Semester 6
BP605T
Immunology, Vaccine Preparation & Hybridoma Technology
This comprehensive unit covers the immune system and its pharmaceutical applications: immunity types (humoral vs cellular), immunoglobulin structure (IgG, IgA, IgM, IgE, IgD), MHC structure and function, hypersensitivity reactions (Types I-IV), immunostimulation and immunosuppression. It then covers vaccine technology — preparation of bacterial vaccines, toxoids, viral vaccines, antitoxins, and storage conditions. Hybridoma technology for monoclonal antibody production is studied, along with blood products and plasma substitutes.
Syllabus & Topics
- 1Types of Immunity – Humoral: Humoral immunity = antibody-mediated immunity by B-lymphocytes. Antigen enters body → processed by APC (antigen-presenting cell) → presented to Th (helper T) cell → Th activates B cell → B cell differentiates into: Plasma cells (secrete antibodies/immunoglobulins) and Memory B cells (long-term immunity). Antibodies neutralize pathogens, opsonize for phagocytosis, activate complement, agglutinate bacteria. Primary response: slow (7-10 days), IgM first, then IgG. Secondary response (memory response): rapid (2-3 days), higher titer, predominantly IgG. Basis of vaccination.
- 2Cellular Immunity: Cell-mediated immunity (CMI) = T-lymphocyte mediated. No antibodies involved. T cells mature in Thymus. Types: (1) Cytotoxic T cells (CTLs, CD8⁺): directly kill virus-infected cells, cancer cells, transplanted cells. Recognize antigen + MHC Class I. (2) Helper T cells (Th, CD4⁺): ‘master regulators’ — secrete cytokines that activate CTLs, B cells, macrophages. Recognize antigen + MHC Class II. Th1 → activate macrophages, CTLs (cellular response). Th2 → activate B cells (humoral response). (3) Regulatory T cells (Tregs): suppress immune response → prevent autoimmunity. CMI important for: intracellular pathogens (TB, viruses), cancer surveillance, transplant rejection.
- 3Immunoglobulin Structure: Basic unit: Y-shaped monomer with 4 polypeptide chains — 2 identical Heavy (H) chains + 2 identical Light (L) chains, connected by disulfide bonds. Each chain has Variable (V) region (antigen binding) and Constant (C) region (effector functions). Fab region (Fragment antigen binding): 2 per antibody — each contains VH-VL (antigen-binding site = paratope). Fc region (Fragment crystallizable): contains CH2-CH3 — binds to Fc receptors on phagocytes, activates complement, determines Ig class. Hinge region: flexible → allows Fab arms to move independently. Five classes based on H chain: IgG (γ), IgA (α), IgM (μ), IgD (δ), IgE (ε). Light chains: κ (kappa) or λ (lambda).
- 4Immunoglobulin Classes: IgG: most abundant (75%), monomer, crosses placenta (neonatal immunity), opsonization, complement activation. 4 subclasses. IgA: secretory antibody — dimer + J chain + secretory component. Found in saliva, tears, breast milk (colostrum), GI/respiratory mucosa. First line of mucosal defense. IgM: largest — pentamer (5 monomers + J chain). First Ig produced in primary response. Best at complement activation and agglutination. Cannot cross placenta. IgE: monomer, lowest serum concentration. Binds mast cells/basophils → mediates Type I hypersensitivity (allergy, anaphylaxis). Defense against parasites. IgD: monomer, found on surface of mature B cells. Role in B cell activation (BCR co-receptor).
- 5MHC (Major Histocompatibility Complex): MHC = group of genes encoding cell surface glycoproteins that present antigens to T cells. In humans: HLA (Human Leukocyte Antigen) complex on chromosome 6. MHC Class I: expressed on ALL nucleated cells. Present endogenous antigens (intracellular — viral, tumor) to CD8⁺ CTLs. Structure: α chain (heavy, 3 domains: α1, α2 form peptide groove; α3 binds CD8) + β₂-microglobulin. MHC Class II: expressed on APCs (macrophages, dendritic cells, B cells) only. Present exogenous antigens (phagocytosed) to CD4⁺ Th cells. Structure: α chain (α1, α2) + β chain (β1, β2; peptide groove = α1+β1; β2 binds CD4). Function: self-nonself recognition → immune response against foreign + MHC determines transplant compatibility → HLA matching crucial for organ transplant.
- 6Hypersensitivity Reactions: Type I (Immediate/Anaphylactic): IgE-mediated. Allergen → IgE → binds mast cell Fc receptors → re-exposure → cross-linking → degranulation → Histamine, Leukotrienes release → vasodilation, bronchoconstriction. Examples: anaphylaxis, asthma, hay fever, urticaria. Treatment: Adrenaline, Antihistamines, Corticosteroids. Type II (Cytotoxic): IgG/IgM binds to cell surface antigens → complement activation or ADCC → cell lysis. Examples: Hemolytic disease of newborn, blood transfusion reactions, autoimmune hemolytic anemia, drug-induced hemolysis. Type III (Immune Complex): Ag-Ab complexes deposit in tissues → complement activation → inflammation. Examples: Serum sickness, SLE (Lupus), Arthus reaction, post-streptococcal glomerulonephritis. Type IV (Delayed/Cell-mediated): T cell-mediated, no antibodies. Takes 24-72 hours. Examples: Tuberculin test (Mantoux), contact dermatitis, transplant rejection, granuloma formation.
- 7Immune Stimulation & Suppression: Immunostimulants: enhance immune response. (1) Specific: vaccines, toxoids. (2) Nonspecific: BCG (Bacillus Calmette-Guérin — activates macrophages), Levamisole, Interferons, Interleukins (IL-2), Colony-stimulating factors (G-CSF — Filgrastim for neutropenia). Adjuvants: substances that enhance immune response to antigen — Alum (aluminum hydroxide/phosphate), Freund’s adjuvant, MF59 (squalene). Immunosuppressants: suppress immune response — needed for transplant rejection prevention, autoimmune diseases. Cyclosporine (inhibits Calcineurin → blocks IL-2), Tacrolimus (FK-506), Azathioprine (antimetabolite), Methotrexate, Corticosteroids, anti-CD3 (Muromonab — OKT3), Mycophenolate mofetil.
- 8Vaccine Preparation – Bacterial & Viral: Bacterial vaccines: (1) Killed/Inactivated: bacteria killed by heat/formalin. Examples: Typhoid (TAB vaccine — killed S. typhi), Cholera, Pertussis (whole cell). (2) Live attenuated: weakened bacteria. BCG (attenuated M. bovis for TB). (3) Subunit: purified antigens — Meningococcal polysaccharide vaccine. Toxoids: bacterial exotoxins treated with formalin (0.3-0.5%, 37°C, 3-4 weeks) → lose toxicity, retain antigenicity. Examples: Tetanus toxoid (TT), Diphtheria toxoid (as part of DPT). Viral vaccines: (1) Live attenuated: OPV (Sabin — oral polio), MMR (measles, mumps, rubella), Varicella. Grown in cell cultures → serial passage → attenuated. (2) Inactivated: IPV (Salk — injectable polio), Rabies, Influenza (inactivated). (3) Recombinant: Hepatitis B (HBsAg in yeast). Antitoxins/Antiserum: antibodies derived from immunized animals (horse/rabbit) → passive immunity. Anti-tetanus serum (ATS), Anti-rabies serum (ARS), Anti-snake venom.
- 9Vaccine Storage & Stability: Cold chain: vaccines are temperature-sensitive biological products. Most require 2-8°C storage (refrigerator). OPV: −20°C (frozen). Reconstituted vaccines must be used within hours. Vaccine Vial Monitor (VVM): time-temperature indicator on vial → color change indicates heat exposure → discard if inner square matches/darker than outer circle. National Cold Chain: from manufacturer → State Vaccine Store (−25 to −15°C: walk-in freezer, ILR) → District → PHC → Outreach (ice-lined refrigerator, cold boxes, vaccine carriers). Stability issues: proteins denature with heat, freeze-sensitive vaccines (DPT, TT, Hep B — contain alum) damaged by freezing → adsorbed vaccines must NOT be frozen.
- 10Hybridoma Technology: Developed by Köhler & Milstein (Nobel Prize, 1984). Purpose: produce Monoclonal Antibodies (mAbs) — identical antibodies from a single B cell clone → highly specific for one epitope. Problem: B cells producing desired antibody die quickly in culture. Myeloma (cancer) cells grow indefinitely but don’t make useful antibodies. Solution: FUSE both → Hybridoma = immortal + antibody-producing. Steps: (1) Immunize mouse with antigen → harvest spleen B cells. (2) Fuse B cells with myeloma cells using PEG (polyethylene glycol) or electrofusion. (3) Select hybridomas in HAT medium (Hypoxanthine-Aminopterin-Thymidine) — unfused B cells die naturally, unfused myeloma cells die (aminopterin blocks de novo pathway, they lack HGPRT for salvage), only hybridomas survive (have HGPRT from B cell). (4) Screen for desired antibody (ELISA). (5) Clone positive hybridomas → expand → produce mAbs.
- 11Monoclonal Antibody Applications & Blood Products: mAb applications: (1) Diagnostics: pregnancy test (anti-hCG), blood typing, ELISA kits, imaging. (2) Therapeutics: Rituximab (anti-CD20 — lymphoma), Trastuzumab (anti-HER2 — breast cancer), Infliximab (anti-TNFα — RA), Bevacizumab (anti-VEGF — cancer), Nivolumab (anti-PD1 — immunotherapy). (3) Research: flow cytometry, immunohistochemistry. Blood products: Whole blood, Packed RBCs, Platelet concentrate, Fresh Frozen Plasma (FFP), Cryoprecipitate (Factor VIII, Fibrinogen), Albumin (volume expander). Plasma substitutes: Dextran (dextran-40, dextran-70), Hydroxyethyl starch (HES), Modified gelatin (Haemaccel), Crystalloids (NS, RL). Used when blood products unavailable or for volume expansion.
Learning Objectives
Humoral vs Cellular: Compare humoral and cell-mediated immunity by cells involved, mediators, and targets.
IgG Structure: Draw and label the structure of IgG showing H/L chains, V/C regions, Fab, Fc, hinge, disulfide bonds.
Hypersensitivity Types: Compare all four types of hypersensitivity by mediator, timing, mechanism, and clinical examples.
Hybridoma Steps: Describe the hybridoma technology step-by-step including HAT selection with rationale.
Vaccine Types: Classify vaccines as live, killed, toxoid, subunit, and recombinant with examples of each.
Exam Prep Questions
Q1. What is the HAT selection principle in Hybridoma technology?
Cells use two pathways for nucleotide synthesis: de novo pathway and salvage pathway (using HGPRT enzyme). Aminopterin in HAT medium blocks the de novo pathway → cells MUST use the salvage pathway to survive. Myeloma cells lack HGPRT gene → cannot use salvage pathway → die in HAT. Normal B cells survive initially but die naturally (limited lifespan in culture). Hybridomas have HGPRT from B cells + immortality from myeloma → ONLY hybridomas survive HAT selection.
Q2. Why are monoclonal antibodies better than polyclonal for diagnostics?
Polyclonal antibodies are a mixture of antibodies from multiple B cell clones → recognize multiple epitopes on an antigen → batch-to-batch variation, cross-reactivity. Monoclonal antibodies are from a SINGLE clone → recognize ONE specific epitope → highly specific, consistent batch-to-batch, no cross-reactivity, can be produced indefinitely from hybridoma. This makes them ideal for: precise diagnostic tests (ELISA, pregnancy tests), therapeutic targeting (cancer cells), and research (flow cytometry).
Q3. What is the difference between a Vaccine and a Toxoid?
Vaccine: contains either whole organisms (live attenuated or killed) or their antigenic components (subunit, recombinant) → stimulates active immunity against the pathogen itself. Toxoid: specifically made from bacterial exotoxins that have been inactivated by formaldehyde treatment → loses toxicity but retains antigenicity → stimulates antibodies against the TOXIN (not the bacterium). Examples: Tetanus toxoid (against Clostridium tetani toxin), Diphtheria toxoid (against C. diphtheriae toxin).