (Fab segments) in the upper part of the "Y"-shaped structure are the key to the antibody's ability to bind to specific antigen epitopes on the surface of the antigen, while the lower rod (Fc segment) is the key to binding to many immune cells and exerting their functions (Image source: China Pharmaceutical University @ Wang Hui teacher courseware)

If that’s not vivid enough, you can imagine it like this:

The explosion-proof fork invented to stop violence (Photo source: Arthub artist official website @沈敬东 public account: 3720艺)

Antibodies work mainly in the following ways:

1. Neutralization effect:

Antibodies directly bind to antigens through the Fab segment, binding to proteins on the surface of the antigen, making these proteins unable to bind to receptors on cells, thereby preventing pathogens from entering cells.

For example, after the novel coronavirus enters the body, the spike glycoprotein (S protein) on its surface needs to bind to the ACE2 receptor (the corresponding receptor of the S protein) on the cell surface before it can enter the cell. Antibodies first bind to the S protein, and after occupying these positions, the virus can no longer use the S protein to invade the cell.

Schematic diagram of antibodies binding to S protein, where the red Y-shaped structure represents the antibody (Image source: Zhihu @跑来跑去的马)

2. Agglutination:

When some pathogens with granular structures coexist with corresponding antibodies, the antibodies will bind to the pathogens like a "bridge", causing multiple pathogens to gather together to form larger clumps, making it easier for phagocytes to engulf the pathogens.

Neutralization (2a, 2b) and agglutination (2c) of antibodies (A) on antigens (B) (Image source: Wikipedia)

3.Regulating effect:

The Fc segment of some antibodies can bind to the corresponding receptors (FcR) on the surface of neutrophils and macrophages, enhancing their ability to phagocytize pathogens.

The Fc segment (D) of the antibody binds to the corresponding receptor (E) on the surface of specific immune cells (C) and promotes phagocytosis (right picture) (Image source: Wikipedia)

Let me add a comment: In daily life, many people are allergic to pollen, dust, etc. This is because the body's immune system regards pollen and other substances as antigens. When immune cells first come into contact with these antigens, they produce an immune response to them, produce a lot of specific antibodies, and the body enters a sensitive state. When these immune cells come into contact with the same antigen again, the antibodies will quickly bind to the antigen, and the other end of the antibody will bind to the surface of mast cells, inducing mast cells to produce a certain reaction and release some substances such as histamine, causing smooth muscle contraction, increased mucus secretion, and allergic symptoms such as asthma.

The mechanism of allergy production, in which the blue Y-shaped structure represents the antibody, the small red balls on it represent the antigen, and the large purple balls represent mast cells. After the antibody binds to the antigen, it binds to the mast cell, inducing it to degranulate and release the substance inside the cell (Image source: Zhihu @跑来跑去的马)

4. Antibody-dependent cell-mediated cytotoxicity:

After the pathogen successfully infects a cell, it will "colonize" the cell and use its resources and production tools to create new viruses (during the production process, some viral coat proteins will be displayed on the cell surface and become recognizable antigens). The corresponding antibody binds to the viral antigen on the surface of the host cell through its Fab segment, and then binds to the Fc receptor expressed on the surface of killer cells (such as NK cells) through the Fc segment, allowing these killer cells to kill the virus-infected cells and eliminate the virus at the same time.

5. Complement activation:

Complement is another special weapon for killing pathogens and plays many important roles in the immune process. With the help of antibodies, the immune system can activate and call upon these weapons.

6. Through the placenta and mucous membrane:

Antibodies can be divided into five types according to their heavy chains: IgA, IgD, IgE, IgG, and IgM. Each type of antibody has different functions. Among them, IgG can enter the baby's blood through the placenta, providing the baby with natural passive immunity. Therefore, although the baby is fragile, the mother's antibodies can protect them. And IgA in immunoglobulin can pass through the mucosa and is the main factor in local mucosal resistance to infection.

Maternal immunity: passive immunity acquired by newborn animals from their mothers (Image source: medicalxpress.com)

In summary, the main ways in which antibodies work are: their Fab segment can recognize and bind to pathogens to make them lose their ability to infect, and the Fc segment can bind to Fc receptors on other immune cells to work with the help of immune cells.

Plasma therapy to cure Ebola

Pathogens invade the body and stimulate the body to produce an immune response. Effector B cells secrete antibodies, which flow through the blood to various parts of the body to help the body defend and eliminate pathogens. After the pathogens are eliminated, the antibodies in the blood do not disappear immediately, but continue to exist for a period of time to consolidate the defense line (after all, the weapons have been manufactured, and there is no need to destroy them immediately even if the war is over).

The yellow slurry obtained by extracting blood from patients who have recovered from a disease and centrifuging to remove blood cells is plasma, which contains a large number of comprehensive antibodies against the disease. After inactivation and other treatments, the plasma can be used to treat patients with the disease. This method is different from conventional vaccination (the purpose of the vaccine is to help the body develop immunity against a disease, that is, active immunity) and is called passive immunity.

Plasma products (Image source: https://www.science.org.au/)

Plasma products were first used to treat diseases in the early 20th century, when scientists used animal models to study the protective and therapeutic effects of plasma from people who had recovered from infectious diseases. The therapy was first used on humans in 1916, when plasma from polio survivors was used to treat acute polio patients, achieving good results. Subsequently, the use of plasma from recovered patients to treat infectious diseases was used in succession for a variety of diseases, such as SARS 17 years ago, MERS a few years ago, and Ebola, which was prevalent in Africa. Convalescent plasma was used for treatment, and it is currently the only way to cure Ebola.

Antiserum containing antibodies (antiserum is serum containing antibodies, which has less protein than plasma) (Image source: https://vmrd.com/)

The initial immune response caused by the new antigen has a 10-day incubation period. After the incubation period, low-affinity IgM antibodies are produced first, followed by low-affinity IgG antibodies, reaching a peak on the 21st day. Only when the same pathogen invades again and the body responds a second time will high-affinity IgG antibodies be produced within 3 to 5 days.

Therefore, the best time to use convalescent plasma therapy is within 10 days of onset, when the body has not yet produced IgG antibodies. By injecting high-affinity, high-concentration IgG antibodies into critically ill patients, passive immunity can be produced to protect the body, thereby preventing the occurrence of late immune factor storms.

Some people may question whether plasma therapy is so effective, so why is it not used on a large scale? For example, can't it be used in large quantities in the treatment of COVID-19?

Since plasma comes from the blood of different people, it may contain other unknown pathogens, and there may be a possibility of cross infection when it is transfused into other patients. In addition, the manufacturing of blood products requires high standards and complicated processes, and there are tens of thousands of patients with new coronary pneumonia, so it is difficult to cover a large area. Therefore, for non-critical patients, symptomatic treatment is still the first choice.

How to make artificial antibodies?

In addition to the antibodies we produce ourselves, there are many situations in medicine and research that require "artificial antibodies". The following introduces the classic methods of antibody preparation and the prospects of using genetic engineering to create new antibodies.

Antigen epitope is a feature of the antigen surface and is the site where antibodies bind. A single antigen can often provide more than one antigen epitope. Antibodies are divided into two types: monoclonal antibodies (mAb) and polyclonal antibodies (pAb) based on the number of antigen epitopes that they can recognize. Although monoclonal antibodies only recognize a single antigen epitope, they have better specificity and directionality; polyclonal antibodies can recognize multiple antigen epitopes (to some extent, they can be understood as a mixture of multiple monoclonal antibodies that recognize different epitopes of a certain antigen), and can better recognize antigens (when used for antigen detection).

Preparation of polyclonal antibodies (Image source: https://courses.lumenlearning.com/)

The preparation method of polyclonal antibodies is relatively simple, which simulates the process of inducing the body to produce immune responses and secrete antibodies after antigens enter the animal body under natural conditions. The main process includes: preparing antigens - selecting experimental animals - animal immunization - taking blood for testing (to verify whether the corresponding antibodies are successfully produced in the animal body) - killing experimental animals and collecting serum - purifying antibodies - identifying the purity and specificity of antibodies, etc.

The preparation process of monoclonal antibodies (Image source: https://www.researchgate.net/)

The preparation of monoclonal antibodies is relatively more technically difficult. After preparing antigens to immunize animals, a single B lymphocyte that produces antibodies is fused with bone marrow tumor cells to obtain hybrid cells that can produce antibodies and proliferate indefinitely, and then produce antibodies. The fused cells are screened and identified by specific methods to obtain positive clones, and then cell culture is performed or the cells are injected into the abdominal cavity of animals (usually mice) for culture, and the cell culture supernatant or mouse ascites is collected and purified to obtain monoclonal antibodies.

These two are very classic antibody preparation methods. The antibodies obtained are from the corresponding experimental animals (non-human), and have very obvious disadvantages in clinical application: antibodies from different species of animals are also "antigens" of the human body, so the antibodies themselves cause the body's immune response. After repeated use, the human body may produce corresponding antibodies (antibodies against this non-human antibody, not the antibodies against specific antigens that we hope to obtain).

Therefore, researchers are currently working hard to humanize antibodies using genetic engineering - replacing animal-derived components with as many human components as possible without affecting the function of the antibody. In addition, in order to expand the use of antibodies, researchers are currently developing new methods, such as antibody multi-target modification and antibody-drug conjugation (antibodies carry drugs to specific target locations to exert their effects).

Catumaxomab (trade name Removab), the first dual-target antibody drug to be marketed (Image source: https://www.helmholtz.de)

Summary——Take home message!

Antibodies are proteins in nature, produced by effector B cells, and are very important members of the immune system. Starting from the fetal period, they (mainly IgG) can protect our health through the placenta. After we grow up and come into contact with a variety of antigens, the corresponding antibodies will be secreted throughout the body to protect our safety. Many vaccines we inject are also designed to produce antibodies against known pathogens through immune responses. (How to develop a new coronavirus vaccine?)

Antibodies can also be used to treat a variety of diseases. When the body encounters a pathogen that its own immune system cannot defeat, it can use the plasma/serum containing the corresponding antibodies from other people or other animals for passive immunization, such as anti-snake venom serum and plasma from patients who have recovered from COVID-19. Antibody therapy for tumors is an important direction of antibody drug therapy. For example, PD-1/PD-L1 antibody drugs have become a star in cancer treatment.

Researchers can now use genetic engineering technology to modify antibodies to make them safer and more effective, and there are more and more examples of using antibodies to treat cancer in clinical practice.

Our own immune system is magical and powerful, and researchers are working all the time to make antibodies more effective.

References:

Flexner S, Lewis PA. EXPERIMENTAL EPIDEMIC POLIOMYELITIS IN MONKEYS. J Exp Med. 1910;12(2):227-55

Cheng Y, Wong R, Soo YO, et al. Use of convalescent plasma therapy in SARS patients in Hong Kong. Eur J Clin Microbiol Infect Dis. 2005;24(1):44-6

Arabi Y, Balkhy H, Hajeer AH, et al. Feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with Middle East respiratory syndrome coronavirus infection: a study protocol. Springerplus. 2015

Kraft CS, Hewlett AL, Koepsell S, et al. The Use of TKM-100802 and Convalescent Plasma in 2 Patients With Ebola Virus Disease in the United States. Clin Infect Dis. 2015;61(4):496-502

Rhoades RA, Pflanzer RG (2002). Human Physiology (5th ed.). Thomson Learning. p. 584. ISBN 978-0-534-42174-8.

Borghesi L, Milcarek C. From B cell to plasma cell: regulation of V(D)J recombination and antibody secretion. Immunol Res. 2006;36(1-3):27-32