Definition of Humoral Immune Response
- Antibody-mediated immunity is another name for humoral immunity. This physiological process, which is a part of both the innate and adaptive immune systems, protects the body from infections and foreign chemicals in extracellular fluids.
- It entails a two-stage, humoral immune response: primary and secondary.
- The primary phase is initiated by the body’s initial interaction with an antigen (surface protein present on the membranes of pathogens); the secondary phase reflects the immune system’s response to repeated contact with the same antigen.
- Several immune cell types offer humoral immunity, which occurs in distinct stages.
- Older vaccines activate the humoral immune response by delivering weakened or deactivated pathogens; more recent vaccine innovations use templates or mRNA sequences to activate the humoral immune response. As a result of the COVID-19 pandemic, several more recent vaccines are in the news.
- Antibody and complement activity are the foundation of humoral immunity. It is intended mostly for:
- Extracellular bacteria, in particular exotoxin-producing bacteria, such as Corynebacterium diphtheriae, Clostridium tetani, etc.
- Bacteria whose pathogenicity results from the presence of polysaccharide capsules (e.g., Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, etc.).
- Certain viruses that affect the respiratory or gastrointestinal systems. Additionally, humoral immunity contributes to the pathophysiology of hypersensitivity reactions and specific autoimmune disorders.
Characteristic pattern for Production of antibodies
Antibody production is the primary characteristic of humoral immune responses. The synthesis of antibodies conforms to the following pattern:
- Lag phase: This is the phase immediately following antigen exposure. Throughout this phase, no antibodies are detectable in the bloodstream.
- Log phase: This is the subsequent phase, which is characterised by a constant increase in antibody titers in the circulation.
- Plateau: This is the balance phase between antibody synthesis and catabolism.
- Phase of decline: This phase is characterised by an increase in the catabolism of antibodies relative to their synthesis, resulting in a decrease in the antibody titer in circulation. There are two forms of humoral immune response: primary and secondary.
- During the primary response, which occurs when an individual encounters an antigen for the first time, the antibody response to that antigen is detectable in the serum following a longer lag period than during the secondary response.
- The serum antibody concentration continues to climb for several weeks before declining; it may reach extremely low concentrations.
- During this first response, a small clone of antigen-specific B lymphocytes and plasma cells is produced.
- The lag period normally lasts between 7 and 10 days, although it can be longer, even weeks, depending on the antigen. For instance, the lag phase for some antigens, such as diphtheria toxoid, can be as lengthy as 2–3 weeks, whereas it can be as short as a few hours for pneumococcal polysaccharide.
- In addition to antigen dose and mode of delivery, the lag period is influenced by the antigen’s dose and route of administration, whether oral or parenteral.
- IgM is generated initially, followed by IgG, IgA, or both. IgM levels tend to decrease more rapidly than IgG levels.
- Due to a second interaction with the same antigen or a closely related “cross-reacting” antigen, the antibody response is often more rapid in the secondary response, months or years after the original reaction.
- Typically, the lag period is extremely brief (only 3–5 days). Antibody levels are also significantly higher than during the main reaction.
- These modifications to the secondary immune response are attributed to the persistence of antigen-specific “memory cells” after the initial encounter with the antigen.
- These memory cells grow extensively in order to generate numerous clones of specific B cells and plasma cells, which mediate the secondary reaction.
- In the subsequent response:
- The amount of IgM produced is qualitatively similar to that produced after the initial interaction with the antigen; however, significantly more IgG is produced, and the level of IgG tends to persist for a significantly longer period of time than in the primary response.
- In addition, these antibodies tend to bind antigen more tightly (i.e., have a higher affinity) and hence dissociate less readily. Due to changes that arise in the DNA that encodes the antigen-binding site, antibody binding is enhanced. This is known as somatic hypermutation.
Fate of Antigen in Tissues
- The route of antigen delivery influences the place of antigen localisation within the body. The majority of antigens given subcutaneously, for instance, are mostly localised in the draining lymph nodes, whereas only a little proportion is present in the spleen.
- In contrast, the majority of intravenously administered antigens are found in the spleen, liver, bone marrow, kidney, and lungs, but not in the lymph nodes.
- Approximately three-quarters of these antigens are broken down and eliminated in the urine by reticuloepithelial cells.
Production of Antibodies
- Typically, the synthesis and manufacture of antibodies relies on the complicated interaction of three cells: (a) macrophages, (b) T cells that provide support, and (c) B cells.
- Antigen-presenting cells (APCs), which include macrophages and dendritic cells, present antigens to immunocompetent cells.
- Many T-cell-dependent antigens, including proteins and erythrocytes, appear to require processing by macrophages prior to the production of antibodies.
- T-cell-independent antigens, however, do not require macrophage engagement in antibody formation. Both macrophages and dendritic cells present the antigen at the cell surface, either in its original form or after processing.
- By adjusting the optimal dose of antigen exposed to lymphocytes to activate immunological responses, macrophages play a crucial role. Antigen fragments emerge on the surfaces of macrophages in combination with class II MHC proteins after processing by macrophages.
- The antigen-class II MHC protein complex interacts to specific helper T cell receptors. These helper T cells then create cytokines that activate B cells, which then produce antibodies that are specific for the antigen.
- Interleukin-2 (T-cell growth factor), interleukin-4 (B-cell growth factor), and interleukin-5 are the activated cytokines (B-cell differentiation factor).
- The activated B cells proliferate clonally and develop into plasma cells, which manufacture particular immunoglobulins (antibodies).
- Neutralization of poisons and viruses and opsonization (coating) of the pathogen, which facilitates its uptake by phagocytic cells, are major activities of antibodies in host defence.
- Certain chemicals (e.g., polysaccharides) can activate B cells without the assistance of T cells, despite the need of helper T cells in the production of antibodies. These are known as T-cell-independent antigens.
- These antigens, however, drive B cells to produce just IgM antibodies and not other types.
- This is because B cells require interleukins 4 and 5 to make IgG, IgA, and IgE by switching classes. Interleukins 4 and 5 are only generated by T helper cells.
- First, B cells identify antigens with their surface IgM, which operates as an antigen receptor; second, they present epitopes to helper T cells in conjunction with class II MHC proteins.
- B cell IgM antigen receptor detects foreign proteins in addition to lipids, carbohydrates, DNA, RNA, etc.
- Class II MHC proteins, on the other hand, present protein fragments to helper T cells. The IgM antigen receptor binds to a wide range of molecules, hence stimulating B cells to generate antibodies against all conceivable molecules.
Theories of antibody formation
There are two distinct ideas about the development of antibodies. These theories are educational and selective.
A. Instructive theory
Theoretical evidence implies that an immunocompetent cell is capable of producing all types of antibodies. The antigen instructs immunocompetent cells to generate complementary antibodies. Two illuminating hypotheses are advanced as follows:
Direct template theory
- This hypothesis was proposed for the first time by Breinl and Haurowitz (1930).
- They hypothesised that a specific antigen or antigenic determinants would serve as a template for antibody folding.
- Consequently, the antibody molecule would acquire a conformation that is complementary to the antigen template.
Indirect template theory
- This theory was initially proposed by Burnet and Fenner (1949).
- They hypothesised that the introduction of antigenic determinants into antibody-producing cells caused a heritable alteration in these cells.
- Antigenic determinant genocopy was integrated into the genome and transmitted to daughter cells.
- This explanation for specificity and secondary responses is no longer accepted, however.
B. Selective theories
The following selective theories were proposed:
Side chain theory
- Ehrlich put out this notion (1898). According to this notion, immunocompetent cells possess surface receptors that can react with antigens that contain complimentary side chains.
- When antigens are injected into a host, they connect with compatible cell receptors. This renders the receptors inactive.
- As a compensatory mechanism, the same sort of receptors that circulate as antibodies are overproduced.
Natural selection theory
- Jerne put up this theory (1955). According to this notion, millions of globulin molecules were generated against all potential antigens throughout foetal development.
- When introduced to the host, the antigen binds selectively to the globulin molecule with the closest complementary fit.
- Antibody-forming cells are stimulated to generate the same type of antibody by globulin containing a combination antigen.
Clonal selection theory
- Burnet (1957) claimed that immunological specificity existed in the cell, but not in the serum, and he provided the most plausible clonal selection theory.
- According to this theory, a vast number of clones of immunologically competent cells (ICCs) containing unique antibody patterns are generated throughout foetal development via somatic mutations of ICCs against all potential antigens.
- This idea proposes that each ICC expresses membrane receptors unique to a particular antigen. This unique receptor specificity is determined prior to antigen exposure of the cell.
- Antigen binding to its specific receptor activates the cell, which then proliferates to form clones and produce the antibody.
- The idea of clonal selection is widely acknowledged and provides a framework for a better understanding of the specificity, immunological memory, and the property of adoptive immunity to recognise self and nonself.
Factors affecting production of antibodies
Numerous variables influence the development of antibodies. These factors are described in greater detail below:
1. Genetic factors
- Antigen-response of an organism is influenced by genetic variables.
- Responders to antigens are known as responders, while nonresponders are known as nonresponders.
- These variations are controlled genetically by the immune response (Ir) gene situated on the short arm of the sixth chromosome.
- Both the embryo and the newborn lack complete immune competence at birth.
- By the age of 5–7 years for IgG and 10–15 years for IgA, the development of lymphoid organs results in full competence.
3. Nutritional status
- Malnutrition has an impact on both humoral and cell-mediated immunity.
- Deficiencies in amino acids and vitamins have been proven to reduce antibody production.
4. Route of antigen
- The route of antigen administration influences the elicitation of an immunological response in a host.
- Parenteral antigen administration causes a stronger immune response than oral or nasal administration.
5. Dose of antigen
- A minimal critical dosage of antigen is required to induce an optimal immune response. Extremely high or low doses do not stimulate the immune system.
- This condition is known as immunological paralysis.
6. Multiple antigens
- When two or more antigens are delivered simultaneously, antibody responses differ.
- Antibody responses to one or more of them may be decreased due to antigenic competition, augmented as shown following vaccination with a triple vaccine (diphtheria, pertussis, and tetanus), or similar.
- Therefore, the composition and relative proportions of various antigens must be meticulously regulated for maximum efficacy.
- Adjuvants are chemicals that boost the immunogenicity of antigens.
- The adjuvants delay the antigen’s release from the injection site and lengthen the antigenic stimulation.
- Among the chemicals used as adjuvants include
- Freund’s incomplete adjuvant (protein antigen incorporated in water phase of water in oil emulsion).
- Freund’s complete adjuvant (incomplete adjuvant along with suspension of killed tubercle bacilli).
- Aluminum can act as a salt for both phosphate and hydroxide.
- Others, including particles of silica, beryllium sulphate, endotoxin, etc.
8. Immunosuppressive agents
Immunosuppressive agents are substances that inhibit the immunological response. They are utilised in transplantation surgery and other cases when lowering of host immunity is necessary. The following agents are involved:
- X-irradiation: Sublethal doses of irradiation are hazardous to reproducing cells and are used to inhibit antibody production by x-irradiation. The manufacturing of antibodies ceases 24 hours after irradiation.
- Radiometric drugs: These include alkylating chemicals (such as cyclophosphamide, nitrogen mustard, etc.) that inhibit the formation of antibodies. When administered for three days, cyclophosphamide fully suppresses the antibody response. It inhibits the replication of B cells specifically.
- Corticosteroids: Corticosteroids are anti-inflammatory medications that reduce the reactivity of both B and T lymphocytes. They modify the maturation of activated cells by inhibiting interleukin synthesis. They inhibit delayed hypersensitivity, but have minimal effect on antibody formation when administered in therapeutic levels for a brief duration.
- Antimetabolites: Antimetabolites include folic acid antagonists (such as methotrexate), purine analogues (6-mercaptopurine and azathioprine), cytosine analogues (cytosine arabinose), and uracil analogues (uracil arabinose) (5-fluorouracil). These chemicals impede DNA and RNA synthesis, preventing cell proliferation and differentiation, which are needed for cellular and humoral immune responses. These are typically employed to prevent graft rejection.
- Antilymphocyte serum: Antilymphocyte serum (ALS) is an antiserum heterogeneously produced against T lymphocytes. The ALS primarily targets lymphocytes in circulation but not lymphocytes in lymphoid organs. In transplantation surgery, it is primarily utilised to prevent graft rejection.
- Antibodies produced by a single clone of cells (e.g., myeloma) are homogenous and referred to as monoclonal antibodies.
- In multiple myeloma, for instance, antibodies are produced by a single clone of plasma cells against a single antigenic determinant; these antibodies are hence monoclonal.
- The monoclonal antibodies differ from polyclonal antibodies, which are heterologous and generated in response to antigen by many clones of plasma cells.
Method of production of monoclonal antibodies
- Kohler and Milstein (1975) were the first to disclose a method for the synthesis of monoclonal antibodies against a desired antigen; they were awarded the Nobel Prize in 1984 for their achievement.
- By fusing myeloma cells with antibody-producing cells, hybridomas are generated that manufacture monoclonal antibodies.
- These hybridomas generate nearly limitless numbers of useful antibodies for research and diagnostics.
- In this process, mouse splenic lymphocytes and mouse myeloma cells are united to create hybrid cells or hybridomas.
- Myeloma cells supply hybrid cells with immortality, while splenic plasma cells are responsible for antibody production.
- These hybridomas can manufacture monoclonal antibodies forever when maintained in culture. In the following techniques, hybridoma cells are created:
- First, an animal (such as a mouse) is inoculated with the target antigen.
- Lymphocytes from the spleen are then merged with mouse myeloma cells lacking the enzyme hypoxanthine phosphoribosyl transferase and cultured in vitro (HPRT).
- The inclusion of some compounds, such as polyethylene glycol, facilitates cell fusion. The fused cells are cultivated in a culture medium (HAT media) that supports the growth of the hybrid cells but not the proliferation of the parent cells.
- Finally, the resultant clones of cells are screened to determine whether or not they produce an antibody to the target antigen.
- These clones are then chosen for cultivation in perpetuity. The hybridomas may be maintained continuously and will manufacture monoclonal antibodies indefinitely.
- Since mouse monoclonal antibodies are unsuitable for therapeutic application, the original technology has been modified to make human monoclonal antibodies, such as chimeric antibodies.
- To cure leukaemia, chimeric antibodies comprised of human constant regions and mouse variable regions are being developed.
- By delivering toxins, such as diphtheria, to tumour cells or by complement-mediated cytotoxicity, chimeric antibodies can also be employed to kill tumour cells.
Function of Antibodies
Antibodies are the first line of defence against pathogens and their products. Actively inducing antibodies in the host with vaccines or passively acquiring them can give instant protection against the infection. For instance, hyperimmunized sera containing ready-made antitoxins against toxins like tetanus, botulism, or diphtheria are administered to promptly neutralise the effects of these toxins in the body. In addition, early in the incubation phase, hyperimmune sera having high titers of particular antibodies are administered to block attachment and multiplication of rabies and hepatitis A and B viruses. The following roles of antibodies can be summarised:
- Antibodies can prevent pathogens from attaching to their targets by binding to the pathogen or foreign substance. Antibodies to bacterial toxins, for instance, can block the toxin from attaching to host cells, rendering it ineffective.
- Similarly, antibody binding to a virus or bacterial pathogen can inhibit infection or colonisation by blocking the pathogen from attaching to its target cell.
- Antibodies that bind to a disease or foreign substance might opsonize it, allowing phagocytic cells to more easily absorb and destroy it.
- The Fc portion of other antibodies binds with Fc receptors on phagocytic cells, facilitating phagocytosis of the pathogen.
- Antibody activation of the complement cascade can result in the lysis of specific bacteria and viruses.
- In addition, certain complement cascade components (e.g., C3b) opsonize pathogens and accelerate their absorption by phagocytic cells via complement receptors.
Tests for Detection of Humoral Immunity
- IgG, IgM, and IgA concentrations in the patient’s serum are the primary approach for detecting humoral immunity.
- For measuring antibodies, radial immunodiffusion and immunoelectrophoresis are extensively used techniques.
Phases of Humoral Immune Responses
The majority of immune responses mediated by antibodies present in plasma, lymph, and tissue fluids are referred to as humoral immune responses. It provides protection from extracellular germs and foreign macromolecules. This immunity is conferred by the transfer of antibodies to the receiver. The activation and effector phases of the immune system’s humoral responses.
These occurrences are as follows:
- An APC, such as a macrophage, ingests the antigen through phagocytosis and degrades it in a lysosome.
- On the macrophage, a T-cell receptor identifies processed antigen bound to a class II MHC protein.
- Cytokines released by TH cells and IL-1 secreted by macrophages drive TH cells to develop and form a clone of B-cell-interacting cells.
- Lymphatic tissue enters an activation phase.
- B-cells are also antigen presenting cells. When antigen binds to a specific IgM receptor, receptor-mediated endocytosis, degradation, and presentation of the processed antigen on class II MHC proteins ensue.
- When a TH cell receptor interacts to an antigen—MHC II complex on a B cell, cytokines are released.
- These cytokines induce the B-cell to generate clones of B-cells.
- Now, these B-cells create plasma cells that secrete antibodies.
Antibodies play vital role in elimination of antigenic agents
- Opsonization is when the antibody binds to the surface epitopes of the antigen, rendering it more susceptible to phagocytosis.
- The antibody molecule can bind to the antigen to form an antigen-antibody complex, which then joins step-by-step with the complement to initiate and promote phagocytosis of the antigen.
- The antibody has the ability to bind toxin molecules produced by microorganisms, rendering them non-toxic.
- Antibodies can render free virus particles incapable of attaching to host cell membranes by interacting with the epitopes on viral particles.
- Binding to prospective pathogens on the surface of mucous membranes to avoid colonisation.
- Confirming antigen specificity by binding to Fc (fragment crystallised) receptors on NK cells or macrophages during antibody-dependent cell-mediated cytotoxicity (ADCC).
- Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Chapter 9, The Humoral Immune Response. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10752/
- Tanaka, T., Couser, W., & Nangaku, M. (2011). The Role of Humoral and Cell-Mediated Adaptive Immune Response. In (Ed.), An Update on Glomerulopathies – Etiology and Pathogenesis. IntechOpen. https://doi.org/10.5772/21937