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Smallpox vaccines: Current and future
Smallpox vaccines: Current and future RUBEN BONILLA-GUERRERO and GREGORY A. POLAND ROCHESTER, MINNESOTA Abbreviations: CCSV ⫽ cell-cultured smallpox vaccine; CDC ⫽ Centers for Disease Control and Prevention; MVA ⫽ modified vaccinia Ankara; NIAID ⫽ National Institute of Allergy and Infectious Diseases; NIH ⫽ National Institutes of Health; NYCBOH ⫽ New York City Board of Health strain of vaccinia virus; NYC-CL ⫽ New York City calf-lymph strain; WHO ⫽ World Health Organization
T
he cases of anthrax that accompanied the postal attacks in 2001 confirm that biologic weapons can and will be used against civilian populations.1 Smallpox has the same potential and has been weaponized to serve as a biologic weapon.2 As a result, the US government has committed substantial resources to assure broad access to the current vaccine and to develop new vaccines to defend against this possibility. In this review, we summarize the production, composition, application, indications and contraindications of the current commercially available smallpox vaccine Dryvax (Wyeth Laboratories, Inc, Madison, NJ). In addition, we highlight innovative features of new smallpox vaccines and consider how advances in vaccine technology may lead to the successful creation of improved smallpox vaccines. BACKGROUND
Like smallpox (variola), vaccinia virus belongs to the family Poxviridae and is a member of the Orthopoxvirus genus.3 Vaccinia is an enveloped, double-stranded DNA virus with a large linear genome that encodes more than 200 proteins.4 Our understanding of its pathobiology and its mechanisms of inducing protective immunity are incomplete. Vaccinia is less virulent than variola, and an immune response to vaccinia provides cross-protection against variola, as confirmed by From the Mayo Vaccine Research Group, the Department of Internal Medicine, and the Program in Translational Immunovirology and Biodefense, Mayo Clinic and Foundation. Submitted for publication July 14, 2003; accepted July 14, 2003. Reprint requests: Gregory A. Poland, MD, Vaccine Research Group, Mayo Clinic, 200 First Street SW, Rochester, MN 55905; e-mail: [email protected]. J Lab Clin Med 2003;142:252-7. Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003/$30.00 ⫹ 0 doi:10.1016/S0022-2143(03)00143-4
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the success of the worldwide smallpox-eradication campaign with vaccinia.5,6 Smallpox was declared eradicated by WHO in 1980.7 After its eradication, variola only existed in research laboratories in a very few countries. In 1977, the last naturally acquired case of smallpox occurred, in Somalia,8 and in 1978, the last reported case of smallpox caused by accidental infection of a laboratory worker in occurred in England.9 CURRENT VACCINE
At the time of this writing, the United States has 15 million doses of the licensed smallpox vaccine Dryvax and 75 million to 90 million doses of a smallpox vaccine manufactured by Aventis-Pasteur (Swiftwater, Pa). Both vaccines are derived from vaccinia extracted from calf lymph. Concerns regarding the availability of the vaccine have been minimized by reports that the current Dryvax smallpox vaccine can still elicit a vigorous immune response when diluted fivefold to vaccinate naı¨ve individuals or even 10-fold for previously vaccinated individuals. As a result, sufficient doses are available now to vaccinate the entire US population.10-13 The original and currently licensed smallpox vaccine Dryvax contains live, attenuated vaccinia virus. This vaccine retains substantial virulence and is therefore responsible for several serious adverse events. Because this live virus requires a competent immune system to elicit an effective response and to contain local replication of the virus, the vaccine cannot be administered to individuals with certain immune disorders. For example, individuals with eczema/atopic dermatitis, including as much as 20% to 30% of the general US population, cannot receive the smallpox vaccine.14 Statistics collected during past vaccination campaign in the United States reveal that death occurs in 1 or 2 recipients per 1 million primary vaccines.15-17 The risk of experiencing any one of the other serious complications
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Table I. Current and future live virus smallpox vaccines Vaccine source
Dryvax, Wyeth Aventis-Pasteur Acam1000 Acam2000 DynPort NIH VaxGen and Kaketsuken
Vaccinia virus strain
Production
Availability
NYCBOH NYCBOH NYCBOH NYCBOH NYCBOH MVA LC16m8
Infected calves Infected calves MRC-5 cells Vero cells MRC-5 cells Chicken-embryo fibroblast cells Rabbit-kidney cells
15 million doses, undiluted 75-90 million doses, undiluted Phase 2 clinical trial Phase 1 clinical trial Phase 1 clinical trial Phase 1 clinical trial Phase 1 clinical trial
is approximately 1 in 14,000. As a result of the eradication of smallpox, research on the smallpox vaccine was halted before the discovery of many modern tools of immunology and cellular and molecular biology. As a result, the specific immunologic mechanisms that might be most effectively targeted to enhance the safety and efficacy of this vaccine have not been precisely defined. Smallpox vaccine, Dryvax, and the Aventis-Pasteur vaccine were not grown under sterile conditions. Instead, the shaved abdominal skin of calves was infected with vaccinia, after which exudate containing the virus was collected from the vesicles that appeared.16 This method of producing smallpox vaccine was commonly used in the United States, as well as in most parts of the world. Dryvax vaccine was made with a lyophilized preparation of live vaccinia virus from NYC-CL, whereas the Aventis-Pasteur vaccine was prepared as a nonlyophilized preparation of glycerinated live vaccinia virus. This strain was originally obtained from NYCBOH after 22 to 28 passages of purification and attenuation.16,18 The lyophilized Dryvax vaccine is reconstituted with a suspension that contains 0.005% brilliant-green dye, 0.25% phenol, and 50% glycerin.19,20 In contrast, the nonlyophilized Aventis-Pasteur vaccine is contained in sealed glass capillary tubes containing sufficient material for a single vaccination. This vaccine is suspended in a solution that contains 0.006% brilliant-green dye, 0.4% phenol, and 40 to 60% glycerin. Glycerin acts as a stabilizer for antibacterial and preservative agents, and glycerin helps the vaccine adhere to the skin surface and permits longer storage of the vaccine by preventing the formation of ice crystals.21 Different methods of vaccination are used with the 2 vaccines. For example, the Aventis-Pasteur vaccine was prepared as a ready-to-use solution, packaged as single-dose capillary-tube units with no more than 200 bacterial organisms per milliliter. Once the vaccine was restored to room temperature, 1 end of the capillary tube was broken off, after which 1 drop of vaccine was placed on the skin surface. The needle end of the tube was then introduced, through the vaccine drop, into the
skin with an up-and-down motion approximately 30 times.21 Although this vaccination method is no longer used, the Division of Microbiology and Infectious Diseases of NIAID, NIH, has been conducting clinical studies of the smallpox vaccine that Aventis-Pasteur donated to the Department of Health and Human Services to evaluate the safety and efficacy of undiluted vaccine and various dilutions of 2 lots of the vaccine with different potencies.18 To date, 685 vaccinia-naı¨ve adults and 15 previously vaccinated adults have been vaccinated in these studies. Additional studies in previously vaccinated adults are being planned (personal communication, Dr Stephen P. Heyse, Office of Clinical Research Affairs, Division of Microbiology and Infectious Diseases, NIAID, NIH, June 2003). The current CDC smallpox-vaccination protocol (intended to be carried out with the Dryvax vaccine) recommends that after vaccine reconstitution, a bifurcated needle is dipped into the vaccine vial, which retains a drop of vaccine of approximately 0.0025 mL containing 2.5 ⫻ 105 plaque-forming units of vaccinia virus. Three percutaneous punctures for a first-time vaccinee and 15 for a repeat vaccinee are made to introduce the vaccine virus into the dermis. A drop of blood should be visible after the punctures are made. If blood is not seen, 3 more punctures are made with the same bifurcated needle (the needle is not introduced into the vaccine vial again).22 Normal vaccinia-virus replication occurs in the cells of the basal layer of the dermis, producing cell death, localized necrosis, and the formation of vesicles containing virus-rich fluid. Approximately 1 week after vaccination, a pustule with erythematous and inflamed tissue surrounding the original vesicle will form. The appearance of additional smaller vesicles, known as satellite lesions, around the vaccination site is not uncommon. After smallpox vaccination, inflammation at the vaccination site usually lasts for about 14 days. A scab begins to form that eventually falls off 21 to 28 days after vaccination.23,24 A vaccinee is considered infectious until the scab falls off. This process, called a vaccine “take,” can be confirmed by the presence of neutralizing antibodies and measurements of cell-mediated immunity.10,11,25 Com-
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mon vaccine-related side effects include enlarged lymph nodes, fever, general malaise, a “robust take” (vaccination site grows to ⬎ 10 cm, with extensive swelling of tissue around the pustule), and extensive erythema, which usually peaks after 8 to10 days.10,19,24 The vaccine lesion usually resolves 2 to 6 days faster in repeat vaccinees.13 During the global eradication program, strains other than NYCBOH were used in the making of smallpox vaccines. One of the most widely used strains in Europe was the Lister strain (also called the Ellestre strain), which was manufactured by means of serial passages of vaccinia through rabbit and sheep skin.20,26 Other, less virulent strains used in the eradication of smallpox, including the LC16m8 strain (the Lister strain, passaged through rabbit-kidney cells), had take rates similar to those of the other strains used.25 A major advantage of the LC16m8 strain was the low number of reported side effects such as fever and general malaise. Among 40,000 vaccinees in Japan, 3 subjects experienced convulsions, 8 had generalized vaccinia, and 1 demonstrated eczema vaccinatum. All these cases were said to be mild.26 VaxGen (Brisbane, Calif) intends to conduct clinical trials of the LC16m8 vaccinia strain, which the company has licensed from Chemo-SeroTherapeutic Research Institute (Kumamoto, Japan).27 Side effects (reactogenicity) varies among the other common vaccinia strains used during the smallpoxeradication campaign. These strains include 1 from the former Soviet Union, EM-63, which is classified as a low-reactogenicity strain and another from China, Temple of Heaven, which is classified as highly reactogenic.26 Despite their great benefits to the world during the smallpox-eradication campaign, these vaccines also have several significant limitations. The nature of the manufacturing process for the United States–produced Dryvax vaccine potentially permitted the introduction of contaminating pathogens or other nonvaccinia proteins into vaccinees. Concerns about the frequency and severity of vaccine-associated complications are shared by the medical community and the general public. Such complications can be classified into 3 categories: (1) local reactions, which include autoinoculation distant from the vaccination site and vaccinia keratitis; (2) generalized reactions such as erythema multiforme, nonspecific rashes, and generalized vaccinia; and (3) mild and severe systemic reactions. Mild reactions, which appear in 20% to 40% of primary vaccinees, include such symptoms as fever, chills, headache, myalgias, and fatigue, which are symptoms of many livevirus vaccines. Severe reactions (which occur much less frequently, 15 to 50 per million vaccinees) include progressive vaccinia, eczema vaccinatum, postvac-
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cinial encephalitis, fetal vaccinia, and myopericarditis.17,24,28-32 Another important limitation of the current smallpox vaccine is the large number of people with medical conditions that preclude use of the vaccine. The current smallpox vaccine is contraindicated in several types of immunodeficiency (human immunodeficiency virus– related, congenital, acquired, any type of organ transplantation, generalized malignancy, autoimmune diseases); any type of immunosuppressive therapy, regardless of the diagnosis (chemotherapy, radiotherapy, antimetabolites, alkylating agents, or immunomodulators); and history of eczema or atopic dermatitis, as well as other skin diseases and lesions (burns, large wounds, shingles, chickenpox, and active herpes or psoriasis). Individuals with severe or moderate acute illness or allergies to polymyxin B, streptomycin, chlortetracycline, neomycin, or phenol should not be vaccinated. Persons with inflammatory eye disease requiring steroid treatment should defer vaccination until completion of therapy and resolution of the condition. In addition, persons with intimate or household contacts who have eczema, atopic dermatitis, or other acute or chronic skin conditions, immunosuppression, or immunodeficiency should not be vaccinated. Persons younger than 18 years and individuals with close household members less than 1 year old should consider deferring vaccination. Pregnant women, women who are breastfeeding, and women who plan to become pregnant in the 4 weeks after the proposed vaccination should not be vaccinated. In March 2003, the Advisory Committee on Immunization Practices and the CDC issued additional recommendations to defer vaccination in individuals with heart disease while the association with cardiac events is being investigated.33 Individuals with previous myocardial infarction, angina, congestive heart failure, cardiomyopathy, stroke or transient ischemic attack, chest pain or shortness of breath with activity, or other heart conditions under the care of a physician are also advised to not receive the vaccine. Vaccination should also be deferred in people with 3 or more of the following risk factors: hypertension, hypercholesterolemia, or diabetes; current cigarette smokers; and those with a first-degree relative who had a cardiac condition before age 50.34 These recommendations were later supported in a recent study by Halsell et al32 reporting 18 confirmed cases of myopericarditis among 230,734 military primary vaccinees and by the CDC’s report of 3 cases among 29,584 civilians as of April 2003.35 Despite the current controversy over safety and antiquated production and application methods, this vaccine was the most efficient and effective tool used by
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the WHO in the eradication of smallpox worldwide. This same vaccine is now being used in the ongoing vaccination programs of the US Department of Defense and for civilian preparedness. The current vaccine could be broadly administered once more in the event of a documented smallpox case or exposure. In the event of such an emergency, recommendations on the administration of vaccine, including that in individuals with contraindications, would be guided by the likelihood of direct exposure to a case of smallpox and infection. NEW VACCINES
Research on the development of improved smallpox vaccines ended with the eradication of smallpox in 1980. Reports from the early 1980s indicate that only the CDC in Atlanta and the Research Institute of Viral Preparations in Moscow were in possession of variola virus.5,36 Concerns that other nations and groups possessed smallpox virus highlighted the susceptibility of US troops and civilians to the virus and the need to develop new vaccine and drug strategies.2 Advances in the fields of immunology, molecular biology, genomics, proteomics, and vaccine biology over the last 2 decades should facilitate the design and production of improved vaccines. For instance, Acambis, Inc (Cambridge, Mass) and DynPort Vaccine Co LLC (Frederick, Md) are creating new smallpox vaccines using tissue and cell-culture based vaccines. These techniques allow the sterile growth of a cell line that will host the vaccinia virus from which the smallpox vaccine is created and ensures the purity of the vaccine by preventing potential contamination while maintaining the immunogenicity of the vaccine. Acambis is developing 2 versions of the smallpox vaccine. ACAM1000 is a live-virus vaccine with a vaccinia strain derived through plaque purification of Dryvax vaccine grown in human diploid embryonic lung cells (MRC-5), and ACAM2000 is a live-vaccinia vaccine derived from ACAM1000 grown in Vero cells.19 Data from Acambis comparing ACAM1000 and Dryvax in mice and monkeys support the conclusion that ACAM1000 is less virulent than Dryvax (written information provided by manufacturer, May 14, 2003). In addition, a recent phase 2 double-blind human clinical trial is reported to have shown that the two vaccines have equivalent abilities to elicit major cutaneous-reaction takes and to induce neutralizing antibody and cell-mediated immunity (unpublished information provided by the manufacturer, May 14, 2003). DynPort has developed a smallpox vaccine, referred to as cell-cultured smallpox vaccine, or CCSV, that is a thrice-plaque-purified vaccinia virus adapted to replicate in MRC-5 cells (information provided by the man-
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ufacturer, March 4, 2003). DynPort has completed CCSV vaccinations involving 350 volunteers (250 vaccinia-naı¨ve, 100 vaccinia-experienced) in a phase 1 clinical trial, and no serious adverse events have been reported. Plaque-reduction neutralization by antibody and cell-mediated immune studies are described by the manufacturer to show that CCSV is comparable to Dryvax. Additional phase 2 and phase 2/3 studies are planned to establish the noninferiority of CCSV to Dryvax, to demonstrate lot-to-lot consistency of the CCSV manufacturing process, and to expand the collection of safety data (information provided by the manufacturer, March 4, 2003). One of the most attenuated vaccinia strains is the MVA, which was created by way of more than 570 passages of the original Ankara strain through chickenembryo fibroblast cultures.26 The MVA strain replicates poorly in humans cells and fails to induce formation of a skin lesion. However, the ability of MVA to induce protection in recent animal models at a rate similar to that seen with Dryvax establishes MVA as a promising candidate in the development of a new smallpox vaccine.37 Phase 1 studies with MVA are being conducted in human subjects at the NIH.19 Moreover, recent publications make reference to older foreign studies, indicating that this highly attenuated smallpox vaccine was safe when administered by way of different routes. No reported complications accompanied inoculations by way of the intracutaneous, subcutaneous, and intramuscular routes among 120,000 human subjects in Turkey in 1974 and in southern Germany in 1978, even among individuals with higherrisk conditions such as eczema.20,38 In a more recent study, McClain et al39 compared Dryvax vaccine and a cell-cultured vaccinia vaccine made by way of several passages of the currently licensed vaccinia vaccine though MRC-5 cells. Their results demonstrate that, at least at the doses used, the MRC-5 cell– derived vaccinia virus, administered intramuscularly or intradermally, induced a lower immune response than did Dryvax administered percutaneously as measured on the basis of cell-mediated immunity and neutralizing antibodies. These authors concluded that smallpox vaccine administered percutaneously induced a greater immune response than that seen when the vaccine was given by way of other routes, suggesting the need to retain percutaneous administration in the testing of new vaccines. NEW TECHNOLOGY FOR USE IN DESIGNING NEW VACCINES
Development of vaccines against potential bioweapons, including smallpox, is of high priority to the federal government. Current techniques such as polymer-
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ase chain reaction have allowed the full sequencing of the variola and vaccinia genomes, and mass spectrometry and peptide isolation and synthesis have enabled the identification of specific vaccinia peptides and their application as immunogenic molecules. These techniques were not available at the time of the WHO smallpox-eradication campaign. Advances in molecular and cell biology have permitted the description of cytokine pathways and the synthesis of cytokines, proteins produced by helper T-cells and accessory cells that have the capability to steer the immune response toward cell-mediated or humoral immune responses. Cytokines may also be beneficial as vaccine adjuvants to boost a less immunogenic, but safer, vaccine to trigger protective immunity.40 Perhaps the most promising technique in the discovery and production of an improved smallpox vaccine involves the use of mass spectrometry. Findings elicited with this technique, in combination with the most recent advances in molecular immunology, suggest that the role of specific peptides presented in the context of HLA molecules to T-cells can be considered one of the most important fundamental drivers of an immune response.41 More recent evidence indicates that class I peptide epitopes of sufficiently high affinity do not necessarily require costimulatory molecules or the presence of helper T-cells to initiate a response.42,43 Newly identified HLA-A*02102–restricted T-cell epitopes (the most common major histocompatibility complex class I allele in human beings) conserved among vaccinia and variola44 might therefore be used in the design of a peptide-based smallpox vaccine. An approach developed by our group is to harness the power and sensitivity of newer mass-spectrometry techniques for the identification of novel antigens that could be used as vaccine candidates.45 By infecting human cell lines with vaccinia virus, we can isolate and, using advanced analytic techniques, identify naturally processed and presented vaccinia peptides eluted from the human leukocyte antigen peptide– binding groove. In turn, these peptides could be used as vaccine candidates — either as peptide-based vaccines or as peptide-adjuvanted vaccine candidates. Vaccinia-derived peptides, which are also common to the variola virus, would allow the potential development of immunologic cross-protection. The major appeal of such peptide-based vaccines is the lack of problems otherwise associated with a live attenuated viral vaccine. Also, very recent developments in quantitative protein/ peptide analysis involving selective-labeling systems, which permit quantification and sequencing of new peptides, will aid in the differentiation of viral peptides from self-peptides on the basis of their isotopic proprieties.46,47 Finally, mass spectrometry yields a qualita-
tive and quantitative analysis of peptides and provides simultaneous parameters of information, such as peptide abundance and structure. This information could be used to understand specific biologic processes such as epitope recognition. The difficulties associated with peptide-based vaccines often include the limited range of antigens recognized, the need to identify safe and effective adjuvants to elicit robust responses, the number of doses required, and the duration of the immune responses, as well as the potential selectivity of these responses (eg, cellular vs humoral, systemic vs mucosal). CONCLUSIONS
The development of a safer and more effective smallpox vaccine remains a challenge. Achieving this goal will certainly require the collaboration of several scientific disciplines to protect people worldwide against the use of variola as a destructive agent of bioterrorism or biologic warfare. Although new vaccines are under production, none is yet available as a licensed product. The quest to develop the next generation of smallpox vaccines, which will provide the benefits of high immunogenicity and efficacy, low reactogenicity and incidence of side effects, and perhaps an easier way of administration compared with the current product, has been undertaken by several groups of investigators in the United States, as well as by several pharmaceutical companies worldwide. Despite the fast pace of these research programs and the availability of federal funds to support this research, the commitment to deliver a efficient and safer smallpox vaccine to the public will take several years. We thank Dr. Thomas P. Monath, chief scientific officer at Acambis, Inc; Dr David J. Clanton, senior scientist at DynPort Vaccine Co; and Dr Stephen P. Heyse, medical officer at the Office of Clinical Research Affairs, Division of Microbiology and Infectious Diseases of the NIAID, NIH, for information included in this manuscript. REFERENCES
1. Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 2002;8:1019-28. 2. Henderson DA. The looming threat of bioterrorism. Science 1999;283:1279-82. 3. Esposito J, Fenner F. Poxviruses, in: Fields virology. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001, p 2885-91. 4. Moss B. Poxviridae: The Viruses and Their Replication, in: Fields virology. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001, p 2849-83. 5. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988. 6. Fenner F. A successful eradication campaign. Global eradication of smallpox. Rev Infect Dis 1982;4:916-30.
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7. World Health Organization. Declaration of global eradication of smallpox. Wkly Epidemiol Rec 1980;55:148. 8. Deria A, Jezek Z, Markvart K, Carrasco P, Weisfeld J. The world’s last endemic case of smallpox: surveillance and containment measures. Bull World Health Org 1980;58:279-83. 9. Wade N. New smallpox case seems lab-caused. Science 1978; 201:893. 10. Frey SE, Couch RB, Tacket CO, Treanor JJ, Wolff M, Newman FK. Clinical responses to undiluted and diluted smallpox vaccine. N Engl J Med 2002;346:1265-74. 11. Frey SE, Newman FK, Cruz J, et al. Dose-related effects of smallpox vaccine. N Engl J Med 2002;346:1275-80. 12. Newman FK, Frey SE, Blevins P, Yan L, Belshe RB. Stability of undiluted and diluted vaccinia-virus vaccine, Dryvax. J Infect Dis 2003;187:1319-22. 13. Frey SE, Newman FK, Yan L, Belshe RB. Response to smallpox vaccine in persons immunized in the distant past. JAMA 2003; 289:3295-9. 14. Engler RJ, Kenner J, Leung DY. Smallpox vaccination: risk considerations for patients with atopic dermatitis. J Allergy Clin Immunol 2002;110:357-65. 15. Lane JM, Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968. N Engl J Med 1969;281:1201-8. 16. Fenner F, Henderson DA, Arita I, Ladnyi ID. Developments in vaccination and control between 1900 and 1966. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988:277-312. 17. Kemper AR, Davis MM, Freed GL. Expected adverse events in a mass smallpox vaccination campaign. Eff Clin Pract 2002;5: 84-90. 18. Aventis Pasteur donates approximately 75 to 90 million doses of smallpox vaccine to US [press release]. Swiftwater, Pa: AventisPasteur USA; March 29, 2002: www.us.aventispasteur.com/ press_releases/Corporate_News.htm. Accessed June 2003. 19. Bartlett J, Borio L, Radonovich L, Mair JS, O’Toole T, Mair M. Smallpox vaccination in 2003: key information for clinicians. Clin Infect Dis 2003;36:883-902. 20. Rosenthal SR, Merchlinsky M, Kleppinger C, Goldenthal KL. Developing new smallpox vaccines. Emerg Infect Dis 2001;7: 920-6. 21. Smallpox vaccine USP [package insert]. Swiftwater, Pa: Connaught Laboratories; March 1976. 22. Wharton M, Strikas RA, Harpaz R, et al. Recommendations for using smallpox vaccine in a pre-event vaccination program. Supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR Recom Rep 2003;52(RR-7):1-16. 23. Vaccinia (smallpox) vaccine. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2001;50(RR-10):1-22. 24. Cono J, Casey CG, Bell DM. Smallpox vaccination and adverse reactions. Guidance for clinicians. MMWR Recom Rep 2003; 52(RR-4):1-28. 25. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. The pathogenesis, pathology and immunology of smallpox and vaccinia. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988:122-67. 26. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox vaccine and vaccination in the intensified smallpox eradication programme. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988:540-90. 27. VaxGen and Kaketsuken announce agreement to make potential-
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ly safer smallpox vaccine for US [press release]. www.vaxgen.com/ pressroom/index.html. Accessed September 18, 2003. Gurvich EB. The age-dependent risk of postvaccination complications in vaccinees with smallpox vaccine. Vaccine 1992;10: 96-7. Bray M, Wright ME. Progressive vaccinia. Clin Infect Dis 2003; 36:766-74. Centers for Disease Control and Prevention. Update: adverse events following smallpox vaccination—United States, 2003. JAMA 2003;289:2060-3. Centers for Disease Control and Prevention. Cardiac adverse events following smallpox vaccination—United States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:248-50. Halsell JS, Riddle JR, Atwood JE, et al. Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. JAMA 2003;289:3283-9. Centers for Disease Control and Prevention. Smallpox (vaccinia) vaccine contraindications. http://www.bt.cdc.gov/agent/smallpox/ vaccination/contraindications-clinic.asp. Accessed June 2003. Centers for Disease Control and Prevention. Supplemental recommendations on adverse events following smallpox vaccine in the pre-event vaccination program: recommendations of the Advisory Committee on Immunization Practices. JAMA 2003;289: 2064. Update: adverse events following smallpox vaccination—United States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:278-82. Breman JG, Henderson DA. Diagnosis and management of smallpox. N Engl J Med 2002;346:1300-8. Drexler I, Staib C, Kastenmuller W, et al. Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines. Proc Natl Acad Sci U S A 2003;100:217-22. Blanchard TJ, Alcami A, Andrea P, Smith GL. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J Gen Virol 1998;79(pt 5):1159-67. McClain DJ, Harrison S, Yeager CL, et al. Immunologic responses to vaccinia vaccines administered by different parenteral routes. J Infect Dis 1997;175:756-63. Schijns VE. Mechanisms of vaccine adjuvant activity: initiation and regulation of immune responses by vaccine adjuvants. Vaccine 2003;21:829-31. Poland GA. Immunogenetic mechanisms of antibody response to measles vaccine: the role of the HLA genes. Vaccine 1999;17: 1719-25. Kedl RM, Kappler JW, Marrack P. Epitope dominance, competition and T cell affinity maturation. Curr Opin Immunol 2003; 15:120-7. Franco A, Tilly DA, Gramaglia I, Croft M, Cipolla L, Meldal M. Epitope affinity for MHC class I determines helper requirement for CTL priming. Nat Immunol 2000;1:145-50. Terajima M, Cruz J, Raines G, et al. Quantitation of CD8⫹ T cell responses to newly identified HLA-A*0201-restricted T cell epitopes conserved among vaccinia and variola (smallpox) viruses. J Exp Med 2003;197:927-32. Poland GA, Ovsyannikova IG, Johnson KL, Naylor S. The role of mass spectrometry in vaccine development. Vaccine 2001;19: 2692-700. Griffin TJ, Aebersold R. Advances in proteome analysis by mass spectrometry. J Biol Chem 2001;276:45497-500. Bakhtiar R, Nelson RW. Mass spectrometry of the proteome. Mol Pharmacol 2001;60:405-15.
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he cases of anthrax that accompanied the postal attacks in 2001 confirm that biologic weapons can and will be used against civilian populations.1 Smallpox has the same potential and has been weaponized to serve as a biologic weapon.2 As a result, the US government has committed substantial resources to assure broad access to the current vaccine and to develop new vaccines to defend against this possibility. In this review, we summarize the production, composition, application, indications and contraindications of the current commercially available smallpox vaccine Dryvax (Wyeth Laboratories, Inc, Madison, NJ). In addition, we highlight innovative features of new smallpox vaccines and consider how advances in vaccine technology may lead to the successful creation of improved smallpox vaccines. BACKGROUND
Like smallpox (variola), vaccinia virus belongs to the family Poxviridae and is a member of the Orthopoxvirus genus.3 Vaccinia is an enveloped, double-stranded DNA virus with a large linear genome that encodes more than 200 proteins.4 Our understanding of its pathobiology and its mechanisms of inducing protective immunity are incomplete. Vaccinia is less virulent than variola, and an immune response to vaccinia provides cross-protection against variola, as confirmed by From the Mayo Vaccine Research Group, the Department of Internal Medicine, and the Program in Translational Immunovirology and Biodefense, Mayo Clinic and Foundation. Submitted for publication July 14, 2003; accepted July 14, 2003. Reprint requests: Gregory A. Poland, MD, Vaccine Research Group, Mayo Clinic, 200 First Street SW, Rochester, MN 55905; e-mail: [email protected]. J Lab Clin Med 2003;142:252-7. Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003/$30.00 ⫹ 0 doi:10.1016/S0022-2143(03)00143-4
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the success of the worldwide smallpox-eradication campaign with vaccinia.5,6 Smallpox was declared eradicated by WHO in 1980.7 After its eradication, variola only existed in research laboratories in a very few countries. In 1977, the last naturally acquired case of smallpox occurred, in Somalia,8 and in 1978, the last reported case of smallpox caused by accidental infection of a laboratory worker in occurred in England.9 CURRENT VACCINE
At the time of this writing, the United States has 15 million doses of the licensed smallpox vaccine Dryvax and 75 million to 90 million doses of a smallpox vaccine manufactured by Aventis-Pasteur (Swiftwater, Pa). Both vaccines are derived from vaccinia extracted from calf lymph. Concerns regarding the availability of the vaccine have been minimized by reports that the current Dryvax smallpox vaccine can still elicit a vigorous immune response when diluted fivefold to vaccinate naı¨ve individuals or even 10-fold for previously vaccinated individuals. As a result, sufficient doses are available now to vaccinate the entire US population.10-13 The original and currently licensed smallpox vaccine Dryvax contains live, attenuated vaccinia virus. This vaccine retains substantial virulence and is therefore responsible for several serious adverse events. Because this live virus requires a competent immune system to elicit an effective response and to contain local replication of the virus, the vaccine cannot be administered to individuals with certain immune disorders. For example, individuals with eczema/atopic dermatitis, including as much as 20% to 30% of the general US population, cannot receive the smallpox vaccine.14 Statistics collected during past vaccination campaign in the United States reveal that death occurs in 1 or 2 recipients per 1 million primary vaccines.15-17 The risk of experiencing any one of the other serious complications
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Table I. Current and future live virus smallpox vaccines Vaccine source
Dryvax, Wyeth Aventis-Pasteur Acam1000 Acam2000 DynPort NIH VaxGen and Kaketsuken
Vaccinia virus strain
Production
Availability
NYCBOH NYCBOH NYCBOH NYCBOH NYCBOH MVA LC16m8
Infected calves Infected calves MRC-5 cells Vero cells MRC-5 cells Chicken-embryo fibroblast cells Rabbit-kidney cells
15 million doses, undiluted 75-90 million doses, undiluted Phase 2 clinical trial Phase 1 clinical trial Phase 1 clinical trial Phase 1 clinical trial Phase 1 clinical trial
is approximately 1 in 14,000. As a result of the eradication of smallpox, research on the smallpox vaccine was halted before the discovery of many modern tools of immunology and cellular and molecular biology. As a result, the specific immunologic mechanisms that might be most effectively targeted to enhance the safety and efficacy of this vaccine have not been precisely defined. Smallpox vaccine, Dryvax, and the Aventis-Pasteur vaccine were not grown under sterile conditions. Instead, the shaved abdominal skin of calves was infected with vaccinia, after which exudate containing the virus was collected from the vesicles that appeared.16 This method of producing smallpox vaccine was commonly used in the United States, as well as in most parts of the world. Dryvax vaccine was made with a lyophilized preparation of live vaccinia virus from NYC-CL, whereas the Aventis-Pasteur vaccine was prepared as a nonlyophilized preparation of glycerinated live vaccinia virus. This strain was originally obtained from NYCBOH after 22 to 28 passages of purification and attenuation.16,18 The lyophilized Dryvax vaccine is reconstituted with a suspension that contains 0.005% brilliant-green dye, 0.25% phenol, and 50% glycerin.19,20 In contrast, the nonlyophilized Aventis-Pasteur vaccine is contained in sealed glass capillary tubes containing sufficient material for a single vaccination. This vaccine is suspended in a solution that contains 0.006% brilliant-green dye, 0.4% phenol, and 40 to 60% glycerin. Glycerin acts as a stabilizer for antibacterial and preservative agents, and glycerin helps the vaccine adhere to the skin surface and permits longer storage of the vaccine by preventing the formation of ice crystals.21 Different methods of vaccination are used with the 2 vaccines. For example, the Aventis-Pasteur vaccine was prepared as a ready-to-use solution, packaged as single-dose capillary-tube units with no more than 200 bacterial organisms per milliliter. Once the vaccine was restored to room temperature, 1 end of the capillary tube was broken off, after which 1 drop of vaccine was placed on the skin surface. The needle end of the tube was then introduced, through the vaccine drop, into the
skin with an up-and-down motion approximately 30 times.21 Although this vaccination method is no longer used, the Division of Microbiology and Infectious Diseases of NIAID, NIH, has been conducting clinical studies of the smallpox vaccine that Aventis-Pasteur donated to the Department of Health and Human Services to evaluate the safety and efficacy of undiluted vaccine and various dilutions of 2 lots of the vaccine with different potencies.18 To date, 685 vaccinia-naı¨ve adults and 15 previously vaccinated adults have been vaccinated in these studies. Additional studies in previously vaccinated adults are being planned (personal communication, Dr Stephen P. Heyse, Office of Clinical Research Affairs, Division of Microbiology and Infectious Diseases, NIAID, NIH, June 2003). The current CDC smallpox-vaccination protocol (intended to be carried out with the Dryvax vaccine) recommends that after vaccine reconstitution, a bifurcated needle is dipped into the vaccine vial, which retains a drop of vaccine of approximately 0.0025 mL containing 2.5 ⫻ 105 plaque-forming units of vaccinia virus. Three percutaneous punctures for a first-time vaccinee and 15 for a repeat vaccinee are made to introduce the vaccine virus into the dermis. A drop of blood should be visible after the punctures are made. If blood is not seen, 3 more punctures are made with the same bifurcated needle (the needle is not introduced into the vaccine vial again).22 Normal vaccinia-virus replication occurs in the cells of the basal layer of the dermis, producing cell death, localized necrosis, and the formation of vesicles containing virus-rich fluid. Approximately 1 week after vaccination, a pustule with erythematous and inflamed tissue surrounding the original vesicle will form. The appearance of additional smaller vesicles, known as satellite lesions, around the vaccination site is not uncommon. After smallpox vaccination, inflammation at the vaccination site usually lasts for about 14 days. A scab begins to form that eventually falls off 21 to 28 days after vaccination.23,24 A vaccinee is considered infectious until the scab falls off. This process, called a vaccine “take,” can be confirmed by the presence of neutralizing antibodies and measurements of cell-mediated immunity.10,11,25 Com-
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mon vaccine-related side effects include enlarged lymph nodes, fever, general malaise, a “robust take” (vaccination site grows to ⬎ 10 cm, with extensive swelling of tissue around the pustule), and extensive erythema, which usually peaks after 8 to10 days.10,19,24 The vaccine lesion usually resolves 2 to 6 days faster in repeat vaccinees.13 During the global eradication program, strains other than NYCBOH were used in the making of smallpox vaccines. One of the most widely used strains in Europe was the Lister strain (also called the Ellestre strain), which was manufactured by means of serial passages of vaccinia through rabbit and sheep skin.20,26 Other, less virulent strains used in the eradication of smallpox, including the LC16m8 strain (the Lister strain, passaged through rabbit-kidney cells), had take rates similar to those of the other strains used.25 A major advantage of the LC16m8 strain was the low number of reported side effects such as fever and general malaise. Among 40,000 vaccinees in Japan, 3 subjects experienced convulsions, 8 had generalized vaccinia, and 1 demonstrated eczema vaccinatum. All these cases were said to be mild.26 VaxGen (Brisbane, Calif) intends to conduct clinical trials of the LC16m8 vaccinia strain, which the company has licensed from Chemo-SeroTherapeutic Research Institute (Kumamoto, Japan).27 Side effects (reactogenicity) varies among the other common vaccinia strains used during the smallpoxeradication campaign. These strains include 1 from the former Soviet Union, EM-63, which is classified as a low-reactogenicity strain and another from China, Temple of Heaven, which is classified as highly reactogenic.26 Despite their great benefits to the world during the smallpox-eradication campaign, these vaccines also have several significant limitations. The nature of the manufacturing process for the United States–produced Dryvax vaccine potentially permitted the introduction of contaminating pathogens or other nonvaccinia proteins into vaccinees. Concerns about the frequency and severity of vaccine-associated complications are shared by the medical community and the general public. Such complications can be classified into 3 categories: (1) local reactions, which include autoinoculation distant from the vaccination site and vaccinia keratitis; (2) generalized reactions such as erythema multiforme, nonspecific rashes, and generalized vaccinia; and (3) mild and severe systemic reactions. Mild reactions, which appear in 20% to 40% of primary vaccinees, include such symptoms as fever, chills, headache, myalgias, and fatigue, which are symptoms of many livevirus vaccines. Severe reactions (which occur much less frequently, 15 to 50 per million vaccinees) include progressive vaccinia, eczema vaccinatum, postvac-
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cinial encephalitis, fetal vaccinia, and myopericarditis.17,24,28-32 Another important limitation of the current smallpox vaccine is the large number of people with medical conditions that preclude use of the vaccine. The current smallpox vaccine is contraindicated in several types of immunodeficiency (human immunodeficiency virus– related, congenital, acquired, any type of organ transplantation, generalized malignancy, autoimmune diseases); any type of immunosuppressive therapy, regardless of the diagnosis (chemotherapy, radiotherapy, antimetabolites, alkylating agents, or immunomodulators); and history of eczema or atopic dermatitis, as well as other skin diseases and lesions (burns, large wounds, shingles, chickenpox, and active herpes or psoriasis). Individuals with severe or moderate acute illness or allergies to polymyxin B, streptomycin, chlortetracycline, neomycin, or phenol should not be vaccinated. Persons with inflammatory eye disease requiring steroid treatment should defer vaccination until completion of therapy and resolution of the condition. In addition, persons with intimate or household contacts who have eczema, atopic dermatitis, or other acute or chronic skin conditions, immunosuppression, or immunodeficiency should not be vaccinated. Persons younger than 18 years and individuals with close household members less than 1 year old should consider deferring vaccination. Pregnant women, women who are breastfeeding, and women who plan to become pregnant in the 4 weeks after the proposed vaccination should not be vaccinated. In March 2003, the Advisory Committee on Immunization Practices and the CDC issued additional recommendations to defer vaccination in individuals with heart disease while the association with cardiac events is being investigated.33 Individuals with previous myocardial infarction, angina, congestive heart failure, cardiomyopathy, stroke or transient ischemic attack, chest pain or shortness of breath with activity, or other heart conditions under the care of a physician are also advised to not receive the vaccine. Vaccination should also be deferred in people with 3 or more of the following risk factors: hypertension, hypercholesterolemia, or diabetes; current cigarette smokers; and those with a first-degree relative who had a cardiac condition before age 50.34 These recommendations were later supported in a recent study by Halsell et al32 reporting 18 confirmed cases of myopericarditis among 230,734 military primary vaccinees and by the CDC’s report of 3 cases among 29,584 civilians as of April 2003.35 Despite the current controversy over safety and antiquated production and application methods, this vaccine was the most efficient and effective tool used by
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the WHO in the eradication of smallpox worldwide. This same vaccine is now being used in the ongoing vaccination programs of the US Department of Defense and for civilian preparedness. The current vaccine could be broadly administered once more in the event of a documented smallpox case or exposure. In the event of such an emergency, recommendations on the administration of vaccine, including that in individuals with contraindications, would be guided by the likelihood of direct exposure to a case of smallpox and infection. NEW VACCINES
Research on the development of improved smallpox vaccines ended with the eradication of smallpox in 1980. Reports from the early 1980s indicate that only the CDC in Atlanta and the Research Institute of Viral Preparations in Moscow were in possession of variola virus.5,36 Concerns that other nations and groups possessed smallpox virus highlighted the susceptibility of US troops and civilians to the virus and the need to develop new vaccine and drug strategies.2 Advances in the fields of immunology, molecular biology, genomics, proteomics, and vaccine biology over the last 2 decades should facilitate the design and production of improved vaccines. For instance, Acambis, Inc (Cambridge, Mass) and DynPort Vaccine Co LLC (Frederick, Md) are creating new smallpox vaccines using tissue and cell-culture based vaccines. These techniques allow the sterile growth of a cell line that will host the vaccinia virus from which the smallpox vaccine is created and ensures the purity of the vaccine by preventing potential contamination while maintaining the immunogenicity of the vaccine. Acambis is developing 2 versions of the smallpox vaccine. ACAM1000 is a live-virus vaccine with a vaccinia strain derived through plaque purification of Dryvax vaccine grown in human diploid embryonic lung cells (MRC-5), and ACAM2000 is a live-vaccinia vaccine derived from ACAM1000 grown in Vero cells.19 Data from Acambis comparing ACAM1000 and Dryvax in mice and monkeys support the conclusion that ACAM1000 is less virulent than Dryvax (written information provided by manufacturer, May 14, 2003). In addition, a recent phase 2 double-blind human clinical trial is reported to have shown that the two vaccines have equivalent abilities to elicit major cutaneous-reaction takes and to induce neutralizing antibody and cell-mediated immunity (unpublished information provided by the manufacturer, May 14, 2003). DynPort has developed a smallpox vaccine, referred to as cell-cultured smallpox vaccine, or CCSV, that is a thrice-plaque-purified vaccinia virus adapted to replicate in MRC-5 cells (information provided by the man-
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ufacturer, March 4, 2003). DynPort has completed CCSV vaccinations involving 350 volunteers (250 vaccinia-naı¨ve, 100 vaccinia-experienced) in a phase 1 clinical trial, and no serious adverse events have been reported. Plaque-reduction neutralization by antibody and cell-mediated immune studies are described by the manufacturer to show that CCSV is comparable to Dryvax. Additional phase 2 and phase 2/3 studies are planned to establish the noninferiority of CCSV to Dryvax, to demonstrate lot-to-lot consistency of the CCSV manufacturing process, and to expand the collection of safety data (information provided by the manufacturer, March 4, 2003). One of the most attenuated vaccinia strains is the MVA, which was created by way of more than 570 passages of the original Ankara strain through chickenembryo fibroblast cultures.26 The MVA strain replicates poorly in humans cells and fails to induce formation of a skin lesion. However, the ability of MVA to induce protection in recent animal models at a rate similar to that seen with Dryvax establishes MVA as a promising candidate in the development of a new smallpox vaccine.37 Phase 1 studies with MVA are being conducted in human subjects at the NIH.19 Moreover, recent publications make reference to older foreign studies, indicating that this highly attenuated smallpox vaccine was safe when administered by way of different routes. No reported complications accompanied inoculations by way of the intracutaneous, subcutaneous, and intramuscular routes among 120,000 human subjects in Turkey in 1974 and in southern Germany in 1978, even among individuals with higherrisk conditions such as eczema.20,38 In a more recent study, McClain et al39 compared Dryvax vaccine and a cell-cultured vaccinia vaccine made by way of several passages of the currently licensed vaccinia vaccine though MRC-5 cells. Their results demonstrate that, at least at the doses used, the MRC-5 cell– derived vaccinia virus, administered intramuscularly or intradermally, induced a lower immune response than did Dryvax administered percutaneously as measured on the basis of cell-mediated immunity and neutralizing antibodies. These authors concluded that smallpox vaccine administered percutaneously induced a greater immune response than that seen when the vaccine was given by way of other routes, suggesting the need to retain percutaneous administration in the testing of new vaccines. NEW TECHNOLOGY FOR USE IN DESIGNING NEW VACCINES
Development of vaccines against potential bioweapons, including smallpox, is of high priority to the federal government. Current techniques such as polymer-
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ase chain reaction have allowed the full sequencing of the variola and vaccinia genomes, and mass spectrometry and peptide isolation and synthesis have enabled the identification of specific vaccinia peptides and their application as immunogenic molecules. These techniques were not available at the time of the WHO smallpox-eradication campaign. Advances in molecular and cell biology have permitted the description of cytokine pathways and the synthesis of cytokines, proteins produced by helper T-cells and accessory cells that have the capability to steer the immune response toward cell-mediated or humoral immune responses. Cytokines may also be beneficial as vaccine adjuvants to boost a less immunogenic, but safer, vaccine to trigger protective immunity.40 Perhaps the most promising technique in the discovery and production of an improved smallpox vaccine involves the use of mass spectrometry. Findings elicited with this technique, in combination with the most recent advances in molecular immunology, suggest that the role of specific peptides presented in the context of HLA molecules to T-cells can be considered one of the most important fundamental drivers of an immune response.41 More recent evidence indicates that class I peptide epitopes of sufficiently high affinity do not necessarily require costimulatory molecules or the presence of helper T-cells to initiate a response.42,43 Newly identified HLA-A*02102–restricted T-cell epitopes (the most common major histocompatibility complex class I allele in human beings) conserved among vaccinia and variola44 might therefore be used in the design of a peptide-based smallpox vaccine. An approach developed by our group is to harness the power and sensitivity of newer mass-spectrometry techniques for the identification of novel antigens that could be used as vaccine candidates.45 By infecting human cell lines with vaccinia virus, we can isolate and, using advanced analytic techniques, identify naturally processed and presented vaccinia peptides eluted from the human leukocyte antigen peptide– binding groove. In turn, these peptides could be used as vaccine candidates — either as peptide-based vaccines or as peptide-adjuvanted vaccine candidates. Vaccinia-derived peptides, which are also common to the variola virus, would allow the potential development of immunologic cross-protection. The major appeal of such peptide-based vaccines is the lack of problems otherwise associated with a live attenuated viral vaccine. Also, very recent developments in quantitative protein/ peptide analysis involving selective-labeling systems, which permit quantification and sequencing of new peptides, will aid in the differentiation of viral peptides from self-peptides on the basis of their isotopic proprieties.46,47 Finally, mass spectrometry yields a qualita-
tive and quantitative analysis of peptides and provides simultaneous parameters of information, such as peptide abundance and structure. This information could be used to understand specific biologic processes such as epitope recognition. The difficulties associated with peptide-based vaccines often include the limited range of antigens recognized, the need to identify safe and effective adjuvants to elicit robust responses, the number of doses required, and the duration of the immune responses, as well as the potential selectivity of these responses (eg, cellular vs humoral, systemic vs mucosal). CONCLUSIONS
The development of a safer and more effective smallpox vaccine remains a challenge. Achieving this goal will certainly require the collaboration of several scientific disciplines to protect people worldwide against the use of variola as a destructive agent of bioterrorism or biologic warfare. Although new vaccines are under production, none is yet available as a licensed product. The quest to develop the next generation of smallpox vaccines, which will provide the benefits of high immunogenicity and efficacy, low reactogenicity and incidence of side effects, and perhaps an easier way of administration compared with the current product, has been undertaken by several groups of investigators in the United States, as well as by several pharmaceutical companies worldwide. Despite the fast pace of these research programs and the availability of federal funds to support this research, the commitment to deliver a efficient and safer smallpox vaccine to the public will take several years. We thank Dr. Thomas P. Monath, chief scientific officer at Acambis, Inc; Dr David J. Clanton, senior scientist at DynPort Vaccine Co; and Dr Stephen P. Heyse, medical officer at the Office of Clinical Research Affairs, Division of Microbiology and Infectious Diseases of the NIAID, NIH, for information included in this manuscript. REFERENCES
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