4 Types of Antigens and Immunization Procedures

Dr. M. N. Gupta

  1. Objectives
  • To learn about various types of antigens we use in experimental immunology and are equally encountered by the immune system
  • What are the various types of Immunization procedures
  • To understand the features of the three types of acquired immunity
  1. Concept Map
  1. Description

Various antigens of microbial origin will be studied in this module. In the previous module we had learnt about innate immunity. Here we are going to learn about acquired immunity. It is of three types: Active, Passive and Adoptive.

 

Microbial organisms have diverse types of antigens. The acquired immunity has three routes.

 

The immunization procedures have to be viewed in the context of why immunization is being carried out.

 

The preferred method of immunization against different infectious agents depends upon the nature of the infectious agents/disease.

 

In a later module we will discuss what makes a particular substance, a good antigen or a bad antigen.

 

In much of experimental immunology, we mostly deal with proteins and their conjugates as experimental antigens Important microbial antigens are proteins or protein conjugates:

  •  Clostridial Toxins
  •  Bacterial flagella
  •  Viral Capsids
  •  Protozoan cell membranes Other examples are:
  •  Snake venoms
  •  Serum proteins
  •  Milk proteins
  •  Hormones

Antibodies themselves constitute important antigens in experimental immunology. All antibodies are immunoglobulins and are proteins/glycoproteins.

Cell wall lipopolysaccharide of gram negative bacteria are endotoxins.

  •  Lipid A is an endotoxin
  •  Lipopolysaccharide O is an antigen

Some bacteria are enclosed by capsules which are either protein or polysaccharide in nature. Capsular antigens are called K-antigens. Pili or fimbriae are called F-antigens.

 

Bacteria flagella consists of protein called flagellin and these antigens are called H-antigens.

 

Proteins secreted by bacteria or obtained by autolysis of bacteria are called endotoxins. The antibody formed against all these bacterial antigens have protective function.

Viral Antigens

  •  The protein coat is called capsid
  •  Capsids are good antigens

Cell surface Antigens of Mammalian Cells

 

Blood Group Antigens: Early unsuccessful blood transfusions between unrelated individuals led to the realization about A and B antigens

Histocompatibility Antigens

 

Nucleated cells have many cell surface antigens. Most important ones are the histocompatibility antigens which provoke strong immune response and are major histocompatibility antigens. These play a very important role in immune responses and will be discussed in considerable detail in the future lectures.

 

Auto Antigens and Super Antigens

 

Auto Antigens

 

Sometimes antibodies are produced in response to normal self antigens. This happens when mechanisms responsible for self tolerance are bypassed. We will discuss this later while discussing tolerance and autoimmune diseases.

 

Super Antigens

 

In the 1970s, Howard M. Johnson and co-workers at the University of Texas showed that bacterial enterotoxin A produced high amount of interferons.

 

It has been found later on that enterotoxins (secreted by some bacteria like S. aureus, S.pyrogens, Pseudomonas sp.) and retroviral proteins can act as super antigens that is, provoke an exceptionally high T-cell response.

 

Some antigens are commonly used in experimental immunology as these are relatively cheap and available in pure form and large quantities.

 

Commonly used Antigens

  •  Endotoxins from E.coli
  •  Bovine serum albumin
  •  Flagellin
  •  Hemocyanin: Blood pigment of keyhole limpet
  •  IgG
  •  Lysozyme
  •  Sheep RBCs
  •  TGAL: A synthetic antigen consisting of Tyr and Glu linked to Ala side chains on a Lys backbone

Types of Immunization

Immunization can be transferred by antibodies or lymphocytes

 

Passive Immunization

  •  Usually IgG is given either intravenously or intramuscularly
  •  Passive immunization is useful for immunocompromised individual who have deficient immune system responses
  •  A well known example is the transfer of antisera from horse against tetanus to humans. It is usual to administer equine (Fab’)2 to minimize its antigenicity
  •  Protection of foetus by maternal antibody and intestinal absorption of colostral antibody are examples of natural passive immunization

This classification now gives us a clearer picture of the various ways the acquired immunity may be obtained. Protection of the foetus by maternal antibodies or by administering artificial immunoglobulins comprise the Passive acquired immunity. Active immunity can be obtained through natural infection or by administering artificial agents like living or dead organisms, metabolic products like toxoids, Genetically engineered antigens and synthetic antigens.

 

Animals used for raising antisera by active Immunization Rabbits, goats, sheep and fowls are generally used The unrelated specie has to be used. A far removed animal/bird from the source of the antigen on the evolutionary tree is the best.

 

Immune response differs from animal to animal. So, initially several animals should be tried.

 

If the volume required is large, goat or sheep are preferred.

 

Expenses like cost of animals and their housing sometime have to be considered

 

Sites of Immunization

 

Subcutanous and intramuscular routes are used. Single site or multisite injections may be used. Multisite injections provoke the immune system by different routes and generally result in better titres.

Choice of antigens

 

Under the best conditions antisera can be raised with antigens in microgram amounts. Mixing of some substances (called adjuvants) with antigens help in obtaining better immunization.

 

Proteins which show less evolutionary divergence between mammalians turn out to be poor antigens. Cyt C, actin and myosin are examples of this.

 

Two approaches to overcome this are:

  •  Raise antibody in fowls
  •  Croslink to form homopolymers or heteropolymers (e.g. with oval bumin)

Even conjugates like sepharose 2B-albumin-casein have been used to improve the antigenicity of casein

Immunization Schedule

 

Non-immune serum before immunization should be collected for running control. Immunization should be repeated at regular intervals minimum after 2-3 weeks. Trial bleeding can track response and help in designing the immunization schedule in individual cases.

 

Preparation of low affinity adsorbents does not require high titre antiserum. Immunological purity is different from biochemical purity. This should be kept in mind while assessing antibody preparation. In general shorter immunization schedules contain fewer antibody to contaminating antigens.

 

Bleeding Methods

 

Trial bleeding should use simple methods like from rabbit ear veins. In case of sheep or goat, venipuncture of the jugular vein works well.. Preparative bleeding may require local anaesthesia. Atleast 2-3 weeks should intervene between successive bleedings.

 

 

Priming immunization and primary response

 

The first immunization is sometime referred to as primary immunization.

 

The humoral response of the immune system in terms of antibody production is called primary response.

 

The primary response itself has four phases

 

During these different phases, both the concentration and nature of immunoglobulin changes.

 

 

Phases of the Primary Response

Latent or lag phase:

 

It is initial 2-3 weeks period after the first immunization. During this period antibody is not detectable in the serum. Obviously, if less sensitive method for antibody detection is used, the lag phase will appear to be longer

 

(b) Exponential Production Phase:

 

The antibody concentration in the serum rises rapidly

 

(c) Steady State:

 

The antibody concentration reaches a steady state and its rate of production and degradation are balanced

 

(d) Declining Phase:

 

Immune response slows down and antibody concentration declines rapidly.

 

Generally only one kind of immunoglobulin called IgM is synthesized during the primary response.

 

If another kind of immunoglobulin IgG is synthesized, its formation is detected only after IgM concentration has nearly declined.

 

Phases of the Secondary Response

 

The lag phase is shorter, generally by half. IgM may or may not be detected. If detected, it is soon replaced by IgG king of antibody. Serum contains much higher amount of antibody. It persists longer, lasting for months or even years. Other kinds of antibodies/ Ig may also be formed.

 

The capacity to give this secondary (also called memory or anamnestic) response may persist for years in humans and serves as their basis for human immunization programs.

 

Summary

  • Immunization may be intentional or accidental
  • Antibody response depends upon the type of antigen
  • Immunization may be active, passive or adoptive
  • In active immunization with experimental animals, immunization schedule, bleeding schedule etc are important
  • Active immunization consists of primary response and secondary response