Immunology_Janeway

Immunology_Janeway. The antigenic components and adjuvants in a vaccine are not approved for use on their own; they are approved only in the context of the specific vaccine in which they are formulated. At present, alum is the only adjuvant that is approved by the FDA in the United States for use in marketed human vaccines, although some other adjuvant–vaccine combinations are undergoing clinical trials. Alum is the common name for certain inorganic aluminum salts, of which aluminum hydroxide and aluminum phosphate are most frequently used as adjuvants. In Europe, in addition to the alum adjuvants, an oil (squalene)-in-water emulsion called MF-59 is used as an adjuvant in a formulation of influenza vaccine and is undergoing evaluation in clinical trials. As we described in Section 3-9, alum seems to act as an adjuvant by stimulating one of the innate immune system’s bacterial sensor mechanisms, NLRP3, thus activating the inflammasome and the inflammatory reactions that are a prerequisite for an effective


Immunology_Janeway

Immunology_Janeway. adjuvant by stimulating one of the innate immune system’s bacterial sensor mechanisms, NLRP3, thus activating the inflammasome and the inflammatory reactions that are a prerequisite for an effective adaptive immune response.


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Immunology_Janeway. Several other adjuvants are widely used experimentally in animals but are not approved for use in humans. Many of these are sterile constituents of bacteria, particularly of their cell walls. Freund’s complete adjuvant is an oil-in-water emulsion containing killed mycobacteria. The peptidoglycan muramyl dipeptide and the glycolipid trehalose dimycolate (TDM) found in mycobacterial cell walls contain much of the adjuvant activity of the whole killed organism. Other bacterial adjuvants include killed B. pertussis, bacterial polysaccharides, bacterial heat-shock proteins, and bacterial DNA. Many of these adjuvants cause quite marked inflammation and so are not suitable for use in vaccines for humans.


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Immunology_Janeway. Many adjuvants seem to work by triggering the innate viral and bacterial sensor pathways in APCs, via TLRs and proteins of the NOD-like receptor family such as NLRP3 (see Chapter 3), and thereby activating them to initiate an adaptive immune response. The TLR-4 agonist lipopolysaccharide (LPS) has adjuvant effects, but its use is limited by its toxicity. Small amounts of injected LPS can induce a state of shock and systemic inflammation that mimics Gram-negative sepsis, raising the question whether its adjuvant effect can be separated from its toxic effects. Monophosphoryl lipid A, an LPS derivative and TLR-4 ligand, partly achieves this, retaining adjuvant effects but being associated with much lower toxicity than LPS. Both unmethylated CpG DNA, which activates TLR-9, and imiquimod, a small-molecule drug that acts as a TLR-7 agonist, can provide adjuvant activity experimentally, but neither is approved as an adjuvant in human vaccines.


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Immunology_Janeway. 16-30 Protective immunity can be induced by DNA-based vaccination.


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Immunology_Janeway. Surprisingly, when bacterial plasmids were used to express proteins in vivo for gene therapy, some were found to stimulate an immune response. Later, it was found that DNA encoding a viral immunogen, when injected intramuscularly in mice, induced antibody responses and cytotoxic T cells that could protect against subsequent infection from the live virus. This response does not seem to damage the muscle tissue, is safe and effective, and, because it uses only a single microbial gene or a stretch of DNA encoding sets of antigenic peptides, does not carry the risk of active infection. This procedure is termed DNA vaccination, and can be carried out in various ways. In one, DNA coated onto minute metal particles can be administered by a gene gun, so that particles penetrate the skin and potentially some underlying muscle, but other approaches, such as electroporation, are also possible. Because of DNA’s stability, DNA vaccination is suitable for mass immunization. One problem with


Immunology_Janeway

Immunology_Janeway. potentially some underlying muscle, but other approaches, such as electroporation, are also possible. Because of DNA’s stability, DNA vaccination is suitable for mass immunization. One problem with DNA-based vaccines, however, is that they are comparatively weak. Mixing in plasmids that encode cytokines such as IL-12, IL-23, or GM-CSF makes immunization with genes encoding protective antigens much more effective. In DNA vaccination, the antigen is produced by cells that are directly transfected, such as skin or muscle, but CD8 T-cell activation requires cross-presentation of the antigen by dendritic cells. Current approaches are identifying how best to transfect DNA into these dendritic cell populations. DNA vaccines are being tested in human trials for malaria, influenza, HIV infection, and breast cancer.


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Immunology_Janeway. 16-31 Vaccination and checkpoint blockade may be useful in controlling existing chronic infections. There are many chronic diseases in which infection persists because of a failure of the immune system to eliminate disease. Such infections can be divided into two groups: those in which there is an obvious immune response that fails to eliminate the organism, and those that seem to be invisible to the immune system and evoke a barely detectable immune response.


Immunology_Janeway

Immunology_Janeway. In the first category, the immune response is often partly responsible for the pathogenic effects. Infection by the helminth Schistosoma mansoni is associated with a powerful TH2-type response, characterized by high levels of IgE, circulating and tissue eosinophilia, and a harmful fibrotic response to schistosome ova in the liver, leading to hepatic fibrosis. Other common parasites, such as Plasmodium and Leishmania species, also cause damage in many patients because they are not eliminated effectively by the immune response. The mycobacterial agents of tuberculosis and leprosy cause a persistent intracellular infection; a TH1 response helps to contain these infections but also causes granuloma formation and tissue necrosis (see Fig. 11.13).


Immunology_Janeway

Immunology_Janeway. Among viruses, hepatitis B and hepatitis C infections are commonly followed by a persistent viral burden and hepatic injury, resulting in eventual death from hepatitis or from hepatocellular carcinoma. Infection with HIV, as we have seen in Chapter 13, also persists despite an ongoing immune response. In a preliminary trial involving HIV-infected patients, dendritic cells derived from the patients’ own bone marrow were loaded with chemically inactivated HIV. After immunization with the loaded cells, a robust T-cell response to HIV was observed in some patients that was associated with the production of IL-2 and IFN-γ (Fig. 16.29). Viral load in these patients was reduced by 80%,


Immunology_Janeway

Immunology_Janeway. Fig. 16.29 Vaccination with dendritic cells loaded with HIV substantially reduces viral load and generates T-cell immunity. Left panel: viral load is shown for a weak and transient response to treatment (pink); the red bar represents individuals who made a strong and durable response. Right panel: CD4 T-cell IL-2 and interferon-γ production for individuals who made a weak or strong response. The production of both these cytokines, indicating T-cell activity, correlates with the response to treatment.


Immunology_Janeway

Immunology_Janeway. Viral load weak response strong response weak response strong response weak response strong response IFN-˜(after 112 days of treatment) IL-2 Effect of dendritic-cell vaccination for HIV on immune function and virus production 0 1 2 3 4 5 0 10,000 20,000 30,000 40,000 50,000 Percentage of HIV-specifc CD4 cells expressing cytokine and in almost half of these patients the suppression of viremia lasted for more than a year. Nonetheless, these responses were not sufficient to eliminate the HIV infection.


Immunology_Janeway

Immunology_Janeway. In the second category of chronic infection, which is predominantly viral, the immune response fails to clear the infection because of the relative invisibility of the infectious agent to the immune system. A good example is herpes simplex virus type 2, which is transmitted venereally, becomes latent in nerve tissue, and causes genital herpes, which is frequently recurrent. The invisibility of this virus seems to be caused by a viral protein, ICP-47, that binds to the TAP complex (see Section 6-3) and inhibits peptide transport into the endoplasmic reticulum in infected cells. Thus, viral peptides are not presented to the immune system by MHC class I molecules. A similar example in this category of chronic infection is genital warts, caused by certain papilloma viruses that evoke very little immune response, particularly a cell-mediated response. As discussed previously, the results of a recent clinical trial showed that using long-peptide vaccines against HPV-16 was effective in


Immunology_Janeway

Immunology_Janeway. little immune response, particularly a cell-mediated response. As discussed previously, the results of a recent clinical trial showed that using long-peptide vaccines against HPV-16 was effective in increasing the strength of cell-mediated immune responses to viral antigens, and in reducing or eliminating precancerous lesions associated with the HPV infection (see Section 16-28). These results are a positive indication that vaccines directed at increasing cell-mediated responses to other pathogens may be similarly effective.


Immunology_Janeway

Summary.

Immunology_Janeway. Summary.


Immunology_Janeway

Immunology_Janeway. Vaccination is arguably the greatest success of immunology, having eradicated or virtually eliminated several human diseases. It is the single most successful manipulation of the immune system so far, because it takes advantage of the immune system’s natural specificity and inducibility. But important human infectious diseases remain that lack effective vaccines. Most effective vaccines are based on attenuated live microorganisms, but such vaccines carry some risk and are potentially lethal to immunosuppressed or immunodeficient individuals. New techniques are being developed to generate genetically attenuated pathogens for use as vaccines, particularly for malaria and tuberculosis. While most current viral vaccines are based on live attenuated virus, many bacterial vaccines are based on components of the microorganism, including components of the toxins that it produces. Protective responses to carbohydrate antigens, which in very young children do not provoke lasting immunity, can


Immunology_Janeway

Immunology_Janeway. components of the microorganism, including components of the toxins that it produces. Protective responses to carbohydrate antigens, which in very young children do not provoke lasting immunity, can be enhanced by conjugation of the carbohydrate to a protein. Vaccines based on peptides, particularly very long peptides, are just emerging from the experimental stage and are beginning to be tested in humans. A vaccine’s immunogenicity often depends on adjuvants that can help, directly or indirectly, to activate antigen-presenting cells that are necessary for the initiation of immune responses. Adjuvants activate these cells by engaging the innate immune system and providing ligands for TLRs and other innate sensors on antigen-presenting cells. The development of oral vaccines is particularly important for stimulating immunity to the many pathogens that enter through the mucosa.


Immunology_Janeway

Summary to Chapter 16.

Immunology_Janeway. Summary to Chapter 16.


Immunology_Janeway

Immunology_Janeway. One of the great future challenges in immunology is to be able to control the immune system so that unwanted immune responses can be suppressed and desirable responses elicited. Current methods of suppressing unwanted responses rely, to a great extent, on drugs that suppress adaptive immunity indiscriminately and are thus inherently flawed. We have seen in this book that the immune system can suppress its own responses in an antigen-specific manner and that, by studying these endogenous regulatory events, it has been possible to devise strategies to manipulate specific responses while sparing general immune competence. New treatments, including many monoclonal antibodies, have emerged as clinically important therapies to selectively suppress the responses that lead to allergy, autoimmunity, or the rejection of grafted organs. Similarly, as we understand more about tumors and infectious agents, better strategies to mobilize the immune system against cancer and infection are becoming


Immunology_Janeway

Immunology_Janeway. or the rejection of grafted organs. Similarly, as we understand more about tumors and infectious agents, better strategies to mobilize the immune system against cancer and infection are becoming possible. To achieve all this, we need to learn more about the induction of immunity and the biology of the immune system, and to apply what we have learned to human disease.


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Immunology_Janeway. Questions. 16.1 Multiple Choice: Which of the following immunomodulators has a similar mechanism to azathioprine? A. Mycophenolate B. Cyclophosphamide C. Abatacept D. Rapamycin 16.2 Matching: Match the following immunomodulating antibodies with their respective mechanism of action. 16.3 True or False: Chimeric antigen receptor (CAR) T cells are cells that have been retrovirally transduced with a tumor-specific T-cell receptor in order to treat a leukemia. 16.4 Multiple Choice: Which statement is false? A. The vaccine Provenge is prepared using the patient’s own antigen-loaded dendritic cells to induce therapeutic antitumor T-cell responses. B. Clinical trials of vaccines against HPV-16 and HPV18 (associated with 70% of cervical cancers) were 100% effective in preventing cervical cancers caused by these viruses.


Immunology_Janeway

Immunology_Janeway. B. Clinical trials of vaccines against HPV-16 and HPV18 (associated with 70% of cervical cancers) were 100% effective in preventing cervical cancers caused by these viruses. C. Cell-based cancer vaccines can use the patient’s tumor as a source of antigens. In order to enhance immunogenicity these can be mixed with adjuvants such as CpG, which binds to TLR-7. 16.5 Multiple Choice: Which of the following treatments against cancer is a checkpoint blockade therapy? (One or more may apply.) A. Ipilimumab (anti-CTLA-4 antibody) B. Trastuzumab (anti-HER-2/neu antibody) C. Rituximab (anti-CD20 antibody) D. Pembrolizumab (anti-PD-1 antibody) E. Sipuleucel-T (patient’s dendritic cells cultured with prostatic acid phosphatase tumor antigen and GM-CSF and reinfused into patient) 16.6 True or False: Chimeric antigen receptor (CAR) T cells can recognize other target molecules besides peptide:MHC complexes.


Immunology_Janeway

Immunology_Janeway. 16.7 Matching: Classify the currently used vaccines of the following organisms as live-attenuated (A), toxin-based (T), killed (K), or conjugate polysaccharide (P). A. ___ Corynebacterium diphtheriae B. ___ H. influenzae type B C. ___ Measles/mumps/rubella (MMR) D. ___ Bacille Calmette–Guérin (BCG) E. ___ Influenza A virus F. ___ Sabin polio vaccine 16.8 Fill-in-the-Blanks: Vaccines have exhibited many phenomena that are beneficial and can be exploited. For example, when an antibody response against a bacterial polysaccharide is desired, it is conjugated to a protein to exploit the phenomenon of ____________________, thus ensuring T-dependent antibody responses. In addition, vaccines may protect against different subtypes of virus, as in the case of influenza, a phenomenon called _____________ immunity. When enough people in a population are vaccinated, ______ immunity is achieved, where even unvaccinated individuals are indirectly protected from infection.


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Immunology_Janeway. 16.9 Short Answer: Explain the three main drawbacks of peptide-based vaccines. 16.10 True or False: All routes of vaccination successfully elicit virtually identical immune responses. 16.11 Matching: Match the adjuvant to the immune receptor it stimulates. A. Alum i. TLR-9 B. Freund’s complete adjuvant ii. TLR-4 C. Lipopolysaccharide iii. NLRP3 D. DNA iv. NOD2 E. Imiquimod v. TLR-7/8 General references. Maus, M.V., Fraietta, J.A., Levine, B.L., Kalos, M., Zhao, Y., and June, C.H.: Adoptive immunotherapy for cancer or viruses. Annu Rev Immunol. 2014, 32:189–225. Feldmann, M.: Translating molecular insights in autoimmunity into effective therapy. Annu. Rev. Immunol. 2009, 27:1–27. Kappe, S.H., Vaughan, A.M., Boddey, J.A., and Cowman, A.F.: That was then but this is now: malaria research in the time of an eradication agenda. Science 2010, 328:862–866. Kaufmann, S.H.: Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010, 33:567–577.


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Immunology_Janeway. Kaufmann, S.H.: Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010, 33:567–577. Korman, A.J., Peggs, K.S., and Allison, J.P.: Checkpoint blockade in cancer immunotherapy. Adv. Immunol. 2006, 90:297–339. Section references. 16-1 Corticosteroids are powerful anti-inflammatory drugs that alter the transcription of many genes. Kampa, M., and Castanas, E.: Membrane steroid receptor signaling in normal and neoplastic cells. Mol. Cell. Endocrinol. 2006, 246:76–82. Löwenberg, M., Verhaar, A.P., van den Brink, G.R., and Hommes, D.W.: Glucocorticoid signaling: a nongenomic mechanism for T-cell immunosuppression. Trends Mol. Med. 2007, 13:158–163. Rhen, T., and Cidlowski, J.A.: Antiinflammatory action of glucocorticoids— new mechanisms for old drugs. N. Engl. J. Med. 2005, 353:1711–1723. Barnes, P.,J.: Glucocorticosteroids: current and future directions. Br. J. Pharmacol. 2011, 163:29–43.


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Immunology_Janeway. Barnes, P.,J.: Glucocorticosteroids: current and future directions. Br. J. Pharmacol. 2011, 163:29–43. 16-2 Cytotoxic drugs cause immunosuppression by killing dividing cells and have serious side-effects. Aarbakke, J., Janka-Schaub, G., and Elion, G.B.: Thiopurine biology and pharmacology. Trends Pharmacol. Sci. 1997, 18:3–7. Allison, A.C., and Eugui, E.M.: Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 2005, 802 Suppl.: S181–S190. O’Donovan, P., Perrett, C.M., Zhang, X., Montaner, B., Xu, Y.Z., Harwood, C.A., McGregor, J.M.,Walker, S.L., Hanaoka, F., and Karran, P.: Azathioprine and UVA light generate mutagenic oxidative DNA damage. Science 2005, 309:1871–1874. Taylor, A.L., Watson, C.J., and Bradley, J.A.: Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit. Rev. Oncol. Hematol. 2005, 56:23–46.


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Immunology_Janeway. Taylor, A.L., Watson, C.J., and Bradley, J.A.: Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit. Rev. Oncol. Hematol. 2005, 56:23–46. Zhu, L.P., Cupps, T.R., Whalen, G., and Fauci, A.S.: Selective effects of cyclophosphamide therapy on activation, proliferation, and differentiation of human B cells. J. Clin. Invest. 1987, 79:1082–1090. 16-3 Cyclosporin A, tacrolimus, rapamycin and JAK inhibitors are effective immunosuppressive agents that interfere with various T-cell signaling pathways. Araki, K., Turner, A.P., Shaffer, V.O., Gangappa, S., Keller, S.A., Bachmann, M.F., Larsen, C.P., and Ahmed, R.: mTOR regulates memory CD8 T-cell differentiation. Nature 2009, 460:108–112. Battaglia, M., Stabilini, A., and Roncarolo, M.G.: Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005, 105:4743–4748.


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Immunology_Janeway. Battaglia, M., Stabilini, A., and Roncarolo, M.G.: Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005, 105:4743–4748. Bierer, B.E., Mattila, P.S., Standaert, R.F., Herzenberg, L.A., Burakoff, S.J., Crabtree, G., and Schreiber, S.L.: Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc. Natl Acad. Sci. USA 1990, 87:9231–9235. Crabtree, G.R.: Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 1999, 96:611–614. Crespo, J.L., and Hall, M.N.: Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2002, 66:579–591.


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Immunology_Janeway. Crespo, J.L., and Hall, M.N.: Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2002, 66:579–591. Fleischmann, R, Kremer, J., Cush, J., Schulze-Koops, H., Connell, C.A., Bradley, J.D., Gruben, D., Wallenstein, G.V., Zwillich, S.H., Kanik, K.S. et al.: Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N. Engl. J. Med. 2012, 367:495–507. Pesu, M., Laurence, A., Kishore, N, Zwillich, S.H., Chan, G, and O'Shea, J.J.: Therapeutic targeting of Janus kinases. Immunol. Rev. 2008, 223:132–142. 16-4 Antibodies against cell-surface molecules can be used to eliminate lymphocyte subsets or to inhibit lymphocyte function. Graca, L., Le Moine, A., Cobbold, S.P., and Waldmann, H.: Antibody-induced transplantation tolerance: the role of dominant regulation. Immunol. Res. 2003, 28:181–191.


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Immunology_Janeway. Graca, L., Le Moine, A., Cobbold, S.P., and Waldmann, H.: Antibody-induced transplantation tolerance: the role of dominant regulation. Immunol. Res. 2003, 28:181–191. Nagelkerke, S.Q, and Kuijpers, T.W.: Immunomodulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all? Front. Immunol. 2015, 5:674. Nagelkerke, S.Q., Dekkers, G., Kustiawan, I, van de Bovenkamp, F.S., Geissler, J., Plomp, R., Wuhrer, M., Vidarsson, G., Rispens, T., van den Berg, T.K. et al: Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages. Blood 2014, 124:3709–3718. Waldmann, H., and Hale, G.: CAMPATH: from concept to clinic. Phil. Trans. R. Soc. Lond. B 2005, 360:1707–1711. 16-5 Antibodies can be engineered to reduce their immunogenicity in humans. Kim, S.J., Park, Y., and Hong, H.J.: Antibody engineering for the development of therapeutic antibodies. Mol. Cell 2005, 20:17–29.


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Immunology_Janeway. Kim, S.J., Park, Y., and Hong, H.J.: Antibody engineering for the development of therapeutic antibodies. Mol. Cell 2005, 20:17–29. Liu, X.Y., Pop, L.M., and Vitetta, E.S.: Engineering therapeutic monoclonal antibodies. Immunol. Rev. 2008, 222:9–27. Smith, K., Garman, L., Wrammert, J., Zheng, N.Y., Capra, J.D., Ahmed, R., and Wilson, P.C.: Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protocols 2009, 4:372–384. Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M.R., Murphy, B.R., Rappuoli, R., and Lanzavecchia, A.: An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 2004, 10:871–875. Winter, G., Griffiths, A.D., Hawkins, R.E., and Hoogenboom, H.R.: Making antibodies by phage display technology. Annu. Rev. Immunol. 1994, 12:433–455. 16-6 Monoclonal antibodies can be used to prevent allograft rejection.


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Immunology_Janeway. 16-6 Monoclonal antibodies can be used to prevent allograft rejection. Kirk, A.D., Burkly, L.C., Batty, D.S., Baumgartner, R.E., Berning, J.D., Buchanan, K., Fechner, J.H., Jr, Germond, R.L., Kampen, R.L., Patterson, N.B., et al.: Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat. Med. 1999, 5:686–693. Li, X.C., Strom, T.B., Turka, L.A., and Wells, A.D.: T-cell death and transplantation tolerance. Immunity 2001, 14:407–416. Londrigan, S.L., Sutherland, R.M., Brady, J.L., Carrington, E.M., Cowan, P.J., d’Apice,A.J.,O’Connell,P.J.,Zhan,Y.,and Lew,A.M.: In situ protection against islet allograft rejection by CTLA4Ig transduction. Transplantation 2010, 90:951–957. Masharani, U.B., and Becker, J.: Teplizumab therapy for type 1 diabetes. Expert Opin. Biol. Ther. 2010, 10:459–465.


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Immunology_Janeway. Masharani, U.B., and Becker, J.: Teplizumab therapy for type 1 diabetes. Expert Opin. Biol. Ther. 2010, 10:459–465. Pham, P.T., Lipshutz, G.S., Pham, P.T., Kawahji, J., Singer, J.S., and Pham, P.C.: The evolving role of alemtuzumab (Campath-1H) in renal transplantation. Drug Des. Dev. Ther. 2009, 3:41–49. Sageshima, J., Ciancio, G., Chen, L., and Burke, G.W.: Anti-interleukin-2 receptor antibodies—basiliximab and daclizumab—for the prevention of acute rejection in renal transplantation. Biologics 2009, 3:319–336. 16-7 Depletion of autoreactive lymphocytes can treat autoimmune disease. Coiffier, B., Lepage, E., Briere, J., Herbrecht, R., Tilly, H., Bouabdallah, R., Morel, P., Van Den Neste, E., Salles, G., Gaulard, P., et al.: CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N. Engl. J. Med. 2002, 346:235–242. References.


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Immunology_Janeway. References. Coles, A., Deans, J., and Compston, A.: Campath-1H treatment of multiple sclerosis: lessons from the bedside for the bench. Clin. Neurol. Neurosurg. 2004, 106:270–274. Edwards, J.C., Leandro, M.J., and Cambridge, G.: B lymphocyte depletion in rheumatoid arthritis: targeting of CD20. Curr. Dir. Autoimmun. 2005, 8:175–192. Yazawa, N., Hamaguchi, Y., Poe, J.C., and Tedder, T.F.: Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease. Proc. Natl Acad. Sci. USA 2005, 102:15178–15783. Zaja, F., De Vita, S., Mazzaro, C., Sacco, S., Damiani, D., De Marchi, G., Michelutti, A., Baccarani, M., Fanin, R., and Ferraccioli, G.: Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 2003, 101:3827–3834. 16-8 Biologic agents that block TNF-α, IL-1, or IL-6 can alleviate autoimmune diseases.


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Immunology_Janeway. 16-8 Biologic agents that block TNF-α, IL-1, or IL-6 can alleviate autoimmune diseases. Guarda, G., Braun, M., Staehli, F., Tardivel, A., Mattmann, C., Förster, I., Farlik, M., Decker, T., Du Pasquier, R.A., Romero, P., et al.: Type I interferon inhibits interleukin-1 production and inflammasome activation. Immunity 2011, 34:213–223. Feldmann, M., and Maini, R.N.: Lasker Clinical Medical Research Award. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nat. Med. 2003, 9:1245–1250. Hallegua, D.S., and Weisman, M.H.: Potential therapeutic uses of interleukin 1 receptor antagonists in human diseases. Ann. Rheum. Dis. 2002, 61:960–967.


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Immunology_Janeway. Hallegua, D.S., and Weisman, M.H.: Potential therapeutic uses of interleukin 1 receptor antagonists in human diseases. Ann. Rheum. Dis. 2002, 61:960–967. Karanikolas, G., Charalambopoulos, D., Vaiopoulos, G., Andrianakos, A., Rapti, A., Karras, D., Kaskani, E., and Sfikakis, P.P.: Adjunctive anakinra in patients with active rheumatoid arthritis despite methotrexate, or leflunomide, or cyclosporin-A monotherapy: a 48-week, comparative, prospective study. Rheumatology 2008, 47:1384–1388. Mackay, C.R.: New avenues for anti-inflammatory therapy. Nat. Med. 2002, 8:117–118. 16-9 Biologic agents can block cell migration to sites of inflammation and reduce immune responses. Boster, A.L., Nicholas, J.A., Topalli, I., Kisanuki, Y.Y., Pei, W., Morgan-Followell, B., Kirsch, C.F., Racke, M.K., and Pitt, D.: Lessons learned from fatal progressive multifocal leukoencephalopathy in a patient with multiple sclerosis treated with natalizumab. JAMA Neurol. 2013, 70:398–402.


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Immunology_Janeway. Clifford, D.B., De Luca, A., Simpson, D.M., Arendt, G., Giovannoni, G., and Nath, A.: Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases. Lancet Neurol. 2010, 9:438–446. Cyster, J.G.: Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 2005, 23:127–159. Idzko, M., Hammad, H., van Nimwegen, M., Kool, M., Muller, T., Soullie, T., Willart, M.A., Hijdra, D., Hoogsteden, H.C., and Lambrecht, B.N.: Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function. J. Clin. Invest. 2006, 116:2935–2944. Kappos, L., Radue, E.W., O’Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L., et al.: A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 2010, 362:387–401.


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Immunology_Janeway. Podolsky, D.K.: Selective adhesion-molecule therapy and inflammatory bowel disease—a tale of Janus? N. Engl. J. Med. 2005, 353:1965–1968. 16-10 Blockade of co-stimulatory pathways that activate lymphocytes can be used to treat autoimmune disease. Fife, B.T., and Bluestone, J.A.: Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways. Immunol. Rev. 2008, 224:166–182. Ellis, C.N., and Krueger, G.G.: Treatment of chronic plaque psoriasis by selective targeting of memory effector T lymphocytes. N. Engl. J. Med. 2001, 345:248–255. Menter, A.: The status of biologic therapies in the treatment of moderate to severe psoriasis. Cutis 2009, 84:14–24.


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Immunology_Janeway. 16-28 Peptide-based vaccines can elicit protective immunity, but they require adjuvants and must be targeted to the appropriate cells and cell compartment to be effective. Alonso, P.L., Sacarlal, J., Aponte, J.J., Leach, A., Macete, E., Aide, P., Sigauque, B., Milman, J., Mandomando, I., Bassat, Q., et al.: Duration of protection with RTS, S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet 2005, 366:2012–2018. Berzofsky, J.A.: Epitope selection and design of synthetic vaccines. Molecular approaches to enhancing immunogenicity and cross-reactivity of engineered vaccines. Ann. N.Y. Acad. Sci. 1993, 690:256–264. Davenport, M.P., and Hill, A.V.: Reverse immunogenetics: from HLA-disease associations to vaccine candidates. Mol. Med. Today 1996, 2:38–45. Hill, A.V.: Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat. Rev. Immunol. 2006, 6:21–32.


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Immunology_Janeway. Hill, A.V.: Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat. Rev. Immunol. 2006, 6:21–32. Hoffman, S.L., Rogers, W.O., Carucci, D.J., and Venter, J.C.: From genomics to vaccines: malaria as a model system. Nat. Med. 1998, 4:1351–1353. Ottenhoff, T.H., Doherty, T.M., Dissel, J.T., Bang, P., Lingnau, K., Kromann, I., and Andersen, P.: First in humans: a new molecularly defined vaccine shows excellent safety and strong induction of long-lived Mycobacterium tuberculosis-specific Th1-cell like responses. Hum. Vaccin. 2010, 6:1007–1015. Zwaveling, S., Ferreira Mota, S.C., Nouta, J., Johnson, M., Lipford, G.B., Offringa, R., van der Burg, S.H., and Melief, C.J.: Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J. Immunol. 2002, 169:350–358. 16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved for use in humans.


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Immunology_Janeway. 16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved for use in humans. Coffman, R.L., Sher, A., and Seder, R.A.: Vaccine adjuvants: putting innate immunity to work. Immunity 2010, 33:492–503. Hartmann, G., Weiner, G.J., and Krieg, A.M.: CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl Acad. Sci. USA 1999, 96:9305–9310. Palucka, K., Banchereau, J., and Mellman, I.: Designing vaccines based on biology of human dendritic cell subsets. Immunity 2010, 33:464–478. Persing, D.H., Coler, R.N., Lacy, M.J., Johnson, D.A., Baldridge, J.R., Hershberg, R.M., and Reed, S.G.: Taking toll: lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol. 2002, 10:S32–S37. Pulendran, B.: Modulating vaccine responses with dendritic cells and Toll-like receptors. Immunol. Rev. 2004, 199:227–250. Takeda, K., Kaisho, T., and Akira, S.: Toll-like receptors. Annu. Rev. Immunol. 2003, 21:335–376.


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Immunology_Janeway. Takeda, K., Kaisho, T., and Akira, S.: Toll-like receptors. Annu. Rev. Immunol. 2003, 21:335–376. 16-30 Protective immunity can be induced by DNA-based vaccination. Donnelly, J.J., Ulmer, J.B., Shiver, J.W., and Liu, M.A.: DNA vaccines. Annu. Rev. Immunol. 1997, 15:617–648. Gurunathan, S., Klinman, D.M., and Seder, R.A.: DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 2000, 18:927–974. Li, L., Kim, S., Herndon, J.M., Goedegebuure, P., Belt, B.A., Satpathy, A.T., Fleming, T.P., Hansen, T.H., Murphy, K.M., and Gillanders, W.E.: Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination. Proc. Natl Acad. Sci. USA 2012, 109:12716–12721. Nchinda, G., Kuroiwa, J., Oks, M., Trumpfheller, C., Park, C.G., Huang, Y., Hannaman, D., Schlesinger, S.J., Mizenina, O., Nussenzweig, M.C., et al.: The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. J. Clin. Invest. 2008, 118:1427–1436.


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Immunology_Janeway. Wolff, J.A., and Budker, V.: The mechanism of naked DNA uptake and expression. Adv. Genet. 2005, 54:3–20. 16-31 Vaccination and checkpoint blockade may be useful in controlling existing chronic infections. Burke, R.L.: Contemporary approaches to vaccination against herpes simplex virus. Curr. Top. Microbiol. Immunol. 1992, 179:137–158. Grange, J.M., and Stanford, J.L.: Therapeutic vaccines. J. Med. Microbiol. 1996, 45:81–83. Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H., and Johnson, D.: Herpes simplex virus turns off the TAP to evade host immunity. Nature 1995, 375:411–415. Lu, W., Arraes, L.C., Ferreira, W.T., and Andrieu, J.M.: Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat. Med. 2004, 10:1359–1365. Modlin, R.L.: Th1–Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 1994, 102:828–832.


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Immunology_Janeway. Modlin, R.L.: Th1–Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 1994, 102:828–832. Plebanski, M., Proudfoot, O., Pouniotis, D., Coppel, R.L., Apostolopoulos, V., and Flannery, G.: Immunogenetics and the design of Plasmodium falciparum vaccines for use in malaria-endemic populations. J. Clin. Invest. 2002, 110:295–301. Reiner, S.L., and Locksley, R.M.: The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 1995, 13:151–177. Stanford, J.L.: The history and future of vaccination and immunotherapy for leprosy. Trop. Geogr. Med. 1994, 46:93–107. The Immunologist's Toolbox A-1. Immunization.


Immunology_Janeway

Immunology_Janeway. The Immunologist's Toolbox A-1. Immunization. Natural immune responses are normally directed at antigens borne by pathogenic microorganisms. The immune system can also be induced to respond to simple nonliving antigens, and experimental immunologists have focused on the responses to these simple antigens in developing our understanding of the immune response. The deliberate induction of an immune response is known as immunization. Experimental immunizations are routinely carried out by injecting the test antigen into the animal or human subject. The route, dose, and form in which antigen is administered can profoundly affect whether a response occurs and the type of response that is produced. The induction of protective immune responses against common microbial pathogens in humans is often called vaccination, although this term originally referred to the induction of immune responses against smallpox by immunizing with the cross-reactive virus, vaccinia.


Immunology_Janeway

Immunology_Janeway. To determine whether an immune response has occurred and to follow its course, the immunized individual is monitored for the appearance of immune reactants. Immune responses to most antigens include soluble factors, such as cytokines and specific antibodies, and cellular responses, such as the generation of specific effector T cells. Monitoring the cytokine and antibody responses usually involves the analysis of relatively crude preparations of antiserum (plural: antisera). Serum is the fluid phase of clotted blood, which, if taken from an individual immunized against a particular antigen, is called antiserum. To study immune responses mediated by T cells, blood lymphocytes or cells from lymphoid organs such as the spleen are tested; T-cell responses are more commonly studied in experimental animals than in humans.


Immunology_Janeway

Immunology_Janeway. Antisera generated by immunization with even the simplest antigen will contain many different antibody molecules that bind to the immunogen in slightly different ways. In addition, antisera contain many antibodies that do not bind at all to the immunizing antigen, because they were present in the individual prior to immunization. These nonspecific antibodies often lead to technical difficulties in using antisera for detecting an immunogen. To circumvent this problem, antibodies that bind to the immunogen can be purified by affinity chromatography using immobilized antigen (see Section A-3). Alternatively, these problems can be avoided by making monoclonal antibodies (see Section A-7).


Immunology_Janeway

Immunology_Janeway. Any substance that can elicit an immune response is said to be immunogenic and is called an immunogen. There is a clear operational distinction between an immunogen and an antigen. Immunogens are substances that elicit an adaptive immune response, whereas an antigen is defined as any substance that can bind to a specific antibody. All antigens therefore have the potential to elicit specific antibodies; however, not all antigens are immunogenic. An example of this distinction is evident when considering protein antigens. In spite of the fact that antibodies against proteins are of enormous utility in experimental biology and medicine, purified proteins are not generally immunogenic. This is because purified proteins lack microbial-associated molecular patterns (MAMPs), and therefore do not elicit an innate immune response. To provoke an immune response to a purified protein, the protein must be administered together with an adjuvant (see below).


Immunology_Janeway

Immunology_Janeway. Certain properties of antigens that favor the initiation of an adaptive immune response have been defined by studying antibody responses to simple natural proteins such as hen egg-white lysozyme, to synthetic polypeptide antigens, and to small organic molecules of simple structure. The study of antibody responses to small organic molecules, such as phenyl arsonates and nitrophenyls, was essential in defining early immunological principles. These molecules do not provoke antibodies when injected by themselves. However, antibodies can be raised against them if the molecule is attached covalently, by simple chemical reactions, to a protein carrier. Such small molecules were termed haptens (from the Greek haptein, to fasten) by the immunologist Karl Landsteiner, who first studied them in the early 20th century. He found that animals immunized with a hapten–carrier conjugate produced three distinct sets of antibodies (Fig. A.1). One set comprised hapten-specific antibodies that reacted


Immunology_Janeway

Immunology_Janeway. early 20th century. He found that animals immunized with a hapten–carrier conjugate produced three distinct sets of antibodies (Fig. A.1). One set comprised hapten-specific antibodies that reacted with the same hapten on any carrier, as well as with free hapten. The second set of antibodies was specific for the carrier protein, as shown by their ability to bind both the hapten-modified and unmodified carrier protein. Finally, some antibodies reacted only with the specific conjugate of hapten and carrier used for immunization. Landsteiner’s studies focused primarily on the antibody response to the hapten, as these small molecules could be synthesized in many closely related forms. He observed that antibodies raised against a particular hapten bind that hapten but, in general, fail to bind even very closely related chemical structures. The binding of haptens by anti-hapten antibodies has played an important part in defining the precision of antigen binding by antibody molecules.


Immunology_Janeway

Immunology_Janeway. bind even very closely related chemical structures. The binding of haptens by anti-hapten antibodies has played an important part in defining the precision of antigen binding by antibody molecules. Anti-hapten antibodies are also important medically because they mediate allergic reactions to penicillin and other compounds that elicit antibody responses when they attach to self proteins (see Section 14-10).


Immunology_Janeway

Immunology_Janeway. Fig. A.1 Antibodies can be elicited by small chemical groups called haptens only when the hapten is linked to an immunogenic protein carrier. Following immunization with a hapten–carrier conjugate, three types of antibodies are produced. One set (blue) binds the carrier protein alone and is called carrier-specific. One set (red) binds to the hapten on any carrier or to free hapten in solution and is called hapten-specific. One set (purple) binds only the specific conjugate of hapten and carrier used for immunization, apparently binding to sites at which the hapten joins the carrier, and is called conjugate-specific. The amount of antibody of each type in this serum is shown schematically in the graphs at the bottom; note that the original antigen binds more antibody than the sum of anti-hapten and anti-carrier antibodies as a result of the additional binding of conjugate-specific antibody.


Immunology_Janeway

Immunology_Janeway. The route by which antigen is administered affects both the magnitude and the type of response obtained. The most common routes by which antigen is introduced experimentally or as a vaccine into the body are into tissue by subcutaneous (s.c.) injection into the fatty layer just below the dermis or by intradermal (i.d.) or intramuscular (i.m.) injection; directly into the bloodstream by intravenous (i.v.) injection or transfusion; into the gastrointestinal tract by oral administration; and into the respiratory tract by intranasal (i.n.) administration or inhalation.


Immunology_Janeway

Immunology_Janeway. Antigens injected subcutaneously generally elicit strong responses, most probably because the antigen is taken up by resident dendritic cells in the skin and efficiently presented in local lymph nodes, and so this is the method most commonly used when the object of the experiment is to elicit specific antibodies or T cells against a given antigen. Antigens injected or transfused directly into the bloodstream tend to induce immune unresponsiveness or tolerance unless they bind to host cells or are in the form of aggregates that are readily taken up by antigen-presenting cells.


Immunology_Janeway

Immunology_Janeway. Antigen administration via the gastrointestinal tract is used mostly in the study of allergy. It has distinctive effects, frequently eliciting a local antibody response in the intestinal lamina propria, while producing a systemic state of tolerance that manifests as a diminished response to the same antigen if subsequently administered in immunogenic form elsewhere in the body (see Chapter 12). This ‘split tolerance’ may be important in avoiding allergy to antigens in food, because the local response prevents food antigens from entering the body, while the inhibition of systemic immunity helps to prevent the formation of IgE antibodies, which are the cause of such allergies (see Chapter 14).


Immunology_Janeway

Immunology_Janeway. The immune response to an antigen is also influenced by the dose of immunogen administered. Below a certain threshold dose, most proteins do not elicit any immune response. Above the threshold dose, there is a gradual increase in the response as the dose of antigen is increased, until a broad plateau level is reached, followed by a decline at very high antigen doses (Fig. A.2). In general, secondary and subsequent immune responses occur at lower antigen doses and achieve higher plateau values, which is a sign of immunological memory. Most proteins are poorly immunogenic or nonimmunogenic when administered by themselves. Strong adaptive immune responses to protein antigens almost always require that the antigen be injected in a mixture known as an adjuvant. An adjuvant is any substance that enhances the immunogenicity of substances mixed with it. Commonly used adjuvants are listed in Fig. A.3.


Immunology_Janeway

Immunology_Janeway. Adjuvants generally enhance immunogenicity in two different ways. First, adjuvants convert soluble protein antigens into particulate material, which is more readily ingested by phagocytic antigen-presenting cells such as macrophages and dendritic cells. For example, the antigen can be adsorbed on particles of the adjuvant (such as alum), be made particulate by emulsification in mineral oils, or be incorporated into the colloidal particles of immune stimulatory complexes (ISCOMs). Second, and more important, adjuvants contain PAMPs that elicit a strong innate immune response. When taken up by phagocytic cells, the PAMPs in the adjuvant stimulate inflammatory cytokine production and induce the activation of the antigen-presenting cell. The activated antigen-presenting cells upregulate abundant levels of co-stimulatory molecules that are important for activating T cells. Activated antigen-presenting cells also upregulate high levels of MHC class I and class II proteins, plus many of the


Immunology_Janeway

Immunology_Janeway. levels of co-stimulatory molecules that are important for activating T cells. Activated antigen-presenting cells also upregulate high levels of MHC class I and class II proteins, plus many of the proteins important for efficient antigen processing and presentation (see Section 3-12). Due to the strong local inflammatory responses induced by adjuvants that contain PAMPs, most of the adjuvants commonly used in experimental animals are not approved for use in humans.


Immunology_Janeway

Immunology_Janeway. The typical antigen dose–response curve shown here illustrates both the influence of dose on a primary antibody response (amounts of antibody produced expressed in arbitrary units) and the effect of the dose used for priming on a secondary antibody response elicited by a dose of antigen of 103 arbitrary mass units. Very low doses of antigen do not cause an immune response at all. Slightly higher doses seem to inhibit specific antibody production, an effect known as low-zone tolerance. Above these doses there is a steady increase in the response with antigen dose until an optimum response is reached; this response persists across a broad range of doses. Very high doses of antigen also inhibit immune responsiveness to a subsequent challenge, a phenomenon known as high-zone tolerance.


Immunology_Janeway

Immunology_Janeway. Fig. A.3 Common adjuvants and their use. Adjuvants are mixed with the antigen and usually render it particulate, which helps to retain the antigen in the body and promotes uptake by macrophages. Most adjuvants include bacteria or bacterial components that stimulate macrophages and dendritic cells, aiding in the induction of the immune response. ISCOMs are small micelles of the detergent Quil A; when viral proteins are placed in these micelles, they apparently fuse with the antigen-presenting cell, allowing the antigen to enter the cytosol. Thus, the antigen-presenting cell can stimulate a response to the viral protein, much as a virus infecting these cells would stimulate an antiviral response. Vaccines designed to elicit responses to purified proteins often include compounds that stimulate pattern recognition receptors, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), or C-type lectin receptors.


Immunology_Janeway

Immunology_Janeway. Nevertheless, some human vaccines naturally contain microbial antigens that can also act as effective adjuvants. For example, purified constituents of the bacterium Bordetella pertussis, which is the causative agent of whooping cough, are used as both antigen and adjuvant in the triplex DPT (diphtheria, pertussis, tetanus) vaccine. In addition, modified TLR ligands, such as monophosphoryl lipid A, a derivative of LPS, or poly(I):poly(C12U), a derivative of polyI:C, are currently included as components of several human vaccines. A-2 Antibody responses.


Immunology_Janeway

Immunology_Janeway. A-2 Antibody responses. B cells contribute to adaptive immunity by secreting antibodies, and the response of B cells to an injected immunogen is usually measured by analyzing the specific antibody produced in a humoral immune response. This is most conveniently achieved by assaying the antibody that accumulates in the fluid phase of the blood, or plasma; such antibodies are known as circulating antibodies. Circulating antibody is usually measured by collecting blood, allowing it to clot, and then isolating the serum from the clotted blood. The amount and characteristics of the antibody in the resulting antiserum are then determined using the assays described below. Because assays for antibody were originally conducted using antisera from immune individuals, they are commonly referred to as serological assays, and the use of antibodies in such testing is often called serology.


Immunology_Janeway

Immunology_Janeway. The most important characteristics of an antibody response are the specificity, amount, isotype or class, and affinity of the antibodies produced. The specificity determines the ability of the antibody to distinguish the immunogen from other antigens. The amount of antibody can be determined in many different ways and is a function of the number of responding B cells, their rate of antibody synthesis, and the persistence of the antibody after production. The persistence of an antibody in the plasma and extracellular fluid bathing the tissues is determined mainly by its isotype or class (see Sections 5-12 and 10-14); each isotype has a different half-life in vivo. The isotypic composition of an antibody response also determines the biological functions these antibodies can perform and the sites in which antibody will be found. Finally, the strength of binding of the antibody to its antigen in terms of a single antigen-binding site binding to a monovalent antigen is termed its affinity;


Immunology_Janeway

Immunology_Janeway. in which antibody will be found. Finally, the strength of binding of the antibody to its antigen in terms of a single antigen-binding site binding to a monovalent antigen is termed its affinity; the total binding strength of a molecule with more than one binding site is called its avidity. Binding strength is important: the higher the affinity of the antibody for its antigen, the less antibody is required to eliminate the antigen, because antibodies with higher affinity will bind at lower antigen concentrations. All these parameters of the humoral immune response help to determine the capacity of that response to protect the host from infection.


Immunology_Janeway

A-3 Affinity chromatography.

Immunology_Janeway. A-3 Affinity chromatography.


Immunology_Janeway

Immunology_Janeway. The specificity of antigen:antibody binding interactions can be exploited for the purification of a specific antigen from a complex mixture, or alternatively, for the purification of specific antibodies from antiserum containing a mixture of different antibodies. The technique employed is called affinity chromatography (Fig. A.4). For purification of an antigen, antigen-specific antibodies are bound, often covalently, to small, chemically reactive beads, which are loaded into a column. The antigen mixture is allowed to pass over the beads. The specific antigen binds; all the other components in the mixture can then be washed away. The specific antigen is then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. Affinity chromatography can also be used to purify antibodies from complex antisera by


Immunology_Janeway

Immunology_Janeway. of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. Affinity chromatography can also be used to purify antibodies from complex antisera by using beads coated with specific antigen. The technique is known as affinity chromatography because it separates molecules on the basis of their affinity for one another.


Immunology_Janeway

Immunology_Janeway. A-4 Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive inhibition assay.


Immunology_Janeway

Immunology_Janeway. Radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA) are direct binding assays for antibody (or antigen); both work on the same principle, but the means of detecting specific binding is different. Radioimmunoassays are commonly used to measure the levels of hormones in blood and tissue fluids, while ELISA assays are frequently used in viral diagnostics, for example, in detecting cases of infection with the human immunodeficiency virus (HIV), which is the cause of AIDS. For both of these methods, one needs a pure preparation of a known antigen or antibody, or both, in order to standardize the assay. We will describe the assay that is used to determine the amount of a specific antigen in a sample, for instance, the amount of HIV p24 protein in a patient’s serum. For this, a preparation of pure antibody specific for the antigen is required. One can also use RIA or ELISA to determine the amount of specific antibody in a mixture, such as serum; in this case, a preparation of


Immunology_Janeway

Immunology_Janeway. of pure antibody specific for the antigen is required. One can also use RIA or ELISA to determine the amount of specific antibody in a mixture, such as serum; in this case, a preparation of pure antigen is needed as a starting point.


Immunology_Janeway

Immunology_Janeway. For the determination of antigen concentration using RIA, pure antibody against the antigen is radioactively labeled, usually with 125I; for ELISA, an enzyme is linked chemically to the antibody. The unlabeled component, which in this case would be the solution containing an unknown amount of antigen, is attached to a solid support, such as the wells of a plastic multiwell plate, which will adsorb a certain amount of any protein. Following this, the labeled antibody


Immunology_Janeway

Immunology_Janeway. Fig. A.4 Affinity chromatography uses antigen–antibody binding to purify antigens or antibodies. To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by altering the pH, which can usually disrupt antibody– antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown). Add anti-A antibody covalently linked to enzyme Wash away unbound antibody sample 1 (antigen A) sample 2 (antigen B) Enzyme makes colored product from added colorless substrate Measure absorbance of light by colored product Fig. A.5 The principle of the enzyme-linked immunosorbent assay (ELISA).


Immunology_Janeway

Immunology_Janeway. Fig. A.5 The principle of the enzyme-linked immunosorbent assay (ELISA). To detect antigen A, purified antibody specific for antigen A is linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells, to which they bind nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins (not shown). The labeled antibody is then added to the wells under conditions that prevent nonspecific binding, so that only binding to antigen A causes the labeled antibody to be retained on the surface. Unbound labeled antibody is removed from all wells by washing, and bound antibody is detected by an enzyme-dependent color-change reaction. This assay allows arrays of wells known as microtiter plates to be read in fiberoptic multichannel spectrometers, greatly speeding the assay. Modifications of this basic assay allow antibody or antigen in unknown samples to be measured as shown in Figs. A.6 and A.25.


Immunology_Janeway

Immunology_Janeway. is added to the well and allowed to bind to the unlabeled antigen under conditions in which nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Antibody binding in RIA is measured directly in terms of the amount of radioactivity retained by the coated wells, whereas in ELISA the binding is detected by a reaction that converts a substrate into a reaction product of a different color (Fig. A.5). The color change can be read directly in the reaction tray, making data collection very easy and providing a quantitative measurement of reaction product concentration; furthermore, ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays. In a variation of this assay, labeled antiimmunoglobulin antibodies can also be used in RIA or ELISA as a second layer, following the binding of unlabeled antibody to unlabeled antigen-coated plates. The use of such a second layer amplifies the signal,


Immunology_Janeway

Immunology_Janeway. antibodies can also be used in RIA or ELISA as a second layer, following the binding of unlabeled antibody to unlabeled antigen-coated plates. The use of such a second layer amplifies the signal, because at least two molecules of the labeled anti-immunoglobulin antibody are able to bind to each unlabeled antibody. As mentioned above, RIA and ELISA can also be carried out in reverse when the goal is to determine the amount of antibody in a solution; in this case, unlabeled antibody is adhered to the plates, labeled antigen is added, and the amount of labeled antigen bound after washing is measured.


Immunology_Janeway

Immunology_Janeway. A modification of ELISA known as a capture or sandwich ELISA (or more generally as an antigencapture assay) is commonly used to detect secreted products such as cytokines. Rather than the antigen being directly attached to a plastic plate, antigen-specific antibodies are bound to the plate. These are able to bind antigen with high affinity, and thus concentrate it on the surface of the plate, even with antigens that are present in very low concentrations in the initial mixture. A separate labeled antibody that recognizes a different epitope from that recognized by the immobilized first antibody is then used to detect the bound antigen.


Immunology_Janeway

Immunology_Janeway. Another variant of the antigen-capture assay, often referred to as a multiplex assay, has been developed to allow quantitation of multiple antigens in a single sample. This technique is often utilized to examine the levels of multiple cytokines in clinical serum samples, or in sera from experimental animals, cases in which it is not feasible to assess each cytokine of interest individually. For this type of assay, small microspheres are differentially labeled with fluorescent dyes that can be distinguished based on their distinct emission spectra. Microspheres labeled with a given fluorescent dye are conjugated to antibodies specific for one antigen, for instance, a single cytokine. The microspheres— up to 100 different microspheres with unique identifiers—are added to the sample to capture the antigen. Bound antigen is then detected using a second antibody that binds the antigen at a distinct site. This second antibody is conjugated to a different fluorescent dye, and the magnitude


Immunology_Janeway

Immunology_Janeway. the antigen. Bound antigen is then detected using a second antibody that binds the antigen at a distinct site. This second antibody is conjugated to a different fluorescent dye, and the magnitude of its fluorescence is a measure of the quantity of bound antigen. The machine that performs this multiplex analysis, the Luminex® analyzer, then measures the amount of fluorescence associated with each differentially labeled microsphere.


Immunology_Janeway

Immunology_Janeway. These assays illustrate two crucial aspects of all serological assays. First, at least one of the reagents must be available in a pure, detectable form in order to obtain quantitative information. Second, there must be a means of separating the bound fraction of the labeled reagent from the unbound, free fraction so that the percentage of specific binding can be determined. Normally, this separation is achieved by having the unlabeled partner trapped on a solid support. Labeled molecules that do not bind can then be washed away, leaving just the labeled partner that has bound. In Fig. A.5, the unlabeled antigen is attached to the well and the labeled antibody is trapped by binding to it. The separation of bound reagent from the free fraction is an essential step in every assay that uses antibodies.


Immunology_Janeway

Immunology_Janeway. RIA and ELISA do not allow one to measure directly the amount of antigen or antibody in a sample of unknown composition, because both depend on the binding of a pure labeled antigen or antibody. There are various ways around Add labeled antigen A Add labeled antigen A + unlabeled antigen 100 50 1 2 0 standard curve Anti-A antibody bound Wash away unbound antigen Count bound label Known concentration of antigen A Mixture containing low concentration of antigen A Amount of competitor added Percent control binding Compete with standard curve curve 1 less inhibition curve 2 Fig. A.6 Competitive inhibition assay for antigen in unknown samples.


Immunology_Janeway

Immunology_Janeway. Fig. A.6 Competitive inhibition assay for antigen in unknown samples. A fixed amount of unlabeled antibody is attached to a set of wells, and a standard reference preparation of a labeled antigen is bound to it. Unlabeled standard or test samples are then added in various amounts and the displacement of labeled antigen is measured, generating characteristic inhibition curves. A standard curve is obtained by using known amounts of unlabeled antigen identical to that used as the labeled species, and comparison with this curve allows the amount of antigen in unknown samples to be calculated. The green line on the graph represents a sample lacking any substance that reacts with anti-A antibodies.


Immunology_Janeway

Immunology_Janeway. this problem, one of which is to use a competitive inhibition assay, as shown in Fig. A.6. In this type of assay, the presence and amount of a particular antigen in an unknown sample are determined by the antigen’s ability to compete with a labeled reference antigen for binding to an antibody attached to a plastic well. A standard curve is first constructed by adding various amounts of a known, unlabeled standard preparation; the assay can then measure the amount of antigen in unknown samples by comparison with the standard. The competitive binding assay can also be used for measuring antibody in a sample of unknown composition by attaching the appropriate antigen to the plate and measuring the ability of the test sample to inhibit the binding of a labeled specific antibody. A-5 Hemagglutination and blood typing.


Immunology_Janeway

Immunology_Janeway. A-5 Hemagglutination and blood typing. The direct measurement of antibody binding to antigen is used in most quantitative serological assays. However, some important assays are based on the ability of antibody binding to alter the physical state of the antigen to which the antibody binds. These secondary interactions can be detected in a variety of ways. For instance, when the antigen is displayed on the surface of a large particle such as a bacterium, antibodies can cause the bacteria to clump, or agglutinate. The same principle applies to the reactions used in blood typing, only here the target antigens are on the surface of red blood cells and the clumping reaction caused by antibodies against them is called hemagglutination (from the Greek haima, blood).


Immunology_Janeway

Immunology_Janeway. Fig. A.7 Hemagglutination is used to type blood groups and match compatible donors and recipients for blood transfusion. Common gut bacteria bear antigens that are similar or identical to blood-group antigens, and these stimulate the formation of antibodies against these antigens in individuals who do not bear the corresponding antigen on their own red blood cells (left column); thus, type O individuals, who lack A and B, have both anti-A and anti-B antibodies, whereas type AB individuals have neither. The pattern of agglutination of the red blood cells of a transfusion donor or recipient with anti-A and anti-B antibodies reveals the individual’s ABO blood group. Before transfusion, the serum of the recipient is also tested for antibodies that agglutinate the red blood cells of the donor, and vice versa, a procedure called a cross-match, which may detect potentially harmful antibodies against other blood groups that are not part of the ABO system.


Immunology_Janeway

Immunology_Janeway. Hemagglutination is used to determine the ABO blood group of blood donors and transfusion recipients. Clumping or agglutination is induced by antibodies or agglutinins called anti-A or anti-B that bind to the A or B blood-group substances, respectively (Fig. A.7). These blood-group antigens are arrayed in many copies on the surface of the red blood cell, causing the cells to agglutinate when cross-linked by antibodies. Because hemagglutination involves the cross-linking of blood cells by the simultaneous binding of antibody molecules to identical antigens on different cells, this reaction also demonstrates that each antibody molecule must have at least two identical antigen-binding sites. A-6 Coombs tests and the detection of rhesus incompatibility.


Immunology_Janeway

Immunology_Janeway. Coombs tests use anti-immunoglobulin antibodies to detect the antibodies that cause hemolytic disease of the newborn, or erythroblastosis fetalis. Anti-immunoglobulin antibodies were first developed by Robin Coombs, and the test for this disease is still called the Coombs test. Hemolytic disease of the newborn occurs when a mother makes IgG antibodies specific for the rhesus or Rh bloodgroup antigen expressed on the red blood cells of her fetus. Rh-negative mothers make these antibodies when they are exposed at delivery to Rh-positive fetal red blood cells bearing the paternally inherited Rh antigen. During subsequent pregnancies, these antibodies are transported across the placenta to the fetus. This normal process is generally beneficial, as it protects newborn infants against infection. However, IgG anti-Rh antibodies coat the fetal red blood cells, which are then destroyed by phagocytic cells in the liver, causing a hemolytic anemia in the fetus and newborn infant.


Immunology_Janeway

Immunology_Janeway. Rh antigens are widely spaced on the red blood cell surface, and so the IgG anti-Rh antibodies do not bind in the correct conformation to fix complement and so do not cause lysis of red blood cells in vitro. Furthermore, for reasons that are not fully understood, antibodies against Rh antigens do not agglutinate red blood cells, unlike antibodies against the ABO blood-group antigens. Thus, detecting anti-Rh antibodies was difficult until anti-human immunoglobulin antibodies were developed. With these, maternal IgG antibodies bound to the fetal red blood cells can be detected after washing the cells to remove unbound immunoglobulin that is present in the fetal serum. Adding anti-human immunoglobulin antibodies against the washed fetal red blood cells agglutinates any cells to which maternal antibodies are bound. This is the direct Coombs test (Fig. A.8), so called because it directly detects antibody bound to the surface of the fetal red blood cells. An indirect Coombs test is used to


Immunology_Janeway

Immunology_Janeway. antibodies are bound. This is the direct Coombs test (Fig. A.8), so called because it directly detects antibody bound to the surface of the fetal red blood cells. An indirect Coombs test is used to detect nonagglutinating anti-Rh antibody in maternal serum: the serum is first incubated with Rh-positive red blood cells, which bind the anti-Rh antibody, after which the antibody-coated cells are washed to remove unbound immunoglobulin and are then agglutinated with antiimmunoglobulin antibody (see Fig. A.8). The indirect Coombs test allows Rh incompatibilities that might lead to hemolytic disease of the newborn to be detected, and this knowledge allows the disease to be prevented (see Section 15-10). The Coombs test is also commonly used to detect antibodies against drugs that bind to red blood cells and cause hemolytic anemia.


Immunology_Janeway

A-7 Monoclonal antibodies.

Immunology_Janeway. A-7 Monoclonal antibodies.


Immunology_Janeway

Immunology_Janeway. The antibodies generated in a natural immune response or after immunization in the laboratory are a mixture of molecules of different specificities and affinities. Some of this heterogeneity results from the production of antibodies that bind to different epitopes on the immunizing antigen, but even antibodies directed at a single antigenic determinant such as a hapten can be markedly heterogeneous, as shown by isoelectric focusing. In this technique, proteins are separated on the basis of their isoelectric point, the pH at which their net charge is zero. By electrophoresing proteins in a pH gradient for long enough, each molecule migrates along the pH gradient until it reaches the pH at which it is neutral and is thus concentrated (focused) at that point. When antiserum containing anti-hapten antibodies is treated in this way and then transferred to a solid support such as nitrocellulose paper, the anti-hapten antibodies can be detected by their ability to bind labeled hapten. The


Immunology_Janeway

Immunology_Janeway. antibodies is treated in this way and then transferred to a solid support such as nitrocellulose paper, the anti-hapten antibodies can be detected by their ability to bind labeled hapten. The binding of antibodies of various isoelectric points to the hapten shows that even antibodies that bind the same antigenic determinant can be heterogeneous.


Immunology_Janeway

Immunology_Janeway. Antisera are valuable for many biological purposes but they have certain inherent disadvantages that relate to the heterogeneity of the antibodies they contain. First, each antiserum is different from all other antisera, even if raised in a genetically identical animal by using the identical preparation of antigen and the same immunization protocol. Second, antisera can be produced in only limited volumes, and thus it is impossible to use the identical serological reagent in a long or complex series of experiments or clinical tests. Finally, even antibodies purified by affinity chromatography (see Section A-3) can include minor populations of antibodies that give unexpected cross-reactions, which confound the analysis of experiments. To avoid these problems, and to harness the full potential of antibodies, it was necessary to develop a way of making an unlimited supply of antibody molecules of homogeneous structure and known specificity. This has been achieved through the production


Immunology_Janeway

Immunology_Janeway. of antibodies, it was necessary to develop a way of making an unlimited supply of antibody molecules of homogeneous structure and known specificity. This has been achieved through the production of monoclonal antibodies from cultures of hybrid antibody-forming cells or, more recently, by genetic engineering.


Immunology_Janeway

Immunology_Janeway. Starting in the 1950s, biochemists in search of a homogeneous preparation of antibody that they could subject to detailed chemical analysis turned to proteins produced by patients with multiple myeloma, a common tumor of plasma cells. It was known that antibodies are normally produced by plasma cells, and because this disease is associated with the presence of large amounts of a homogeneous gamma globulin called a myeloma protein in the patient’s serum, it seemed likely that myeloma proteins would serve as models for


Immunology_Janeway

Immunology_Janeway. Fig. A.8 The Coombs direct and indirect anti-globulin tests for antibody against red blood cell antigens. An Rh– mother of an Rh+ fetus can become immunized to fetal red blood cells that enter the maternal circulation at the time of delivery. In a subsequent pregnancy with an Rh+ fetus, IgG anti-Rh antibodies can cross the placenta and damage the fetal red blood cells. In contrast to anti-Rh antibodies, maternal anti-ABO antibodies are of the IgM isotype and cannot cross the placenta, and so do not cause harm. Anti-Rh antibodies do not agglutinate red blood cells, but their presence on the fetal red blood cell surface can be shown by washing away unbound immunoglobulin and then adding antibody against human immunoglobulin, which agglutinates the antibody-coated cells. Anti-Rh antibodies can be detected in the mother’s serum in an indirect Coombs test; the serum is incubated with Rh+ red blood cells, and once the antibody has bound, the red blood cells are treated as in the direct


Immunology_Janeway

Immunology_Janeway. can be detected in the mother’s serum in an indirect Coombs test; the serum is incubated with Rh+ red blood cells, and once the antibody has bound, the red blood cells are treated as in the direct Coombs test.