Chapter 18 APPLIED IMMUNOLOGY P. G. REDDY

 

18.1 Summary of Major Objectives

After studying this chapter one should be able to answer questions on the applied aspects of development of immunity in the fetus and neonate, failure of passive transfer, kinds of immunization, including different types of vaccines and their relative merits, the use of adjutants in vaccines. It should also help in the development of a concise knowledge on immunosuppressive and anti-inflammatory drugs. Finally, the student should be familiar with some immunologic assays that are commonly used to measure humoral and cell-mediated immunity. 

18.2 INTRODUCTION

Cellular and molecular aspects of the immune response are complex and show complex interactions with various humoral, cell-mediated and active components of the reticuloendothelial system. Some of the more important components are:

• a. T, B lymphocytes and aggressive killer cells
• b. effecter products such as immunoglobulins and cytokines
• c. Phagocytic cells
• d. activated complement systems causing immune lysis and immune adherence
• e. interaction of MHC molecules in the recognition of antigen and development of immune response

18.2.1 Kinds of Immunity

1. Active immunity- a specifically developed immunity by the host (long lived).

• a. natural - recovery from a clinical or subclinical infection may confer resistance to subsequent disease manifestations to that same infectious agent.
• b. artificial - stimulation of resistance factors by administering attenuated or nonviable immunogens (Vaccines).

2. Passive- the transfer of resistant factors produced in a donor to a susceptible host (short lived).

• a. natural - transfer of specific immunoglobulins via colostrum or the placenta.
• b. artificial - the injection of specific immunoglobulins into the susceptible subject.

    

18.2.2 Ontogeny of the acquired immunity

Progressive development of specific acquired immunity starts during fetal life. In case of bovine fetus lymphocytes develop at 42 days of fetal life and antibodies specific to certain viruses develop as early as 120 days. Cell-mediated immune responses against certain organisms have been observed at birth.

18.2.3 Ontogeny of the lymphoid/leucocyte system in the fetal lamb

Upon birth:

• The host response is selective to antigens.
• Potential antigens within the mother are sometimes transferred and can lead to suppression (Bovine virus diarrhea infection in fetal life) or enhancement of immune responses.
• The use of passive immunity interferes with active immunity in the neonate (maternal antibodies obtained thro' colostrum interfere with vaccination in neonatal life).
• Colostral Ig is absorbed and is dependent on time (age) of the neonate. Newborn should receive colostrum immediately after birth. Enhances phagocytosis by PMN's. Immunoglobulins from milk are not absorbed but provide local protection.

Transfer of Ig from the mother to the fetus depends on the type of placenta. In ruminants, pigs, and horses there is no transplacental transfer. They obtain maternal antibodies thro' colostrum. In dogs and cats, there is only about 5% transfer thro' placenta. 

Absorption of colostral antibodies

• declines rapidly after birth - almost none after 24 hours. It is important that animals receive adequate amount of colostrum in the first 6 hours after birth.

Failure of absorption of colostral antibodies is known as FAILURE OF PASSIVE TRANSFER (FPT). In neonatal foals and calves, serum IgG concentration less than 500 mg/dl may be considered as FPT - Correlates with susceptibility to microbial infection in the early post-natal period.

Reasons for FPT:

• 1) Insufficient colostrum,
• 2) inadequate intake and
• 3) failure of intestinal absorption. (please refer to page 179 of Vet. Immunology by Tizard (III ed).

18.2.3 Categories of vaccine preparations:

Ideal Vaccine:

safe (no adverse side effects), effective, should give prolonged immunity, cheap, stable, adaptable to mass vaccination.

Killed vaccines:

Whole organisms killed by heat or chemical means (formaldehyde, propiolactone). Retain immunogenicity.

Subunit vaccines:

Extracts or cellular fractions (the use of individual protective antigens of an organism); efficacy is usually higher and safer than with whole organisms.

Toxoids:

usually inactivated by formalin. Retain immunogenicity are highly efficacious. Eg. Tetanus toxoid.

Live attenuated viral vaccines:

have lost capacity to cause disease but retain immunogenicity. Prepared by passage thro' unnatural hosts; repeated passage in embryonated eggs and tissue cultures.

18.2.4 Live-attenuated versus killed vaccines

LIVE KILLED

• Duration of immunity long short
• Dose mostly single multiple
• amount of antigen in vaccine small large
• possible inclusion of viable + -
• adventitious agents
• possible reversion to virulence + -
• contraindications (pregnancy, + -
• Immunosuppression)

New types of vaccines:

Main advantage: reduction of harmful side effects caused by reversion to virulence of live-attenuated organism;

Recombinant DNA vaccines:

Introduction of genes coding for appropriate proteins into vaccine strains of bacteria or viruses by genetic engineering techniques.

Examples: Bacterial vector: Salmonella Virus vector: Vaccinia

Immunogenic rabies glycoprotein gene has been incorporated into vaccinia virus. 

Synthetic peptide vaccines:

Once the amino acid sequence of an epitope concerned in protective immunity is known it can be chemically synthesized and used as a vaccine. Eg. synthetic peptide vaccine for foot & mouth virus based on the virus protein VP1.

Anti-idiotype vaccines:

Antigen injected into an animal induces the formation of antibodies (idiotype). When this antibody is injected into another animal it provokes the formation of anti-idiotype. Binding site on an anti-idiotype has the same shape as the antigen that induced the idiotype. Thus, the use of an anti-idiotype vaccine should provoke a protective response.

18.2.5 Vaccination strategy: 

Route:

• 1) subcutaneous or intramuscular injections - for establishing a high titer of circulating antibodies where there is a systemic spread of organism or toxin.
• 2) intranasal or aerosolized vaccine: for respiratory infections.
• 3) oral vaccines: mainly for enteric diseases. 

Time of vaccination:

• Maternal antibodies interfere with successful vaccination in newborn animals. Vaccinate young animals atleast twice- second injection at 15 weeks of age in small animals and at 6 months in large animals.

  Killed vaccines: every 6 months or 1 year.

  Live-attenuated: every 2 or 3 years.

   Adjutants (Latin adjuvare - to help).

• Substances when given with an antigen, enhances antigen-specific immune response.
• Usually incorporated in or injected simultaneously with an antigen; all vaccines contain adjutants.
• Delays the release and degradation of an antigen (depot formation); allows more time for immune system to respond.

Examples: 

• Incomplete Freund's adjutant: is composed of a water-in-oil emulsion to which antigen is incorporated. Mainly induces humoral immunity.
• Complete Freund's adjutant: A water-in-oil emulsion with killed Mycobacterium incorporated in the aqueous phase. Induces both humoral and cell-mediated responses. Not acceptable in food animals not only because of the mineral oil but also because killed mycobateria will interfere with testing of the animals for TB. 

  Aluminum hydroxide (commonly used in veterinary vaccines), Alum, Aluminum phosphate, saponin.

Other new adjutants: muramyl dipeptide, levamisloe, interleukins and ISCOM's (immune-stimulated complexes). 

18.3 Anti-Inflammatory & Immuno-Suppressive Drugs

To manage immune mediated diseases. 

• Anti-inflammatory: Corticosteroids
• Non-steroidal anti-inflammatory agents
• Megesterol acetate 
• Cytotoxic drugs: Alkylating agents, thiopurines, vinka alkaloids, gold salts, cyclosporin A.
•  Coticosteroids:

Main effects:

lymphopenia, chemotactic and other functions of inflammatory cells inhibited, decreased formation of prostaglandins, leukotrienes and thromboxanes.

Products:

Prednisone, prednisolone, dexamethasone, betamethasone.

Non-steroidal anti-inflammatory agents:

Aspirin, salicylic acid derivatives, ibuprofen.

Inhibit cyclooxygenase pathway of arachidonic acid metabolism, inhibit formation of prostaglandins and thromboxanes.

Megesterol acetate:

corticosteroid-like activity; anti-inflammatory in cat but not in dog. 

18.4 Cytotoxic Drugs 

18.4.1 Alkylating agents:

Cross-link DNA and interfere with cell division; inhibit lymphocyte-mediated reactions. Cyclophosphamide: used in dogs and cats - more commonly in rheumatoid arthritis; less frequently in autoimmune skin diseases, systemic lupus erythematosus (SLE) etc.

Thiopurines:

inhibit enzyme systems required for DNA synthesis; inhibit natural killer cell activity.Azathioprine; 6-mercaptopurine - used in combination with others in autoimmune skin diseases, SLE, rheumatoid arthritis.

Vinka alkaloids:

act by binding to tubulin, a protein that is a key component of cellular metabolites. Vincristine, Vinblastine: mild immunosuppressants.

Gold salts:

used in humans for many years in rheumatoid arthritis; inhibits lysosomal enzyme function in macrophages; inhibits CMI and HI responses.

Sodium aurothioglucose, sodium thiomalate.

Cyclosporin A:

derived from fungus Trichoderma polysporum. Binds to specific subpopulations of lymphocytes thus interfering with their functions.

18.4.2 Laboratory Detection of Humoral and Cell-Mediated Responses

18.4.2.1Humoral Immunity:

• -Zinc sulfate turbidity tests for measuring serum antibodies.
• -Immunoprecipitation- the simplest and most direct means of demonstrating soluble antigen-antibody reactions. Examples: double diffusion (agar-gel immunodiffusion; AGID), immunoelectrophoresis and radioimmunoassay (RIA), and single- radial immunodiffusion (SRID).
• -other assays using antigen-antibody reactions: enzyme-linked immunosorbent assay, immunoblotting, radioallergosorbent assay (RAST), immunofluorescence (direct and indirect).
• -Chromatography - widely used in the purification of proteins, Isolation of immunoglobulins and molecular weight determinations. Example: affinity, molecular sieving.
• -Agglutination reactions - involve a particulate antigen. Examples: bacterial agglutination, direct and passive hemagglutination, hemagglutination inhibition and passive hemolysis.
• -Complement reactions - involve the activation of complement by the classical or alternate pathways. Examples : indirect complement fixation with avian serum, passive hemolysis.

 18.4.2.2 Cell-mediated immunity:

• - Skin tests - ability to detect cutaneous hypersensitivity to an injection. The inability of a sensitized host to respond is called energy. Hyperactive animals should be retested with higher concentrations of the antigen. Examples: tuberculin skin test; Maguire Test.
• - Lymphocyte activation and function tests- mitogen-induced lymphocyte proliferation test, mixed lymphocyte reaction, cytotoxicity assay.
• -counting T4 and T8 cells and their ratio by flow cytometry.
• - Intracellular Killing by Phagocytes: an attempt to determine efficiency of phagocytosis in in vitro killing. Example: Nitroblue-tetrazolium dye reduction test.