At the end of 2019, COVID-19, a "black swan", suddenly visited human beings, bringing profound impact on everyone's life. In the absence of vaccines and specific drugs at present, plasma therapy for convalescent patients has a good effect on severe COVID-19 patients. It has been included in the Diagnosis and Treatment Program of novel coronavirus Pneumonia in China and has been used for reference by many countries. This therapy mainly uses the antibodies produced in the recovered person's body to neutralize the virus in the patient's body, prevent the virus from infecting cells, and thus play an emergency treatment role. Antibodies are not only the "star" in the fight against infectious diseases, but also the "hardcore" in tumor immunotherapy. It is worth mentioning that from the discovery of the earliest anti serum to the birth of the first monoclonal antibody, the participation of animals is indispensable. Understanding and exploring animal antibody resources will provide more help for human health.
The discovery of antibodies - the debut of the Nobel Prize in Physiology or Medicine
Let's first take a look at how antibodies are discovered. In 1796, British rural doctor Edward Jenner discovered that vaccination with cowpox could prevent smallpox, thus pioneering the development of immunology. In the 19th century, scientists represented by French chemist Louis Pasteur and German doctor Robert Koch proved the pathogenic theory of infectious diseases and invented various techniques for isolating and cultivating pathogens as well as vaccines. However, due to the limitations of the scientific level at that time, it was not clear why vaccination could make people resistant to infectious diseases? How does the immune system function?
In 1890, Emil Adolf von Behring from the Koch Institute in Germany and Saburo Kitasato from Japan published an article in the German Medical Journal, explaining how to use anti serum to prevent and treat diphtheria and tetanus. They first referred to the factor in serum that can neutralize toxins as antitoxin, which is what we call antibodies today, and thus opened the curtain on antibody research.
In 1901, Belin was awarded the first Nobel Prize in Physiology or Medicine for his research contributions in the field of anti serum. This was the first year that the Nobel Prize was awarded and also the "debut" of the Nobel Prize in Physiology or Medicine.
Types of antibodies
The chemical essence of antibodies is immunoglobulin, whose monomeric structure consists of four polypeptide chains (two heavy chains and two light chains). Conventional antibodies are divided into five categories (1gG, IgM, IgA, IgE, IgD), but due to genetic and evolutionary differences, the types of antibodies in various animals vary, and some even differ greatly. Let's step into the world of animal antibodies together.
Essence in Egg Yolk - IgY Antibody of Chicken
Eggs not only have high nutritional value, but also contain abundant immune active substances. In 1962, researchers from the University of Cambridge in the UK discovered that immunized hens could transfer antibodies from their serum to their yolk, known as immunoglobulin of yolk (IgY) antibodies. In addition to IgY, there are also IgM and IgA antibodies in the chicken body, but no IgE or IgD have been found.
Chicken IgY is functionally similar to conventional IgG, but there are differences in molecular structure. Compared with IgG, although IgY is also composed of two heavy chains (H chain) and two light chains (L chain), the heavy chain of IgY does not have a hinge region, but is composed of one variable region (V region) and four constant regions (CH1-CH4), while conventional IgG only has three constant regions (Figure 1). IgY molecules also contain abundant hydrophobic molecules, making them more suitable for "immersing" in lipid rich yolk environments.
Due to its stable chemical properties and relatively convenient production, IgY does not require animal blood collection. Antibodies can be obtained by collecting eggs, and the cost is relatively low. It has been widely used in the fields of disease diagnosis, drug and health product development in humans and animals. In terms of diagnosis, due to structural differences, IgY does not bind to Fc receptors in mammals, nor does it cross react with other antibodies from mammalian sources, which is of great significance in immune detection and diagnosis; In terms of immunotherapy, due to the good activity of oral yolk antibody products after passing through the gastrointestinal tract, researchers at home and abroad have developed hundreds of highly effective IgY antibodies against Helicobacter pylori, hand foot mouth disease, enteroviruses, hormones, enzymes, and more. The US FDA has approved yolk antibodies as a health food additive for infants and young children or for middle-aged and elderly people. Japanese scientists have extracted IgY antibodies against Streptococcus mutans from eggs and added them to beverages, chocolate, cheese, gummies, cocoa bars, ice cream, and other foods to prevent dental caries. The related products have been launched on the market. In addition, due to the serious abuse of antibiotics, many European countries have proposed to ban the addition of antibiotics in animal feed. China has required a comprehensive ban on the use of antibiotics in animal feed since January 1, 2020. Considering the function of IgY in disease prevention and treatment, this has brought new opportunities for research and development of IgY.
Camel antibodies with 'shortcomings'
In 1989, Belgian scientists discovered that camel (dromedary camel) serum not only contained IgG class conventional antibodies consisting of two heavy chains and two light chains, but also a special antibody lacking light chains and containing only heavy chains, called heavy chain antibodies (HCAbs). Later research found that HCAbs were commonly present in the serum of camelids in the subfamily Camelidae of the order Artiodactyla, including Bactrian camels, Asian Bactrian camels, South American alpacas, Bactrians, and alpacas.
HCAbs naturally lack the light chain and CH1 constant region, and their antigen binding site is only formed by the variable domain of the heavy chain, VHH (variable domain of the heavy chain), which is a single structural domain (Figure 1). The molecular size is close to the nanometer level, so it is also known as a single domain antibody or nanobody. Research has found that the germline genes encoding VHH have high homology with the VH gene II family of human IgG. The surface of VHH is only about 10 amino acids different from human VH, and VHH has a longer complementarity determining region (CDR), which can provide a larger contact surface for antigen antibody interactions. Especially its extended CDR3 can form a protruding circular structure, which can specifically recognize and bind to hidden antigen epitopes (such as the active center of enzymes), while traditional antibody Fab fragments and single chain variable fragments (scFv) can usually only recognize sites located on the antigen surface. The above characteristics enable HCAb to have broad and strong antigen binding ability even in the absence of light chains.
Compared with other antibodies, the molecular weight of HCAbs is relatively small (about 15 KDa), while the molecular weight of conventional IgG is about 150 KDa, and the molecular weight of single chain antibodies is about 30 KDa. Small molecule HCAbs have stronger tissue permeability and are easier to pass through the blood-brain barrier. In addition, HCAbs are easy to express and modify, have good water solubility, strong stability, and weak immunogenicity, making them have broad application prospects in basic research and drug development. Ablynx Biopharmaceutical Company in the United States has developed single domain antibodies against 220 diseases using camelids. The first new drug approved by the US FDA in 2019, Cabrivi (caplacizumab), is a bivalent anti hemophilia factor single domain nanobody used to treat adult acquired thrombotic thrombocytopenic purpura (aTTP), which has previously been approved in the European Union.
Unique Shark Antibody IgNAR
The shark, also known as the 'wolf in the sea', is the most ferocious fish in the ocean. Due to the strong anti infection and anti-cancer abilities of sharks, people speculate that there must be some magical immune substance in their bodies. Scientists have discovered IgM and IgW in sharks. In 1995, American scientists discovered a new antibody in the neck shark, which is different from IgM and IgW, has high homology with T cell antigen receptors, and has the characteristics of immunoglobulin. Therefore, it was named immunoglobulin new antigen receptor (IgNAR).
Similar to HCAbs in camels, IgNAR has a smaller molecular weight (12-15 kDa) and lacks light chains, but the two still have structural differences. IgNAR is a homodimer composed of two heavy chains, which exist in both secretory and membrane-bound forms in sharks. The heavy chain of secretory IgNAR molecules contains one variable region (VNAR) and five constant regions (CH1-CH5) (Figure 1). The molecular weight of VNAR is only about 12 kDa, which is about 20% smaller than VHH. It is currently the smallest natural antigen binding domain known in the animal kingdom, and its unique molecular characteristics have become a hot topic of concern for antibody reagent developers.
In addition, IgNAR can maintain biological activity in shark blood containing 350 mM urea and 1000 mOsm osmotic salt ions. In addition to its good stability, IgNAR also has advantages such as high affinity, good solubility, strong tissue penetration ability, low immunogenicity, and ease of humanized modification. Currently, it has received widespread attention in fields such as disease diagnosis, drug development, and food science.
Figure 1: Structural patterns of different animal antibodies
Cow antibodies with "special skills"
In addition to the unusual IgY of chickens, HCAbs of camels, and IgNAR of sharks, researchers have discovered a special IgM antibody in cattle that has an ultra long CDR H3 region (over 60 amino acids) composed of a long "stalk" and a "knob" formed by disulfide bonds at the top (Figure 2), which is significantly different from the structure of conventional IgM or IgG antibodies. This special antibody of cattle has strong antigen recognition ability and high affinity, becoming a key "weapon" for cattle to resist pathogen infections.
Scientists are curious about why the unique molecular structure of CDR H3 can be formed in cattle. A US research team conducted in-depth analysis of its coding sequence and analyzed its crystal structure. They found that the "sphere" on the ultra long CDR H3 is mainly responsible for binding to antigens, and its sequence changes directly affect the type and strength of IgM binding to antigens. The authors speculate that this ultra long CDR H3 may be formed by cows to resist the threat of pathogenic microorganisms in vivo and in vitro. The above research was published in the journal Cell in 2013. In 2017, researchers from the Scripps Research Institute in the United States published a paper in Nature stating that they synthesized an envelope protein that can simulate the HIV shell and injected the protein into four cows. On the 42nd day after immunization, all four cows produced neutralizing antibodies against HIV, while only about 1% of HIV infected individuals in the population can produce this antibody, which usually takes five years after infection with the virus. Previously, researchers conducted similar studies on rabbits and monkeys, but were unable to induce broad-spectrum neutralizing antibodies against HIV. The ultra long CDR H3 region possessed by bovine antibodies may be related to the formation of broad-spectrum neutralizing antibodies against HIV. This research shows that cattle are not only an ideal tool for studying AIDS vaccine. Meanwhile, it can also serve as a novel model animal for studying neutralizing antibodies against other complex pathogens.
Figure 2: CDR H3 "extra long" structure in bovine antibodies (from Haakenson, et al 2018)
During the long process of life evolution, animal immune systems tend to become more diverse and complex. At present, the animal antibodies that humans can recognize are still very limited, and further exploration of more animal antibody resources is needed to expand the systematic understanding and scientific utilization of animal antibodies.
Currently, the threat of COVID-19 to human health has not yet ended. Scientists need to conduct in-depth basic research on the immune mechanism of COVID-19, while also increasing the development and application of antibody drugs. Especially by utilizing modern molecular biology techniques and combining the advantages of yolk antibodies, nanobodies, and animal antibodies, more accurate COVID-19 diagnostic reagents and more efficient antibody therapy drugs have been developed, making animal antibodies more outstanding in the fight against the COVID-19 epidemic.
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