EX2119
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01426
`
`1
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`Purposes of Immunization
`
`Infectious Disease Prevention
`
`Traditional vaccines have used immunization processes to
`protect an individual against potential future exposure to an
`infectious agent. Even when the process is not 100% effi-
`cacious, a sufficient proportion of the population is pro—
`tected and “herd immunity” is created. In such a case, the
`population is protected against subsequent infections of in-
`dividuals and endemic persistence of the infectious agent.
`When an infectious agent infects one individual, another
`susceptible host is not encountered in the immediate envi-
`ronment and transmission of the organism cannot occur be-
`fore the transmissible stage of the life cycle of the infection
`is passed.
`A large number of diseases exist for which there is no
`effective preventive vaccine, usually because the traditional
`protocol of immunization—with a live attenuated organism,
`an inactivated organism, or antigenic components of an or-
`ganism combined with aluminium-based adjuvants—has
`not been demonstrated to be safe and effective. In many
`cases, complex immune interactions with the infectious
`agents may suppress or moderate the host response. In these
`situations, a simple antibody response against primary sur-
`face antigens is not effective. Other strategies used to evade
`elimination by the host immune system include the follow-
`ing: antigenic variation, latency of infection, ability to rep-
`licate intracellularly or within immunoprivileged sites,
`immune suppression induced through mediators or infection
`and destruction of crucial immune cells, and integration of
`viral DNA into the host cell genome. The process of induc-
`ing responses against poorly immunogenic antigens has im-
`proved in recent years, particularly for polysaccharide
`antigens such as Streptococcus pneumoniae conjugate vac—
`cines, wherein lack of immunogenicity has been overcome
`by conjugation with larger, more immunogenic molecules
`(Moreau 1999).
`
`The efficacy of standard vaccines has typically been
`measured by the degree of humoral response. Protection
`against disease for which many next-generation vaccines
`are currently being developed depends on cell-mediated im—
`munity (CMII) and specific responses in tissues such as
`
`
`
`‘Abbreviations used in this article : ALVAC-FIV, canarypox virus-based
`feline immunodeficiency virus; BCG, Bacille Calmette—Guérin; CMI, cell-
`mediated immunity; CpG motifs, sections of olgonuceotide with a high
`concentration of cytosine-guanine dinucleotides, more prevalent in pro-
`karyotic cells; CTL, cytotoxic T lymphocyte; DTH, delayed-type hyper-
`sensitivity; ELISA, enzyme—linked immunosorbent assay; FIV, feline
`immunodeficiency virus; HIV, human immunodeficiency virus; HLA, hu-
`man lymphocyte antigen; Ig, immunoglobulin; IL, interleukin; KLH, key-
`hole limpet hemocyanin; LT, heat-labile enterotoxin; MAb, monoclonal
`antibody; MHC, major histocompatibility complex; SCID, severe com-
`bined immunodeficient; SPF, specific pathogen-free; TCR, T cell receptor;
`TLR, toll—like receptor; TNF, tumor necrosis factor; YAC, yeast artificial
`chromosome.
`
`mucosa of the lung, gut, or reproductive tract. Tuberculosis
`is a typical example for which new protocols add adjuvants
`to induce type 1 responses, and new routes (e.g., oral vac-
`cination) are used to induce a lung mucosal CMI response
`by targeting the gut mucosa (Doherty et al. 2002).
`Several additional strategies are currently being used to
`produce vaccines for infections against which traditional
`vaccines have not been successful. Examples of strategies
`used to induce effective immunity without adverse conse-
`quences include subcomponent vaccines, peptide—based
`vaccines, recombinant DNA vaccines, chimeric vectors, and
`alternative routes of administration. These strategies are de-
`scribed further in the respective sections of the text below.
`
`Maternal and Fetal Immunization
`
`Neonates are particularly susceptible to infectious disease
`and are very dependent on maternal antibodies for protec-
`tion until they can develop and mount their own immune
`responses. Neonatal vaccination is complicated by the ma-
`ternal antibodies and the immaturity of the immune system,
`but the advent of conjugate vaccines and greater under-
`standing of the immune system are providing opportunities
`for its application (Marchant and Newport 2001). Routine
`prophylactic immunization has focused to date on protect-
`ing neonates after the maternal antibodies wane. Maternal
`
`immunization, in which the dam is immunized against a
`specific disease to develop high levels of antibodies to pass
`on to her newborn, has been practiced for many years in
`veterinary medicine and is being seriously investigated as
`an approach in human medicine (Lehmann et al. 2003).
`Placental anatomy and the process of antibody transfer vary
`greatly between species. Nonhuman primate species have a
`placental anatomy and physiology most closely linked to
`humans, which makes them appropriate for this type of
`research.
`
`The concept of fetal immunization has been demon-
`strated successfully in various animal models. Fetal immu-
`nization with a protein antigen has been studied in baboons,
`and a single DNA immunization against a truncated form of
`glycoprotein D of bovine herpesvirus—l
`into the amniotic
`fluid of the oral cavity has resulted in high serum antibody
`titers and cell—mediated immune response in lambs (Gerdts
`et a1. 2002).
`
`Production of Antitoxin
`
`Immunization has been used to produce antiserum since the
`18905. The antiserum produced is rich in antibodies against -
`the specific antigen inoculated. These processed antisera or
`extracted immunoglobulins are used to treat life-threatening
`conditions such as rabies infection, diphtheria,
`tetanus,
`botulinum intoxication, and venomous snakebites. Anti-
`
`toxin and antivenom are generally manufactured from an-
`tibody-rich serum produced by horses 'or other large
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`animals. Multiple booster inoculations of small quantities of
`toxin or venom 'are used to induce the production of a hy-
`perimmune response. The booster inoculations produce a
`large quantity of antibodies that also have a greater affinity
`for the inciting antigen.
`
`Cancer Treatment
`
`The hypothesis of using vaccines to stimulate the immune
`system to prevent and treat cancer has been considered for
`more than a century. In Table l, the applications of vaccines
`in cancer therapy are listed. Bacille Calmette-Guérin
`(BCGI) is used around the world to treat bladder cancer
`(Kassouf and Kamat 2004). Vaccines may also be used to
`prevent infection by pathogens known to predispose indi-
`viduals to cancer such as hepatitis B virus or Chlamydia
`spp. (Moingeon 2001).
`Several immunological vaccines have been tested in
`clinical trials for treatment of melanoma. Whole cell vac-
`
`cines (allogeneic and autologous cellular vaccines) com—
`prise a broad spectrum of antigenic targets. Ganglioside
`vaccines have been prepared with defined purified antigens
`that may allow for a specific type of immune response
`(Guthman et al. 2004). Other types of cancer vaccines under
`development include DNA vaccines, heat shock protein—
`based vaccines, peptide vaccines, and dendritic cell vac-
`cines (Wolochok and Livingston 2001).
`The majority of current research on cancer vaccines is
`focused on tumor—associated vaccines. In theory, cancer
`vaccines should provide a specific immune response against
`the primary tumor and result in strong immune memory to
`prevent recurrence. Antigenic differences between normal
`and malignant cells provide the basis of cancer vaccines to
`stimulate tumor-specific immune responses. Advances in
`molecular characterization of tumors have identified tumor-
`
`associated antigens that are potential targets for use in cancer
`immunization protocols (Conroy et al. 1996; Moingeon 2001).
`Immunological approaches in cancer vaccines are var—
`ied. The normal immune response is not sufficient to eradi—
`cate tumor cells, and cancer cells may escape detection via
`
`secretion of immunosuppressive factors, down regulation of
`antigen expression, major histocompatibility complex mol—
`ecules, or lack of costimulation. Vaccines may be developed
`that transfer genes of immune-stimulant cells and produce
`appropriate cytokines or that manipulate antigen—presenting
`cells such as dendritic cells. Direct injection of immature
`dendritic cells into tumors is a novel approach being used to
`induce an immune response based on the processing and
`presenting of existing antigens from apoptotic cells (Kim et
`al. 2004a,b). DNA vaccines may also be used after conven—
`tional treatments to eliminate metastasis (El-Aneed 2004).
`
`Other Therapeutic Vaccines
`
`Vaccines are under development for a number of chronic
`infectious and degenerative diseases. Human immunodefi-
`ciency virus (HIVI) is one example. Whole virus, killed
`virus, and recombinant vaccines have been examined for
`this purpose but have not shown sufficient efficacy. Vac—
`cines using recombinant live virus vectors appear to have
`promise, demonstrating both safety and a cytotoxic lympho—
`cyte response (Rocha et al. 2004).
`A highly effective vaccine exists that can prevent hepa-
`titis B, and research is now focused on a therapeutic vac-
`cine. Inducing Thl responses to the hepatitis B core antigen,
`has been demonstrated to be important in recovery from
`disease and infection. Peptide vaccines including B cell- and
`T cell-inducing peptide epitopes have been tested in animal
`models and clinical trials (Arnon and Ben-Yedidia 2003).
`Core antigen loaded nanoparticles are also being developed
`as a strategy to induce an appropriate therapeutic immune
`response (Chong et al. 2005).
`Vaccines are being developed to treat other disease con-
`ditions, including tuberculosis, parasitic disease, and gastric
`ulcers (Arnon and Ben—Yedidia 2003; Sela et a1. 2002).
`Neurodegenerative conditions such as Huntington’s and
`Alzheimer’s diseases, in which there is an abnormal accu-
`mulation of protein aggregates, are also potential candidates
`for .vaccine treatment (Sela et al. 2002). Nonspecific meth-
`ods of treating these and other neurodegenerative disorders
`
`Table 1 Possible application of vaccines against cancer3
`
`Comments
`Modality
`Status
`
`Vaccines (prophylactic or therapeutic
`against pathogens predisposing to
`specific cancers
`Therapeutic (adjuvant setting)
`
`Ongoing clinical studies—
`hepatitis B vaccine available
`
`Ongoing clinical trials
`
`Therapeutic (metastatic disease)
`
`Ongoing clinical trials
`
`Other targets include oncogenic
`papilloma viruses, hepatitis C,
`Helicobacter pylori
`Aim is to prevent recurrence after
`surgical removal
`Aim is to control and maintain quality of
`life
`
`Prophylactic
`
`Aim is to prevent high-risk healthy people
`from developing cancer
`
`
`Theoretical
`
`éAdapted from Moingeon P. 2001. Cancer vaccines. Vaccine 19:1305-1326.
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`include using vaccines to boost the aging immune system
`(Schwartz and Kipnis 2004).
`
`Therapeutic Antibodies
`
`Monoclonal antibodies (MAbsl) were originally proposed
`for use in a variety of chronic inflammatory diseases and for
`preventing transplant organ rejection. MAb therapy has
`been used to induce immunosuppression and prevent organ
`rejection. OKT3, the first MAb approved for this indication,
`inhibits T cell responses by targeted binding of the pan-T
`cell marker CD3. More recently approved humanized neu—
`tralizing MAbs target the interleukin (IL‘)-2 receptor
`or-chain.
`
`Antibodies protect organisms by binding and neutraliz-
`ing active molecules, enabling phagocytosis, and activating
`complement. By capitalizing on some of these functions to
`inhibit the activity of proinflammatory molecules, they can
`serve as a useful tool in the potential treatment of chronic
`inflammatory disease. Antitumor necrosis factor or (TNFOLI)
`MAbs have been used in the treatment of rheumatoid ar—
`
`thritis. Anti-TNFOL MAbs developed from a mousezhuman
`chimerized antibody is currently licensed as Inflixmab.
`Clinical studies have confirmed efficacy with improved
`clinical responses and arresting of joint degeneration. Stud-
`ies are also ongoing to investigate anti-IL—l and lL—6 MAb
`in rheumatoid arthritis (Andreakos et al. 2002).
`Antl-TNFOL therapy has also been studied in other chronic
`immune and inflammatory conditions such as Crohn’s disease,
`spondyloarthropathies, juvenile arthritis, and psoriasis. Clini-
`cal trials have shown promising response in these diseases.
`Studies investigating the role of antibodies in the treatment
`and prevention of prion diseases have also shown promise.
`In vitro studies demonstrate that cultures appear to be rid of
`the agent, but animal studies are difficult to conduct due to
`the long incubation of the disease (Sela et al. 2002).
`Murine MAbs, although effective, may be poorly toler-
`ated in humans as multidose therapeutics. Chimeric anti—
`body technology followed by humanization may provide
`antibodies that are still immunogenic. Fully human antibod-
`ies are preferred, but to date, there has been limited success
`in developing human B cell hybridomas. Transgenic mouse
`technologies have allowed the introduction of transgenes on
`yeast artificial chromosomes (YACsl) into the mouse germ-
`line, generating mice with larger portions of the human
`immunoglobulin (Ig') loci. XenoMouse® contain large
`base-sized YACs from which IgG MAbs have a diverse
`human adult-like repertoire with the CDR3 regions more
`similar in length to human than to mouse (Houdebine 2002;
`Kellermann and Green 2002).
`
`Diagnostic Reagents
`
`Immunization for antibodies has been used to produce re-
`agents for diagnostic tests that depend on antibodies as part
`
`of the detection systems, such as radioimmune assays and
`enzyme-linked immunosorbent assays (ELISAsl). Many of
`the diagnostic tools used to identify proteins or examine the
`immune system depend on monoclonal or polyclonal anti-
`bodies to bind a specific molecule. Detection of the bound
`complex occurs through light scattering or tagging of the
`antibody with radioisotopes, fluorescent molecules, or en-
`zymes to elicit a color change.
`
`Treatment of Allergies
`
`Immunization with food proteins at an appropriate level can
`sensitize animals and cause conditions that mimic human
`
`food allergies. One example is a protocol successfully used
`in dogs to induce peanut and other nut allergies by 6 mo of
`age. Animals are inoculated s.c. with 1 ug of protein extract
`in alum, first at birth and then at 3, 7, and ll wk of age,
`immediately after modified live virus vaccinations (Teuber
`et al. 2002). Specific allergen immunotherapy has been ef-
`fective in treating rhinitis and anaphylaxis. Immunostimu-
`latory DNA has been studied in models of allergen-induced
`airway inflammation and has shown promising results in
`mice (Silverman and Drazen 2003; Walker and Zuany-
`Amorium 2001).
`
`Research Models
`
`As a technique, immunization continues to be used exten-
`sively and has benefited from the large number of currently
`available animal models with well-defined immune cell de—
`
`ficiencies and from the increasing availability of immune
`modulators such as interleukins. These tools have helped to
`define more completely how the immune response is con—
`trolled. Initially, mice deficient in specific lymphocyte
`populations (e.g., natural killer cell-deficient mice, nude
`athymic mice, and severe combined immunodeficient
`(SCIDI) mice) became available and were widely used.
`More recently, transgenic mice have provided opportunities
`to study the immune system in even greater detail. For
`example, the ability of an adjuvant to activate toll—like re-
`ceptors (TLRsl) is derived from a lack of effect on mice
`deficient in TLR-4.
`
`In addition to the use of immunization techniques to
`study the immune system itself, immunization is frequently
`used to block biological reactions using antibodies. This
`application has been frequent in reproductive research in
`which neutralizing particular peptides, proteins, or cell sur-
`face antigens are used to study reproductive physiology and
`the etiology of specific diseases.
`
`Autoimmunity and Degenerative Disease Models
`
`Immunization is used to generate models of diseases that
`have an autoimmune basis. A list of examples is included
`in Table 2. Many degenerative inflammatory conditions,
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`from diabetes to multiple sclerosis, are understood to have
`a misdirected immune response that induces pathology.
`Combinations of immunizations to mimic the conditions
`and animal models with similar immune alterations are used
`
`to understand and develop therapies for these conditions.
`For example, a systemic lupus erythematosus—like syn—
`drome has been established in mice by inoculation with
`active chromatin (Li et al. 2004). Multiple sclerosis-like
`experimental allergic encephalitis has also been established
`using immunization.
`Immunization protocols are also used to investigate po-
`tential therapies. To treat multiple sclerosis-like inflamma-
`tory disease in a mouse model, one successful protocol uses
`autologous attenuated autoreactive T cells to induce an im—
`mune response specifically against autoreactive cells in or-
`der to attenuate the condition (Stinissen et al. 1996). Beta
`crystalline autoantibodies can contribute to the development
`of cataracts. In mice, oral administration of lens homog-
`enate combined with immunization against beta-crystallins
`in adjuvant has been shown to suppress anti-beta crystalline
`antibody production (Sueno et al. 1997).
`
`Screening Potential Vaccines and Adjuvants
`
`As part of the initiative to find and introduce new vaccines
`and new adjuvants, it is necessary to develop methods to
`determine and optimize the in vivo response to new candi-
`date agents. Bringing a potential agent from discovery to
`clinical use is an exercise than can take many years and
`consume many millions of dollars. Screening is the part of
`
`the process that can differentiate between many potential
`antigens, multiple combinations of antigens, and different
`adjuvant matrices, in an effort to choose those agents with
`the most potential for success. Immunization protocols in
`animals are useful for this process because they incorporate
`the biological complexities of the immune system that may
`be predictive of the result in the final host as well as being
`predictive of adverse secondary affects. For example, a for—
`eign peptide that is nonimmunogenic in a mouse may be
`more likely to be nonimmunogenic in a human. The pre-
`dictive ability of one animal species for another is not com-
`plete. The mouse response to DNA vaccination has been
`very successful, but such success has not translated as pre—
`dictably in other mammalian species such as dogs and hu-
`mans (Kutzler and Weiner 2004).
`With an allowance for the constraints of interpretation,
`animal models have been very useful for screening. Proteins
`can present with many different antigenic sites. Screening is
`used to determine which sites will most likely provide the
`wanted immune response. The human lymphocyte antigen
`(HLA‘) transgenic mouse expresses human major histo—
`compatibility complex (MHCI) molecules and is an ex-
`ample of a transgenic mouse strain that has been used for
`screening a battery of antigen candidates for potential hu-
`man CMI responses (Firat et al. 1999).
`
`Quality Control/Testing
`
`Animal immunization is required for testing and quality
`control of vaccines before release to the market. Testing is
`
`Table 2 Examples of autoimmune degenerative diseases with immunization-induced models
`
`
`
` Disease Model Animal Reference (see text)
`
`
`
`
`
`C57BL6/J mouse
`
`Experimental autoimmune
`encephalitis induced by
`multiple antigen peptide
`myeiin oligodendticyte
`giycoprotein 35-55
`(MOG35-55)
`Inoculation with active chromatin Mouse
`
`Rat adjuvant arthritis
`Collagen-induced arthritis
`
`Fiat
`Mouse
`
`Guinea pig
`
`Costa et al. 2003
`
`Li et al. 2004
`
`Ku et al. 1993
`Chiocchia et al. 1993;
`Nagler-Anderson et al. 1986
`Breeling et al. 1988
`
`Multiple sclerosis
`
`Systemic lupus
`erythematosus
`Rheumatoid arthritis
`Autoimmune disease
`
`Ulcerative colitis
`
`Interstitial cystitis
`
`Autoimmune uveitls
`
`Athelosclerosis
`
`Carageenan model enhanced
`by immunization with
`Bacteroides vulgatus
`Bladder homogenate and
`complete Freund’s adjuvant
`immunization
`
`lnterphotoreceptor retinoid
`binding protein
`Immunization with heat shock
`protein 65
`
`Rat
`
`Luber-Narod et al. 1996
`
`C57BL/6 mouse
`
`Willbanks et al. 1997
`
`Afek et al. 2000
`
`Low-density lipoprotein
`receptor-deficient
`(LDL-RD) mice
`
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`mandated by regulatory bodies to ensure identity, purity,
`safety, and efficacy (antigenicity or potency) of each batch
`of vaccine. The purpose of a potency test is to ascertain that
`the batch being tested is capable of an adequate biological
`response and has a potency that reaches a level demon—
`strated to be protective in humans. The purpose of an anti-
`genicity or imrnunogenicity test is to compare consistency
`of manufacturing, using antibody levels as a measure that
`each batch contains the same amount of immunogen as the
`standard batch. Alternative methods for more precisely
`measuring the biochemical quality or purity of antigens
`avoid the need for an in vivo immunization protocol for
`some vaccines. For many older vaccines, the precise immu-
`nogens or biochemical determinants of potency have not
`been isolated.
`
`Unlike typical immunization protocols that use a prime-
`boost strategy, the protocol for imrnunogenicity testing is
`often limited to a single dose because the first response is
`most capable of discriminating the amount and quality of
`the immunogen. A booster response is influenced by other
`factors, and the results can be less discriminating in detect-
`ing differences between two different vaccine lots. In all
`cases, it is important to have supporting data to establish the
`appropriate animal species, method of analysis, and speci—
`fications. For traditional vaccines, clearly defined testing
`protocols are often established by federal licensing agen-
`cies. For new vaccines, appropriate in Vivo and in vitro tests
`must be established as a critical part of the development
`plan. These tests are often linked with the clinical efficacy
`trials.
`
`A typical vaccine potency test measures the level of
`protection either against a direct challenge, using a known
`quantity of infectious organisms, or through an indirect step,
`such as exposing toxin to neutralizing serum before chal-
`lenge. The determination of potency in these cases is gen-
`erally made through a series of dilutions that are compared
`with dilutions of a standard reference prepared under the
`same conditions. Based on this comparison, a quantitative
`result can be assigned in accordance with units assigned to
`the reference vaccine. Once sufficient experience is ob—
`tained to demonstrate the consistency of the production and
`testing systems, it is possible to move to a single dilution
`test that demonstrates the consistent achievement of a mini—
`mum value (Akkermans et al. 1996). This process limits the
`number of animals required.
`Some challenge protection test methods (e. g., the mouse
`pertussis intracerebral challenge test, also known as the
`Kendrick test) remain in use because they have been linked
`to field or clinical trial data (Standfast 1958). As a refine-
`ment of these challenge potency tests, in vitro methods are
`being introduced to assess antibody levels by serological
`assays such as ELISA or toxin neutralization assays that
`have demonstrated correlation with the potency tests. Many
`regulatory jurisdictions are beginning to accept this refine—
`ment for tetanus and diphtheria testing (Sesardic et al.
`1999).
`Tests using immunizations are also conducted to ensure
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`that the detoxification part of the vaccine manufacture pro-.
`cess is complete. The current pertussis toxin detection test
`involves immunization with a pertussis toxin-containing
`vaccine to induce a sensitized state before challenge with
`histamine. An in vivo Chinese hamster ovary test is pro-
`posed to replace the mouse sensitization test used to detect
`the presence of pertussis toxin in acellular pertussis vac-
`cines, but the consistency of correlation between the in Vivo
`and in vitro tests is still under discussion (Kataoka et al.
`2002).
`The animals most commonly used in quality control
`testing protocols are guinea pigs, mice, rats, and nonhuman
`primates. Generally, an i.p. or so. injection is used, both
`because an appropriate response can be obtained and be-
`cause a large number of repeatable inoculations can gener—
`ally be performed.
`Many current vaccines are combinations of antigens.
`Tests that have been established for each of the individual
`valences are applied to these vaccines. It is important to
`ensure that no components of the multivalent vaccine inter-
`fere with the testing. The intensity of the antibody response
`to each individual antigen may vary between mouse strains.
`For a single vaccine valence,
`inbred strains produce the
`most repeatable result whereas an outbred stock that re—
`sponds sufficiently to all valences may be ideal for measur—
`ing the response to a multivalent vaccine. Several inbred
`strains could also be used to obtain the maximum response
`to each valence. Work is also ongoing to assess the feasi—
`bility of reducing the number of animals required by using
`the in vitro assessment of the immunization response to
`enable investigators to use the same animal for several com-
`ponents of the multivalent vaccine (e.g., diphtheria, tetanus,
`and polio in guinea pigs). Immunization of combination
`vaccines in a single animal to detect the response to multiple
`antigens has also been used to study potential immunologi-
`cal interferences (Sesardic al. 1999).
`For some of the newer vaccines intended to develop a
`Th1 rather than an antibody response, in vitro tests are being
`developed to assess the effectiveness of the immunization.
`One example is the establishment of a cytokine profile after
`antigen stimulation of cells in vitro (Metz et al. 2002).
`Immunization inducing CMI is also used to test products
`such as the purified protein derivative used in tuberculosis
`testing. Animals are immunized with a standard so. injec-
`tion with a sensitizing agent (BCG) followed by i.d. injec-
`tion of test samples. The diameter of the reaction induced by
`the delayed-type hypersensitivity (DTHI) response is mea—
`sured around the injection site. DTH is an in vivo assay of
`cell-mediated immune function that directly reflects the
`functions of Th1 lymphocytes.
`
`Inducing Immune Suppression Through
`Regulatory Cell Modulation
`
`Regulatory T cells represent 5 to 10% of the peripheral
`CD-4 T cell population. They inhibit immune responses that
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`are potentially harmful and help to regulate pathogen-
`specific responses by inhibiting or enhancing Th1 or Th2
`responses during infections. Modulation of these cells has
`the potential to treat conditions in which an immune re-
`sponse has an adverse effect (e.g., autoimmune diseases,
`allergies, and tissue graft rejection) or treat infections in
`which a “latent state” has developed from successful inhi-
`bition of the host response. The ability to inhibit or induce
`specific regulatory T cell activity appropriately likely in-
`cludes a combination of appropriate antigens, adjuvants,
`and costimulatory molecules (Burdin 2005; McGuirk and
`Mills 2002).
`
`epididymis-specific protein (Orand et al. 2004) has proved
`to be a successful technique to prevent conception in nu-
`merous animal models (e.g., mice, rats, guinea-pigs, and
`nonhuman primates). Immunization against zona pellucida
`glycoproteins has been investigated in marmosets (Aitken et
`al. 1996). Although the results have been mixed, this re-
`search was useful for demonstrating the impact, both posi-
`tive (long-term infertility) and negative (premature decline
`in primordial follicles), of immunization. Success of oral
`immunization against rabies in wildlife has also opened the
`possibility of similar protocols for wildlife antifertility im-
`munization (Stohr and Meslin 1997).
`
`Immunotoxicology
`
`Treatment of Addictions
`
`Immunotoxicity is a field of expanding interest. Included in
`this field are studies using immunization techniques to as—
`sess the potential immunogenicity of biological drug prod-
`ucts and chemicals. Standardized methods for assessing
`potential effects of chemicals on animal immune responses
`have been developed. One example is a rat model using a
`T cell-dependent antigen (keyhole limpet hemocyanin
`[KLH1]) inoculated id. or iv. to assess the immunosuppres-
`sive potential of coinoculated chemicals (Gore et al. 2004).
`Protocols have been developed in the rat that demonstrate
`the effect of age, gender, strain, and site of antigen inocu-
`lation in immunogenicity protocols, using lead as an immu-
`notoxin and KLH response as a read-out (Bunn et al. 2001 ).
`Another example comprises adjuvant arthritis models,
`which are useful for evaluating potential side effects of
`immunostimulation, including new adjuvants. The usual in-
`tent of immunization is to create a very specific response to
`a specific antigen. Nonspecific responses can be useful be-
`cause they may enhance the level of total response to a
`given antigen. However, when the nonspecific response is
`too great, it can initiate or exacerbate adverse interactions of
`the body’s immune system against itself, manifesting as
`conditions such as rheumatoid arthritis. Exacerbation of ad-
`
`juvant—induced arthritis models can measure whether there
`is sufficient enhancement of the nonspecific response to
`induce an adverse condition (Chiocchia et al. 1993).
`Consideration of the potential immunotoxicity of new
`biologics and drugs is now required prior to regulatory ap—
`proval. The US Center for Drug Evaluation and Research
`has made available a guidance document on this subject
`(CDER 2002).
`
`Fertility Control
`
`Immunization as a means of birth control is of interest for
`
`human and certain animal population management. A con-
`siderable amount of recent activity has been devoted to
`examining the impact of immunization against sperm, egg,
`or hormonal antigens (Delves et al. 2002). Immunization
`against sperm antigen (Diekman and Herr 1997) or testis/
`
`Preclinical research using immunization techniques in ani-
`mal models has demonstrated sufficient progress and has
`led to ongoing human clinical trials for both nicotine and
`cocaine addiction. The objective of these studies is to gen—
`erate specific antibodies that will bind with the drug and
`prevent its entry into the brain. The protocols generally
`involve immunization with a nicotine or cocaine antigen,
`hapten—conjugated peptides, or larger irnmunogenic mol-
`ecules such as KLH or cholera toxin (Carrera et al. 2001;
`Cerny et al. 2002; Sanderson et al. 2003). Other drugs for
`which similar immunization approaches are being investi-
`gated include phencyclidine, methamphetamine, and heroin
`(Kantak 2003).
`
`Immunizing Agents
`
`DNA Immunization
`
`DNA immunization involves the direct introduction of plas-
`mid DNA encoding an antigenic protein, which is then ex-
`pressed within cells Of the organism. Immunization with
`plasmid DNA-encoding antigenic proteins elicits both anti-
`body and cell-mediated immune responses. The introduc-
`tion of DNA can be accomplished by simple i.m. or i.d.
`injections, as well as by propelling DNA—coated gold par-
`ticles into various tissues, preferentially the epidermis (Par-
`tidos 2003). DNA vaccination is applicable to a variety of
`pathogens and is a useful method for enhancing immune
`responses. Most of the work on DNA vaccines has been
`conducted in mice. These vaccines have been demonstrated
`
`to protect mice from developing against tuberculosis, severe
`acute respiratory syndrome, and smallpox. The vaccines
`have the potential to induce prolonged antigenic stimula-
`tion, and because plasmids contain many sections of olgo-
`nuceotide with a high concentration of cytosine—guanine
`dinucleotides (CpG1 motifs), which are more prevalent in
`prokaryotic cells motifs, they may also have an inherent
`adjuvant effect. A summary of recent viral disease animal
`models for DNA vaccines is available in the literature
`
`(Davis and McCluskie 1999).
`
`Volume 46, Number 3
`
`2005
`
`247
`
`7
`
`

`

`Protection against viral pathogens is improved when
`DNA plasmid inoculation is followed by a booster of the
`same encoded antigen expressed in recombinant viral vec-
`tors (Hanke and McMichael 1999; Ramsay et al. 1997).The
`premise is that boostering avoids a cytotoxic T lymphocyte
`(CTLI) response that is too narrow. The results have been
`promising even in nonhuman primate immunization studies
`for diseases such as HTLV—l (Kazanji et a1. 2001) and HIV
`(Puaux et