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It is hoped that HIV patients' own immune responses can be strengthened by vaccines so they will not have to rely exclusively on antiretroviral drugs.
Vaccines stimulate the body's immune system to prevent or control specific diseases. Vaccines against major human infectious diseases such as pertussis, diphtheria, tetanus, and tuberculosis were developed in the early 1900s, and those for polio, mumps, measles, and rubella were not licensed until the 1960s. Because of the widespread use and effectiveness of antibiotics, and the low profit margins and liabilities associated with vaccine products, interest in vaccines diminished in the 1980s and early 1990s. The recent renaissance in vaccines is fueled by multiple factors. New developments in modern biotechnology and immunology have made it possible to produce novel antigens and create new vaccine technologies. The recent licensures of high-profile vaccine products not only offered major contributions to human health but also achieved significant commercial success for the developers. These include Prevnar, a pneumococcal glycoconjugate vaccine; Gardasil, a human papilloma virus (HPV) vaccine; Rotarix and Rotateq, rotavirus vaccines; and Zostavax, the herpes zoster vaccine. Other factors include concerns over possible pandemic flu outbreaks and bioterrorism, the emergence of antibiotic-resistant pathogens, and increased funding from both private and public sectors. The current vaccine market is estimated at about $10 billion worldwide and is expected to double in the next five years.
Vaccines currently licensed in the US are listed in Table 1.1 Vaccines can be divided into four broad categories: live attenuated vaccines, inactivated vaccines, subunit vaccines, and conjugated vaccines. Live vaccines are usually weakened viruses or bacteria that are no longer capable of causing diseases but can still stimulate immune response in the hosts. Good examples are MMR II, Flumist, and Rotateq. Inactivated vaccines are composed of killed virus or bacteria, such as the polio virus vaccine and the rabies vaccine. Subunit vaccines are made from one or more components of a microorganism. Examples are the Streptococcus pneumoniae polysaccharides vaccines and the recombinant Hepatitis B vaccines. Conjugated vaccines are prepared by conjugating a subunit to a carrier protein or other immuno-stimulating agents. An example is Prevnar (7-valent capsular polysaccharides of S. pneumoniae conjugated to a carrier protein CRM197).
Table 1. Vaccines licensed in the US
Significant efforts have been undertaken to identify and develop new vaccine technologies (Table 2).2–3 Inactivated pathogens, recombinant proteins or peptides (with or without adjuvants) are inefficient in inducing T-cell response. Plasmid DNA-based vaccines elicit strong antibody and T-cell responses in animals. Attempts to enhance immune responses to DNA vaccines in humans have been made using new formulations with cationic lipids, electroporation, or the incorporation of cytokine genes in the construction, with promising results. Viral–vector based vaccines tested in humans include attenuated pox viruses (vaccinia or avipox viruses) or adenovirus. Lipopeptides, presenting peptide antigens in association with a lipid moiety, have also been synthesized and tested as candidate vaccines against HBV, HPV, and HIV-1 infections to induce T-cell responses. Transgenic plants, expressing antigens from various pathogens, have been evaluated for vaccination through the oral route, and generated encouraging results when tested in animals. Monocyte-derived dendritic cells, loaded with antigens (presented as peptides, proteins, RNA or recombinant viruses), have generated good results in cancer patients.
Table 2. New vaccine technologies
No single vector alone can elicit optimal immune responses in humans. Thus, there is a trend to use multiple vectors as part of mixed immunization regimens. In such heterologous prime–boost vaccination schemes, the antigen is presented to the immune system using a "priming" vector. A second vector is used as a booster to present the very same antigen. Collectively, such mixed immunization regimens have been shown to elicit stronger antibody, T-cell, and cytotoxic T lymphocyte responses. Association between DNA and pox viruses or vaccinia and canarypox appears to be particularly promising in inducing effective immune responses.
To reduce reactogenicity, most of the new vaccines under development are based on well-defined molecular immunogens, as opposed to whole attenuated or inactivated pathogens. These "molecular vaccines" encompass proteins, peptides, lipopeptides, plasmid DNA, or recombinant viruses based on viral vectors know to be safe to humans. Such vaccines are generally not as immunogenic as traditional vaccines and require adjuvants to induce a more potent and persistent immune response. New vaccine targets often require induction of strong cellular responses, including T helper cells and sometimes cytotoxic T lymphocytes in addition to antibodies. Conventional adjuvants based on aluminum salts predominately induce antibody responses.4–5
Discovering new adjuvants is crucial for the development of vaccines that require cell–mediated responses. The recent shift from an empirical to a rational approach to adjuvant development was made possible by an increased understanding of the control mechanism in the immune system, and of the interplay between the innate and the acquired immune response. In particular, the role of toll-like receptors (TLRs) in recognizing pathogen-associated molecular patterns and the ability to stimulate these receptors using a range of new agonists of varying specificities has significantly advanced the adjuvant field.6
Several TLR agonists have been studied as vaccine adjuvants in clinical studies. The main feature shared by all TLR agonists is their ability to deliver a potent activation signal to antigen presenting cells. CpG oligodeoxynucleotide (ODN), a TLR9 agonist, was shown to be a potent adjuvant of both humoral and cell-mediated immunity in human studies. Monophosphoryl lipid A (MPL), a TLR4 agonist, has been shown to induce both antibody and T-cell responses. It was licensed as an adjuvant for hepatitis B vaccine nonresponders in Europe in 2005.
Great progress has been made toward the development of vaccines against emerging infectious pathogens (e.g., HIV and HSV); cancers; diabetes; rheumatoid arthritis; multiple sclerosis; Alzheimer disease; allergies to either tree pollen, grass pollen, or house dust mites; hypertension; and cholesterol management. Some of these are highlighted below.
Flu Vaccines—Seasonal and Pandemic
The currently licensed flu vaccines are trivalent inactivated virus particles containing at least 15 μg of hemagglutinin polypeptide from each of the three strains selected for that year. They are manufactured using chicken eggs and have several disadvantages: insufficient supply because of limited manufacturing capability, long lead time (>6 months) from identification of strains to start of vaccine distribution, a cold-chain requirement for distribution, short shelf-life (cannot be stockpiled), and needle delivery. Additionally, healthy eggs may not be available due to avian flu.
Cell culture based vaccines are under active development and have several potential advantages: greater controllability, higher yield, faster manufacturing, and no reliance on chicken eggs.7 The first cell culture based flu vaccine (Optaflu) was approved in Europe in June 2007.
Another area of active research to improve current flu vaccines is antigen-sparing through the use of adjuvants and more efficient deliveries. The use of aluminum adjuvant has been shown to reduce the amount of antigens required by at least two-fold.8 An influenza vaccine with an oil-in-water emulsion adjuvant (MF59) is licensed in Europe.9 The co-administration of flu vaccines with GM-CSF or IL-2 in lipid vesicles also has been investigated.10
DNA-based flu vaccines expressing M2 and NP influenza proteins are currently under development. They have the advantages of rapid manufacturing in high volumes (due to a short lead time), long shelf -life (so they can be stockpiled), no need for cold-chain distribution, and possible needle-free delivery.
A universal influenza vaccine(with cross-protective antigens), targeting all "A" strains of the virus, is in Phase 1 clinical development in the US. "A" strains historically have been responsible for influenza pandemics. This vaccine targets both seasonal and pandemic influenza strains, and eliminates the annual rush to re-engineer and manufacture sufficient vaccine supplies. The vaccine focuses on the M2 protein of the influenza A virus, the extracellular part (M2e) of which is highly conserved in all A strains. The conserved M2e domain is genetically fused to a hepatitis B virus core protein (HBc) and expressed in E. coli.
The FDA approved the first US vaccine for humans against the highly pathogenic avian influenza H5N1 virus in April 2007. Other avian influenza viruses, like H7N7 and H9N2, are being evaluated as vaccine targets for a possible pandemic avian flu outbreak.
Despite 25 years of extensive efforts in developing HIV vaccines, no licensed vaccine is available today. The difficulties include high genetic variability of the virus, lack of knowledge of immune correlates of protection, inability to generate broadly neutralizing antibodies, absence of relevant and predictive animal models, and the complexities related to the preparation and conduct of multiple, large-scale clinical trials.11–12
Early investigations were mostly focused on inactivated virus, live-attenuated virus, and recombinant proteins. Although these candidates showed immunogenicity and protection in animal models, clinical studies have not demonstrated protection against the disease in humans. Recent clinical trials are more focused on plasmid DNA and recombinant live vectors, and have used either simple or multiple antigens from both structural and nonstructural HIV proteins to invoke a T-cell response.11 A DNA vaccine using a codon-optimized gp140 from a primary (non-recombinant) HIV-1 subtype C isolate was shown to induce both humoral and cell-mediated responses13 although constructs containing Gag-protease, Tat-Rev-Nef, Tat, and Gag were all shown to induce cell-mediated response.14–17 A canarypox virus vector (ALVAC) has been developed by Sanofi-Pasteur and evaluated in multiple clinical studies.18 Recent clinical studies used a propagation-defective alphavirus replicon derived from an attenuated strain of Venezuelan equine encephalitis (VEE) virus and expressed the Gag gene from a South African HIV-1C isolate.19
Therapeutic vaccination for HIV is also an active area of research and development.20–21 It is hoped that the HIV patients' own anti-HIV immune responses can be strengthened by these vaccines so that the patients will not have to rely exclusively on antiretroviral drugs.22 Several recent reports showed preliminary positive results using a recombinant canarypox vaccine and dendritic cells loaded with heat-inactivated autologous HIV-1.21,23 Other studies used plasmid DNA with various adjuvants and delivery systems.
The concept of therapeutic vaccines against cancers has been around for many decades, starting with the nonspecific immunostimulatory approaches first used by William Coley24 and progressing to more tumor-specific approaches such as autologous and allogenic tumor cell vaccines, and heat-shocked proteins.25 Considerable advances have been made over the past few years in developing cancer vaccines using whole-cells, proteins or peptides, plasmid DNA, and viral vectors with new adjuvants.26
Natural or synthetic peptides and recombinant proteins as vaccines targeting tumor-associated antigens, have shown promising results in humans. A peptide vaccine targeting melanoma melanocyte differentiation antigens (MART-1, gp100, and tyrosinase), was shown to elicit immune responses and prolong relapse-free survival.27
Tumor cell vaccines can be generated from either the patients' own tissues (autologous) or other sources (allogeneic). An autologous tumor cell-based vaccine against melanoma, conjugated to a hapten (dinitrophenyl), demonstrated improved disease-free and overall survival as compared to historical controls.28 It is currently undergoing Phase 1 and 2 clinical studies. Canvaxin has been one of the most promising allogeneic melanoma vaccines investigated to date. It is an irradiated whole-cell vaccine derived from three different melanoma cell lines, and administered with BCG as adjuvant. These cell lines were screened to ensure the greatest likelihood that the vaccine would contain an antigen common to each recipient's tumor. Canvaxin was shown to express at least 20 distinct melanoma associated antigens, immune responses to which have been associated with survival in vaccine recipients.29
Dendritic cell based vaccines are under active development. Once dendritic cells are generated, they can be loaded with tumor antigens through the addition of tumor antigens to the culture media, through incubation with autologous or allogeneic tumor lysate, through gene modification with tumor antigen cDNA or autologous tumor mRNA, or through creation of tumor cell–dendritic cell hybrids. Provenge, a vaccine against prostate cancer developed by Dendreon, is prepared from a patient's own monocytes and then loaded with a tumor antigen (a fusion protein of full-length PAP and GM-CSF). In hormone-refractory prostate cancer patients treated with the vaccine, the median survival was significant longer than placebo.30
Plasmid DNA and viral vector based vaccines have been actively tested in clinical trials. Intramuscular or intradermal plasmid DNA administration is attractive because gene-transduction of the recipient's cells will result in continuous production of the tumor antigen. Recombinant viral vectors are commonly used to enhance gene transduction efficiency over plasmid DNA. A significant challenge of this strategy is the presence of pretreatment antiviral antibodies, which can neutralize the effects of subsequent treatment.
Preventive cancer vaccines are mostly in the early research stage. New breakthroughs will depend on an increased understanding of what tumor-specific antigens are expressed during the initial tumor development stage.
Malaria infection occurs in more than 30% of the world's population, almost exclusively in developing countries, and results in one million deaths annually. Most cases of the disease in humans are caused by four different species of the malarial parasite. There is currently no vaccine available for the parasite pathogens that infect humans, despite extensive efforts. The complex lifecycle of the malaria parasite contributes to the complexity of developing an effective vaccine. The parasite is spread by insect vectors that go through different stages and forms (intracellular, extracellular, sexual, and asexual) as they grow in the blood and tissues (primarily the liver) of the human hosts. Malaria is difficult to grow in large quantities outside the natural host.3
Malaria vaccine development has been focused on subunit vaccines targeting either the pre-erythrocytic or erythrocytic stage of the parasite lifecycle.31 The vaccine that is currently most advanced in development is the RTS,S vaccine in an AS02 adjuvant. It comprises portions of the circumsporozoite protein (CSP) linked to components of the hepatitis B surface antigen such that immunogenic particles are formed. The vaccine (given three doses over several months) provided protection in 41% of the individuals in sporozoite challenge studies. Subsequent field trials showed significant protection in adults and recently in children. This vaccine is believed to act mainly through antisporozoite antibodies, but perhaps also through T-cells that target infected hepatocytes.
The current tuberculosis (TB) vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) provides efficient protection against TB in newborns, but does not prevent the establishment of latent TB or reactivation of pulmonary disease in adults. Current vaccine development focuses on two approaches: (1) Replacing BCG with a more effective vaccine or (2) boosting BCG with a booster vaccine which takes advantage of BCG priming vaccination in childhood, and which is given to increase the immune response and prolong immunity to an adult population.32,33 Several vaccines are in various stages of early clinical development. rBCG30 is a recombinant BCG vaccine (to replace GCG) under clinical studies as a BCG replacement. It overexpresses the surface antigen Ag85B, which appears to increase immune response to this important antigen. MVA85A is a modified vaccine virus Ankara (MVA) strain expressing antigen 85A (to boost BCG). It induces strong immune responses, particularly in previously BCG-vaccinated individual. Ag85B-ESAT6 is made up of two secreted antigens Ag85B and ESAT6. It has shown promise both parenterally and through the mucosal route.
New technologies include recombinant cell culture methodologies, transgenic systems to produce edible vaccines, new conjugation methods, delivery systems free of needles, disposable manufacturing technologies and stable formulations that will enable developing countries to transport or store the vaccines at ambient temperatures. DNA-based vaccines are cost-effective, can be made faster, are stable at higher temperatures, can be delivered by needle free systems and highly suitable not only for developing countries but also to respond to pandemic outbreaks or bioterrorism, given the shorter scale-up time. The development of vaccines for Alzheimer's, cancer, drug addiction, HIV, multiple sclerosis, tropical diseases, and autoimmune disorders such as diabetes, lupus erythematosus, and arthritis will be the future focus for the scientific community and biotechnology companies globally. The opportunities, possibilities, and market potential are so great that many new players will enter the field to shape the changing landscape of vaccine development.
Hank Liu, PhD, is the associate director of manufacturing sciences and technologies at Wyeth Biotech, Pearl River, NY 845.602.2043, firstname.lastname@example.org.
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