Factors affecting Immunogenicity

Updated: 5/26/2023

An immunogen is a substance that can induce an immune response in a host organism. The immune response may involve the production of antibodies, the activation of T cells, or both. The immune response is specific to the immunogen and can provide protection against future exposure to the same or similar immunogens.

Not all substances are immunogenic, however. Some substances may be recognized as foreign by the host but fail to elicit a significant immune response. These substances are called antigens. Antigens can bind to antibodies or T cell receptors but cannot stimulate their production. Antigens may become immunogenic if they are conjugated with an immunogenic carrier molecule, such as a protein.

The ability of a substance to act as an immunogen depends on several factors, such as its foreignness, molecular size, chemical nature, heterogeneity, physical form, and susceptibility to antigen processing and presentation. These factors affect the recognition, uptake, processing, and presentation of the substance by the antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells. APCs are responsible for capturing and displaying antigens to the T cells and B cells, which then initiate the adaptive immune response.

In this article, we will discuss how these factors affect the immunogenicity of a substance and provide some examples of immunogens and antigens. We will also explain some exceptions to these factors and how they relate to the concept of self and non-self in immunology.

One of the most important factors that affect immunogenicity is the degree of foreignness of the antigen. Foreignness refers to how different the antigen is from the host`s own molecules. The immune system is designed to recognize and eliminate foreign substances that may pose a threat to the host`s health. Therefore, an antigen that is highly foreign to the host will elicit a stronger immune response than an antigen that is similar or identical to the host`s molecules.

For example, bovine serum albumin (BSA) is a protein that is found in the blood of cows. When BSA is injected into a cow, it does not trigger an immune response because it is recognized as self by the cow`s immune system. However, when BSA is injected into a rabbit, it induces a strong immune response because it is recognized as non-self by the rabbit`s immune system. The rabbit`s immune system produces antibodies and T cells that bind to and eliminate BSA.

The degree of foreignness of an antigen depends on several factors, such as:

The evolutionary distance between the host and the source of the antigen. Antigens from distantly related species are more foreign than antigens from closely related species. For instance, human proteins are more foreign to mice than to monkeys.
The structural similarity between the antigen and the host`s molecules. Antigens that share common motifs or domains with the host`s molecules are less foreign than antigens that have unique or novel structures. For example, human insulin is less foreign to mice than human growth hormone.
The exposure history of the host to the antigen. Antigens that have been previously encountered by the host are less foreign than antigens that are novel or rare. For instance, chicken egg albumin is less foreign to humans who eat eggs regularly than to humans who never eat eggs.

The degree of foreignness of an antigen influences not only the magnitude but also the quality of the immune response. Antigens that are highly foreign tend to induce a balanced immune response that involves both humoral and cellular immunity. Antigens that are moderately foreign tend to induce a predominantly humoral immune response that involves mainly antibody production. Antigens that are weakly foreign tend to induce a predominantly cellular immune response that involves mainly T cell activation.

In summary, foreignness is a key factor that determines how immunogenic an antigen is. The more foreign an antigen is to the host, the more likely it is to trigger a strong and balanced immune response. However, there are some exceptions to this rule, which will be discussed in the next section.

Foreignness is a key factor that determines the immunogenicity of a substance. However, there are some exceptions to this rule. Some macromolecules that are highly conserved across different species have low immunogenicity, even when they are injected into a different animal. On the other hand, some self-components that are normally hidden from the immune system have high immunogenicity, even when they are injected into the same animal.

One example of a conserved macromolecule is collagen, which is the main structural protein in connective tissues. Collagen has a similar amino acid sequence and structure in many animals, such as mammals, birds, reptiles and fish. Therefore, collagen from one animal is not recognized as foreign by the immune system of another animal. This makes collagen a weak immunogen that does not elicit a strong immune response.

Another example of a conserved macromolecule is cytochrome c, which is a protein involved in cellular respiration. Cytochrome c has a highly conserved sequence of 104 amino acids that is almost identical in all eukaryotes and some prokaryotes. Cytochrome c from one organism is not very different from cytochrome c from another organism. Therefore, cytochrome c is also a weak immunogen that does not induce a strong immune response.

One example of a self-component that is hidden from the immune system is sperm, which are the male reproductive cells. Sperm are produced in the testes and stored in the epididymis, where they are protected from the immune system by a blood-testis barrier. Sperm do not encounter the immune system until they are released during ejaculation. Therefore, sperm are not exposed to immune tolerance mechanisms that prevent self-reactivity. This makes sperm a strong immunogen that can trigger an immune response when injected into the same animal or another animal.

Another example of a self-component that is hidden from the immune system is corneal tissue, which is the transparent layer that covers the front of the eye. Corneal tissue does not have blood vessels or lymphatic vessels, which means that it does not receive any immune surveillance or protection. Corneal tissue also does not express MHC molecules, which are required for antigen presentation and recognition by T cells. Therefore, corneal tissue is not subject to immune tolerance mechanisms that prevent self-reactivity. This makes corneal tissue a strong immunogen that can induce an immune response when transplanted into the same animal or another animal.

These examples show that foreignness is not the only factor that affects immunogenicity. Other factors, such as molecular size, chemical nature, physical form and susceptibility to antigen processing and presentation, also play important roles in determining how well a substance can elicit an immune response.

Most antigens that elicit an immune response are foreign to the host, meaning they are not normally present in the body. However, there are some exceptions to this rule, where self-components, or molecules that are normally present in the host, can also trigger an immune reaction. Two examples of such self-components are sperm and corneal tissues.

Sperm are the male reproductive cells that carry the genetic material to fertilize the egg. They are produced in the testes and stored in the epididymis, where they are protected from the immune system by a physical barrier called the blood-testis barrier. This barrier prevents the exposure of sperm antigens to the circulating lymphocytes and antibodies. However, if this barrier is breached due to injury, infection, or vasectomy, sperm antigens can escape into the blood and lymphatic system and induce an immune response. This can result in sperm autoantibodies, which can impair fertility by reducing sperm motility, agglutinating sperm cells, or blocking sperm-egg binding.

Corneal tissues are the transparent layers of cells that cover the front of the eye and refract light to focus on the retina. They are avascular, meaning they lack blood vessels, and they have a low expression of major histocompatibility complex (MHC) molecules, which are essential for antigen presentation to T cells. These features allow the cornea to avoid inflammation and maintain its clarity and function. However, they also make the cornea vulnerable to immune rejection when transplanted from one individual to another. Corneal transplantation is a common procedure to restore vision in patients with corneal diseases or injuries. However, even though the donor and recipient are matched for blood type and MHC compatibility, there is still a risk of graft rejection due to the presence of minor histocompatibility antigens or alloantigens on the corneal cells. These antigens can activate T cells and B cells in the recipient and initiate an immune attack against the graft.

Therefore, sperm and corneal tissues are examples of self-components that are strongly immunogenic under certain conditions. They illustrate that immunogenicity is not only determined by foreignness, but also by other factors such as physical barriers, vascularization, and antigen presentation.

The effect of molecular size on immunogenicity

Molecular size is one of the factors that affects the immunogenicity of a substance. Immunogenicity is the ability of a substance to induce an immune response in the body. Generally, larger molecules are more immunogenic than smaller ones, because they have more epitopes or antigenic determinants that can be recognized by the immune system.

Epitopes are the specific regions on the antigen that bind to the antibodies or T cell receptors. The more epitopes a molecule has, the more likely it is to trigger an immune response. However, not all epitopes are equally immunogenic. Some epitopes may be hidden or inaccessible due to the conformation or folding of the molecule, or may be masked by other molecules. Therefore, the immunogenicity of a molecule depends not only on its size, but also on its shape and structure.

The most active immunogens tend to have a molecular mass of 14,000 to 6,00,000 Da, usually >1,00,000 Da. For example, tetanus toxoid, egg albumin, and thyroglobulin are highly antigenic substances with molecular mass ranging from 150,000 to 660,000 Da. These molecules have multiple and diverse epitopes that can stimulate both humoral and cellular immunity.

On the other hand, substances with molecular mass less than 5,000 to 10,000 Da are poor immunogens. For example, insulin (5,700 Da) is either non-antigenic or weakly antigenic when injected into animals. This is because insulin has only a few epitopes that are similar among different species and may not be recognized as foreign by the immune system. Moreover, insulin is a soluble and stable molecule that does not form aggregates or complexes that can enhance its immunogenicity.

However, there are some exceptions to the rule of molecular size. Some small molecules can become immunogenic when they are attached to larger carrier molecules. These small molecules are called haptens and they act as incomplete antigens. Haptens alone cannot elicit an immune response, but they can bind to antibodies or T cell receptors when coupled with a carrier molecule. For example, penicillin (334 Da) is a hapten that can cause allergic reactions in some individuals when it binds to proteins in the body.

Therefore, molecular size is an important factor that influences the immunogenicity of a substance, but it is not the only one. Other factors such as foreignness, chemical nature, heterogeneity, physical form, and susceptibility to antigen processing and presentation also play a role in determining how well a substance can induce an immune response in the body.

As mentioned earlier, molecular size is one of the factors that affect immunogenicity. Generally, substances with a molecular mass of more than 1,00,000 Da are highly antigenic, meaning they can elicit a strong immune response when introduced into the body. Some examples of such substances are:

Tetanus toxoid: This is a modified form of the toxin produced by the bacterium Clostridium tetani, which causes tetanus. The toxoid is used as a vaccine to prevent tetanus by inducing the production of antibodies that neutralize the toxin. The molecular mass of tetanus toxoid is about 1,50,000 Da.
Egg albumin: This is a protein found in egg white that has many functions, such as binding water, maintaining viscosity and providing nutrition. Egg albumin is often used as a model antigen in immunological studies because it is readily available and easy to purify. The molecular mass of egg albumin is about 4,50,000 Da.
Thyroglobulin: This is a glycoprotein that is synthesized and stored in the thyroid gland. It serves as a precursor for the production of thyroid hormones, which regulate metabolism and growth. Thyroglobulin is also an autoantigen, meaning it can trigger an immune response against itself in some autoimmune diseases, such as Hashimoto`s thyroiditis and Graves` disease. The molecular mass of thyroglobulin is about 6,60,000 Da.

The reason why these substances are highly antigenic is that they have several features that make them more recognizable and accessible to the immune system. These features include:

Complexity: These substances have a high degree of structural and chemical diversity, which allows them to present multiple epitopes (regions that bind to specific antibodies or T-cell receptors) to the immune system. This increases the chances of activating different types of immune cells and generating a broad and specific immune response.
Solubility: These substances are soluble in water or body fluids, which means they can easily diffuse and reach the lymphatic system, where they encounter antigen-presenting cells (APCs) that process and present them to T-cells. This facilitates the initiation and amplification of the immune response.
Aggregation: These substances tend to form aggregates or clumps when exposed to certain conditions, such as heat, pH or salt concentration. Aggregation enhances immunogenicity by increasing the size and visibility of the antigens, making them more susceptible to phagocytosis (ingestion) by APCs and more stimulatory to B-cells.

Therefore, substances with a molecular mass of more than 1,00,000 Da are highly antigenic because they have properties that favor their recognition and presentation by the immune system.

As mentioned earlier, molecular size is one of the factors that affect immunogenicity. Generally, substances with molecular mass less than 5,000 to 10,000 Da are poor immunogens. This is because they are too small to be recognized by the immune system as foreign and to elicit an adequate immune response. Such substances are called haptens.

Haptens are molecules that can bind to antibodies but cannot induce their production. They need to be attached to a larger carrier molecule, such as a protein or a polysaccharide, to become immunogenic. The carrier molecule provides the necessary size and complexity for the hapten-carrier complex to be processed and presented by antigen-presenting cells (APCs) to T cells and B cells.

For example, insulin is a peptide hormone with a molecular mass of 5,700 Da. It is either non-antigenic or weakly antigenic when injected into animals. However, when insulin is conjugated to bovine serum albumin (BSA), a protein with a molecular mass of 66,000 Da, it becomes highly immunogenic and can induce the production of anti-insulin antibodies.

Another example is penicillin, a drug with a molecular mass of 334 Da. It is not immunogenic by itself, but it can bind covalently to proteins in the body and form penicillin-protein complexes that can trigger allergic reactions in some individuals. The penicillin acts as a hapten and the protein acts as a carrier.

Haptens can also be used to study the specificity and diversity of antibodies. By attaching different haptens to the same carrier molecule, one can generate antibodies that recognize only the hapten part of the complex and not the carrier part. This shows that antibodies can discriminate between very small differences in molecular structure.

In summary, substances with molecular mass less than 5,000 to 10,000 Da are poor immunogens because they are too small to be detected by the immune system. They need to be coupled to larger carrier molecules to become immunogenic. Such substances are called haptens and they can bind to antibodies but cannot induce their production.

The chemical nature and heterogeneity of an antigen affect its ability to elicit an immune response. Antigens are mainly proteins and some are polysaccharides, which are complex molecules composed of different types of amino acids or sugars. The more diverse and varied the chemical structure of an antigen is, the more likely it is to be recognized as foreign and non-self by the immune system.

One way to measure the heterogeneity of an antigen is to compare its number of epitopes with its molecular mass. Epitopes are the specific regions on an antigen that bind to antibodies or T cell receptors. A high ratio of epitopes to molecular mass indicates a high degree of heterogeneity and immunogenicity. For example, a protein with 10 epitopes and a molecular mass of 100,000 Da has a ratio of 0.0001, while a protein with 100 epitopes and a molecular mass of 1,000,000 Da has a ratio of 0.0001. The latter protein is more heterogeneous and immunogenic than the former.

Another way to assess the heterogeneity of an antigen is to compare its composition with that of the host. Antigens that share common amino acids or sugars with the host are less immunogenic than those that have unique or rare components. This is because the immune system has developed tolerance to self-antigens and does not react to them. For example, synthetic homopolymers (multiple copies of a single amino acid or sugar) tend to lack immunogenicity regardless of size, while heteropolymers (composed of different amino acids or sugars) are usually more immunogenic than homopolymers. Similarly, antigens that have aromatic radicals (such as benzene rings) in their structure are more immunogenic than those that do not, because aromatic radicals are less common in biological molecules and confer rigidity and stability to the antigen.

In summary, the chemical nature and heterogeneity of an antigen influence its immunogenicity by affecting its recognition by the immune system. Antigens that are more complex, diverse, and distinct from the host are more likely to induce an immune response than those that are simple, uniform, and similar to the host.

Homopolymers are molecules that consist of multiple copies of the same monomer unit, such as amino acids or sugars. Heteropolymers are molecules that consist of different monomer units, such as proteins or polysaccharides. The chemical nature and heterogeneity of antigens affect their immunogenicity, or the ability to elicit an immune response.

In general, heteropolymers are more immunogenic than homopolymers, regardless of their size. This is because heteropolymers have more structural diversity and complexity, which allows them to interact with a wider range of receptors on the immune cells. Heteropolymers can also form more epitopes, or antigenic determinants, that can be recognized by specific antibodies or T cells.

Homopolymers, on the other hand, tend to lack immunogenicity because they have a repetitive and simple structure that is not easily distinguished from self-molecules by the immune system. Homopolymers may also have low solubility or stability, which reduces their exposure to the immune cells. Homopolymers may require additional factors, such as adjuvants or carriers, to enhance their immunogenicity.

An example of a homopolymer that is weakly immunogenic is poly-L-lysine, which is composed of multiple L-lysine amino acids. Poly-L-lysine has a linear and positively charged structure that does not form many conformations or interactions with the immune cells. An example of a heteropolymer that is highly immunogenic is tetanus toxoid, which is derived from the toxin produced by Clostridium tetani bacteria. Tetanus toxoid has a complex and folded structure that contains multiple epitopes that can bind to different antibodies or T cells.

Therefore, the comparison between homopolymers and heteropolymers shows that the chemical nature and heterogeneity of antigens are important factors that affect their immunogenicity. Heteropolymers are generally more immunogenic than homopolymers because they have more structural diversity and complexity that can stimulate the immune system.

One of the key factors that affect immunogenicity is the ability of an antigen to be processed and presented by antigen-presenting cells (APCs) to T cells. T cells are essential for the initiation and regulation of both humoral and cell-mediated immune responses. However, T cells cannot recognize antigens in their native form. They need to interact with antigens that have been degraded into smaller fragments (peptides) and displayed on the surface of APCs along with major histocompatibility complex (MHC) molecules. MHC molecules are a group of proteins that bind to peptides and present them to T cells. There are two types of MHC molecules: MHC class I and MHC class II. MHC class I molecules present peptides derived from intracellular antigens, such as viruses or tumors, to cytotoxic T cells (CD8+). MHC class II molecules present peptides derived from extracellular antigens, such as bacteria or parasites, to helper T cells (CD4+).

Therefore, antigens that cannot be processed and presented by MHC molecules are poor immunogens, as they fail to activate T cells. For example, D-amino acids are stereoisomers of naturally occurring L-amino acids. As APCs can only process and present L-amino acids, D-amino acids are poor immunogens. Similarly, lipids and nucleic acids are generally poor immunogens, as they are not efficiently processed and presented by MHC molecules.

On the other hand, antigens that are easily phagocytosed by APCs are generally more immunogenic, as they can be efficiently processed and presented by MHC class II molecules. Phagocytosis is a process by which APCs engulf and digest large particles or microorganisms. Phagocytosed antigens are degraded into peptides within lysosomes and then transported to the cell surface where they associate with MHC class II molecules. This allows the APCs to activate helper T cells, which in turn stimulate B cells to produce antibodies and cytotoxic T cells to kill infected cells.

Some examples of antigens that are readily phagocytosed by APCs are bacteria, fungi, protozoa, and some viruses. These antigens are usually particulate, insoluble, or aggregated, which makes them more recognizable by phagocytic receptors on APCs. Moreover, these antigens often have other features that enhance their immunogenicity, such as foreignness, molecular size, chemical nature, and heterogeneity.

In summary, susceptibility to antigen processing and presentation is a crucial factor that determines the immunogenicity of an antigen. Antigens that can be processed and presented by MHC molecules are more likely to elicit an immune response than those that cannot. Antigens that are easily phagocytosed by APCs are generally more immunogenic than those that are not.

Phagocytosis is the process by which certain cells, such as macrophages and dendritic cells, engulf and digest foreign particles, bacteria, or other antigens. Phagocytosis plays a crucial role in the initiation and regulation of immune responses, as it allows the antigen-presenting cells (APCs) to process and present the antigens to T cells and B cells.

Antigens that are easily phagocytosed are generally more immunogenic than those that are not. This is because phagocytosis enhances the exposure of the antigens to the immune system and facilitates their processing and presentation. Phagocytosed antigens can be degraded into smaller peptides that bind to major histocompatibility complex (MHC) molecules on the surface of APCs. These MHC-peptide complexes can then be recognized by T cells, which activate B cells to produce antibodies or mediate cellular immunity.

Some factors that affect the phagocytosis of antigens are:

The size and shape of the antigen: Larger and irregular-shaped antigens are more likely to be phagocytosed than smaller and spherical ones.
The surface charge and hydrophobicity of the antigen: Antigens that have a negative charge or are hydrophobic tend to be more phagocytosed than those that have a positive charge or are hydrophilic.
The presence of opsonins: Opsonins are molecules that bind to the surface of antigens and enhance their phagocytosis by APCs. Examples of opsonins are antibodies, complement proteins, and lectins.

Therefore, phagocytosis is an important factor that influences the immunogenicity of antigens. Antigens that are easily phagocytosed can elicit stronger and more diverse immune responses than those that are not. However, phagocytosis alone is not sufficient to induce immunogenicity; other factors such as foreignness, molecular size, chemical nature, heterogeneity, and physical form also play a role in determining the immunogenic potential of antigens.

The role of adjuvants in enhancing immunogenicity

Adjuvants are substances that are added to antigens to increase their immunogenicity. They can act by various mechanisms, such as:

Protecting the antigen from degradation and prolonging its exposure to the immune system
Increasing the antigen uptake and processing by antigen-presenting cells
Stimulating the production of cytokines and co-stimulatory molecules that activate T-cells and B-cells
Inducing inflammation and recruiting immune cells to the site of injection
Modulating the type and quality of the immune response (e.g. Th1 vs Th2, humoral vs cellular)

Some examples of adjuvants are:

Aluminum salts (alum): The most widely used adjuvant in human vaccines. They form a depot of antigen at the injection site and induce a Th2-biased response.
Oil-in-water emulsions (e.g. MF59, AS03): They enhance antigen delivery to lymph nodes and stimulate innate immunity and cytokine production.
Liposomes: They are spherical vesicles composed of phospholipids that can encapsulate antigens and deliver them to antigen-presenting cells.
Toll-like receptor (TLR) agonists (e.g. CpG oligodeoxynucleotides, monophosphoryl lipid A): They mimic microbial components and activate TLRs on antigen-presenting cells, leading to enhanced antigen presentation and co-stimulation.
Saponins (e.g. QS-21): They are plant-derived glycosides that form complexes with cholesterol in cell membranes and increase membrane permeability and antigen uptake.

Adjuvants can improve the immunogenicity of antigens that are otherwise weak or non-immunogenic, such as synthetic peptides, recombinant proteins, or subunit vaccines. They can also reduce the dose and number of injections required to achieve protective immunity. However, adjuvants can also have adverse effects, such as local reactions, systemic toxicity, or unwanted immune responses. Therefore, the choice and use of adjuvants should be carefully evaluated for each antigen and target population.

Factors affecting Immunogenicity

Introduction to the properties of Immunogen

The role of foreignness in immunogenicity

Exceptions to foreignness: collagen and cytochrome c

Self-components that are strongly immunogenic: sperm and corneal tissues

The effect of molecular size on immunogenicity

Highly antigenic substances with molecular mass >1,00,000 Da

Poor immunogens with molecular mass less than 5,000 to 10,000 Da

The impact of chemical nature and heterogeneity on immunogenicity

Comparison between homopolymers and heteropolymers

The influence of physical form on immunogenicity

Susceptibility to antigen processing and presentation

The influence of phagocytosis on immunogenicity

The role of adjuvants in enhancing immunogenicity

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