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Vaccine Comparison

ACAM1000 ACAM2000 CCSV Dryvax MVA-BN Smallpox DNA Vaccine
Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information
  • Vaccine Ontology ID: VO_0004089
  • Type: Replication competent virus
  • Preparation: ACAM1000 was purified from Dryvax by sequential plaque selection to isolate clone (Weltzin et al., 2003).
  • Virulence: By most measures, ACAM 1000 is less virulent than Dryvax, the existing human smallpox vaccine (Weltzin et al., 2003).
  • Description: Dryvax supplies could be stretched by dilution. As vaccine supplies would still be insufficient, a new vaccine derived from Dryvax that is suitable for modern manufacture in cell culture at a large scale must be developed and clinically tested. The new vaccine forms the basis for the United States government's strategic vaccine stockpile for biodefense, and other countries are taking a similar course of action (Weltzin et al., 2003).
    Clinical trials have been conducted using the NYCBH-derived ACAM1000 vaccinia virus-based vaccines. ACAM1000 was similar to Dryvax in its ability to induce immune responses and in reactogenicity in phase I trials (Parrino et al., 2006).
  • Product Name: Smallpox (Vaccinia) Vaccine, Live
  • Tradename: ACAM2000
  • Manufacturer: Acambis, Inc
  • Vaccine Ontology ID: VO_0000003
  • CDC CVX code: 75
  • Type: Replication competent virus
  • Status: Licensed
  • Location Licensed: USA (License #1733)
  • Host Species for Licensed Use: Human
  • Preparation: ACAM2000 was prepared from ACAM1000 master seed stock and produced in Vero cells to address the need for rapid large-scale vaccine production (Parrino et al., 2006).
    Specifically, ACAM2000 was manufactured by infecting Vero cells grown on microcarriers under serum-free conditions with the P9 production virus inoculum at an MOI of 0.01–0.2. Virus particles were purified and concentrated. The resulting concentrated bulk vaccine was formulated by dilution with a buffer containing stabilizers to a final potency of 1.0–5.0 × 108 pfu/mL, filled into vials containing 0.3 mL (Monath et al., 2004).
  • Virulence: It has long been known that vaccinia strains differ with respect to neurovirulence in infant mice. Clones 1, 3, and 5 and the uncloned virus had virulence properties that were unacceptable for consideration as vaccine candidates. The same four viruses that had exhibited excessive virulence in rabbit skin were significantly more neurovirulent than Dryvax1 (p < 0.05, Kaplan—Meier survival distribution, log rank test), whereas clones 2, 4, and 6 were similar to Dryvax1 or less virulent. The more virulent viruses also replicated to higher titer in mouse brain. In these initial experiments Clone 2 did not appear to be attenuated with respect to neurovirulence, but subsequent studies with larger numbers of animals showed significantly higher survival distribution compared to Dryvax1. Clone 2 (renamed ACAM1000) was selected as the candidate for further development, based on its similarity to Dryvax1 in pock formation in rabbit skin but its lower neurovirulence in mice and monkeys. Seed viruses and vaccine produced from each bioreactor run were tested for neurovirulence in suckling mice, using Dryvax1 as a comparator. Plaque-purified vaccinia virus lines were shown to differ significantly in neurovirulence for mice, in their ability to evoke immune responses against the inserted gene product, and in their HindIII restriction maps. The variant viruses often exhibit reduced infectivity and reduced virulence for mice. We found biological and molecular heterogeneity among 6 clones derived from Dryvax1, with some clonal subpopulations (e.g. Clone 3) having dramatically higher virulence and changes at the genomic level. The degree of neurovirulence for suckling mice was used to distinguish vaccine strains with low, moderate, or high pathogenicity (Vilesova et al., 1985). The new vaccine has advantages over first generation vaccines, since it has been produced to modern manufacturing and control standards, is free from adventitious agents , and does not contain subpopulations of virus with undesirable virulence properties (Monath et al., 2004).
    In addition, mice immunized with MVA were protected against lethal infection with a more virulent form of vaccinia virus altered to coexpress IL-4. IL-4 diminishes the cytolytic capacity of CD81 T cells, resulting in delayed viral clearance and increased virulence (Parrino et al., 2006).
  • Storage: After reconstitution, ACAM2000 vaccine may be administered within 6 to 8 hours if kept at room temperature (20-25°C, 68-77°F); it should then be discarded as a biohazardous material. Unused, reconstituted ACAM2000 vaccine may be stored in a refrigerator (2-8°C, 36-46°F) up to 30 days, after which it should be discarded as a biohazardous material (FDA: ACAM2000).
  • Contraindication: Individuals with severe immunodeficiency who are not expected to benefit from the vaccine (FDA: ACAM2000).
  • Description: The benefits of cloning appeared to outweigh the recognized risk that a clonal virus population may differ biologically from the ‘genetic swarm’ represented by the animal-skin vaccine. Because it would not be possible to conduct field tests for efficacy, the new vaccine would need to match the licensed vaccine (Dryvax®) as closely as possible in preclinical tests for safety, immunogenicity, and protective activity and in clinical trials for safety and immunogenicity (Monath et al., 2004).
    Clinical trials have been conducted using the NYCBH-derived ACAM2000 vaccinia virus-based vaccine. On the basis of animal studies, ACAM2000 is believed to be less neurovirulent than Dryvax. ACAM2000 was similar to Dryvax in its ability to induce immune responses and in reactogenicity in phase I trials. During phase II and phase III clinical trials, cases of myopericarditis were associated with both ACAM2000 and Dryvax in vaccinia-naive volunteers (Parrino et al., 2006).
  • Vaccine Ontology ID: VO_0004090
  • Type: Replication competent virus
  • Preparation: CCSV was derived from Connaught Laboratories Master Seed number 17633 (originating from the New York City Board of Health vaccinia strain), adapted to replicate in MRC-5 cells, and plaque-purified three times. Cells were infected in ten-layer Nunc cell factories, incubated at 37°C for 3 days, and harvested by trypsinization. Infected cells were sonicated to release intracellular virus. The crude virus bulk was purified and concentrated by ultracentrifugation through a 36% sucrose cushion, and the resulting virus pellet was resuspended in 1 mmol/L Tris buffer (pH 9·0). The undiluted final vaccine material was formulated in 2% human serum albumin to give a concentration of 1×108 pfu per mL and was subsequently lyophilised. Lyophilised vials were stored at –20°C before use, reconstituted with 50% glycerin and 0·25% phenol in sterile water for injection, and used within 24 h after dilution (Greenberg et al., 2005).
  • Virulence: (Greenberg et al., 2005)
  • Description: Cell-cultured smallpox vaccine (CCSV) is a replication-competent vaccinia virus vaccine derived from a master seed stock originating from the NYCBH strain. In 2002, a phase I clinical trial conducted in 350 healthy vaccinia-naive and vaccinia-immune adults evaluated the safety, reactogenicity, and immunogenicity of CCSV and Dryvax. Among the study groups, 100 volunteers were assigned to receive various dilutions of CCSV. There were no statistically significant differences between the CCSV and Dryvax groups comparing humoral and cellular immune responses and rates of adverse events. At a delivered dose 50 times lower than the approved Dryvax dose, CCSV was still immunogenic and had a take rate of 100% (Parrino et al., 2006).
  • Product Name: Smallpox Vaccine, Dried, Calf Lymph Type
  • Tradename: Dryvax
  • Manufacturer: Wyeth Pharmaceuticals Inc
  • Vaccine Ontology ID: VO_0000035
  • CDC CVX code: 75
  • Type: live vaccinia virus vaccine
  • Status: Licensed
  • Location Licensed: USA (License #0003)
  • Host Species for Licensed Use: Human
  • Vector: test
  • Preparation: This vaccine is derived from NYCBH strain. It is lyophilized calf lymph and comes with a diluent containing 50% glycerol and 0.25% phenol in sterile water for injection, USP (Parrino et al., 2006).
  • Immunization Route: percutaneous (scarification)
  • Virulence: Dryvax was used to vaccinate military personnel and a select civilian population beginning in 2002. In these highly screened individuals, there were fewer adverse events than anticipated on the basis of the historical data, and no cases of progressive vaccinia or eczema vaccinatum. However, a new finding of cardiac complications have become a cause for concern. Although European and Australian literature from the 1950s, 1960s, and 1970s reported fatal and nonfatal postvaccinial cardiac complications, such reports were rare in the United States. At the time, this difference was believed to have been related to the less reactogenic strain of vaccinia virus used in the United States. However, the findings from the military and civilian vaccination programs indicate those with cardiac disease should not receive vaccinia in nonemergent settings. Of the 38,885 civilian smallpox vaccines administered between 2002 and 2003, there were 21 cases of myopericarditis and 10 ischemic cardiac events, of which 2 were fatal. In the military program as of June 2006, there were more than 1 million vaccinations and 120 cases of myopericarditis. The 16 cases of ischemic heart disease were consistent with rates in unvaccinated military recruits of the same age. The investigation of 8 fatalities after vaccination determined 1 death from an acute lupuslike illness may have a causal relationship to vaccine. In addition, vaccination was thought possibly to contribute to the sudden death of a 26-year-old military recruit 16 days after he received smallpox and influenza vaccinations. However, autopsy revealed myocarditis with parvovirus B in the cardiac muscle and no evidence of vaccinia virus (Parrino et al., 2006).
  • Storage: 2° to 8°C (36° to 46°F) (FDA: Dryvax).
  • Approved Age for Licensed Use: 18 and older
  • Contraindication: The vaccine should not be administered to anyone with known hypersensitivity to any component of the vaccine, individuals who have eczema and women who may be or want to become pregnant (FDA: Dryvax).
  • Description: Dryvax is the only US Food and Drug Administration (FDA)–licensed vaccine in the United States. Vaccination of the general public stopped in the US in 1972, and production of this vaccine stopped in 1982. Recent studies were performed evaluating clinical and immunologic responses to diluted vaccine in volunteers who had not previously been immunized to determine whether this stock vaccine could safely be diluted to provide more available doses. At dilutions of 1:5 or 1:10 (107 plaque-forming units [pfu]), the vaccine retained its potency and was able to elicit adequate immune responses (Parrino et al., 2006).
  • Vaccine Ontology ID: VO_0004097
  • Type: Highly attenuated clone
  • Preparation: MVA-BN has been derived via additional passages in serum free chicken embryo fibroblast (CEF) cultures, and is replication incompetent in mammalian cell lines, avirulent even in immune compromised hosts, highly immunogenic in mammalian animal models, and may be administered both s.c. and i.m. The vaccine was produced by IDT under Good Manufacturing Practice (GMP) conditions and provided by Bavarian Nordic as a liquid frozen product stored at −80 °C. Doses of 2 × 106, 2 × 107, 2 × 108 TCID50/ml were formulated in 10 mM Tris, 140 mM NaCl, pH 7.4. The vaccine was thawed and 0.5 ml were administered to subjects to deliver a dose of 106, 107, 108 TCID50, respectively (Vollmar et al., 2006).
  • Virulence: (Vollmar et al., 2006)
  • Description: MVA-BN (IMVAMUNE) was developed from the Modified Vaccinia Ankara strain (MVA) that was used as a priming vaccine prior to administration of conventional smallpox vaccine in a two-step program and shown to be safe in more than 120,000 primary vaccinees in Germany and used as a veterinary vaccine to protect against several veterinary orthopoxvirus infections (Vollmar et al., 2006).
  • Vaccine Ontology ID: VO_0004096
  • Type: DNA
  • A27L gene engineering:
    • Type: Protein
    • Detailed Gene Information: Click Here.
  • A33R from Monkeypox virus (strain: Zaire-96-I-16) gene engineering:
    • Type: Protein
    • Detailed Gene Information: Click Here.
  • B5R from Monkeypox virus (strain: Zaire-96-I-16) gene engineering:
  • L1R from Monkeypox virus Zaire-96-I-16 gene engineering:
    • Type: Protein
    • Detailed Gene Information: Click Here.
  • Preparation: The 4pox DNA vaccine contained two IMV-specific genes (L1R and A27L) and two EEV-specific genes (A33R and B5R) (Hooper et al., 2004).
  • Virulence: (Hooper et al., 2004)
  • Description: DNA vaccine strategies have been investigated in animal models. A DNA vaccine composed of 4 vaccinia virus genes protected rhesus macaques from severe disease, with the animals exhibiting mild clinical and laboratory abnormalities, after challenge with a lethal dose of monkeypox virus. When vaccinated with a single gene (L1R), macaques developed severe, but not fatal, disease. Heterologous prime-boost strategies have also been evaluated. Priming BALB/c mice with DNA vaccine resulted in greater immune responses after boosting with live vaccinia virus compared with controls (Parrino et al., 2006).
Host Response Host Response Host Response Host Response Host Response Host Response

Human Response

  • Host Strain: Healthy adults 18-29 yrs.
  • Vaccination Protocol: ACAM1000 for clinical testing was produced at pilot lot scale (750,000 doses) according to current Good Manufacturing Practices. A randomized, double-blind clinical study was carried out under an Investigational New Drug application approved by the United States Food and Drug Administration to evaluate the safety, tolerability and immunogenicity of ACAM1000 in 60 healthy adults, without prior smallpox vaccination. On day 0, 30 eligible subjects received inoculation of ACAM1000 by 15 strokes of a bifurcated needle. The vaccine formulation contained 108 PFU/ml. Subjects took daily oral temperature, completed a symptom diary and returned to the clinic on days 3, 7, 10, 15, 30 and 45 and after 6 months for evaluation. The primary endpoint was the proportion of subjects developing a major cutaneous reaction ('take') on day 7 and/or day 10. The primary statistical method was a test of noninferiority of ACAM1000 to Dryvax intended to rule out a 20% difference in take rates. Based on a one-tailed test of noninferiority, with a significance level of 0.05 and power of 80%, and assuming that the common rate of major cutaneous reaction is 90%, 30 subjects per arm of the trial would be required to rule out an ACAM1000 rate of response of 70% or less. Secondary endpoints were neutralizing antibody and T cell responses on days 0 and 45. Peripheral blood mononuclear cells (PBMC) were evaluated by CTL, IFN-gamma ELISPOT and lymphoproliferation assays (Weltzin et al., 2003).
  • Persistence: Subjects took daily oral temperature, completed a symptom diary and returned to the clinic on days 3, 7, 10, 15, 30 and 45 and after 6 months for evaluation. It is expected to confer lifelong immunity (Weltzin et al., 2003).
  • Side Effects: No serious adverse events were reported, and no subject was withdrawn from the study because of an adverse event. All 60 subjects experienced at least one adverse event related to the local cutaneous infection with vaccinia virus. Minimal changes in body temperature were noted. Two subjects experienced atypical healing at the vaccination site. No cardiac adverse events occurred, despite recent reports of myopericarditis. The trial was not powered sufficiently to detect the rare serious adverse events associated with smallpox vaccines (Weltzin et al., 2003).
  • Efficacy: The rate of successful vaccination was 100% (30 of 30 subjects) for ACAM1000 and 97% (29 of 30 subjects) for Dryvax (Table 1). By the prescribed statistical test, ACAM1000 was not inferior in immunogenicity to Dryvax (P < 0.001) (Weltzin et al., 2003).

    T-cell memory to smallpox declines slowly over time, with a half-life of 8–15 years, whereas serum antibody responses (and B-cell memory) to smallpox are maintained essentially for life with little or no observable decline. The protection afforded by smallpox vaccination shows that >90% of vaccinees are protected against lethal smallpox (normally 30% mortality in unvaccinated individuals) for at least 60 years post-vaccination (Slifka, 2004).
  • Description: Dryvax supplies could be stretched by dilution. As vaccine supplies would still be insufficient, a new vaccine derived from Dryvax that is suitable for modern manufacture in cell culture at a large scale must be developed and clinically tested. Safety, tolerability and immunogenicity of ACAM1000 was evaluated based upon comparable results with Dryvax (Weltzin et al., 2003).

Human Response

  • Host Strain: Healthy adults aged 18–29 years.
  • Vaccination Protocol: Clinical development of ACAM2000 commenced with a Phase 1 open-label trial in 100 healthy adults without prior smallpox vaccination. The primary endpoint was the proportion of subjects with a major cutaneous reaction assessed at any time-point from Day 7 (±2) through Day 15 (±2). Fifty-six percent of subjects were male. The majority (89%) were Caucasian; the remaining subjects were African-American (7%), Asian (3%), or Hispanic (1%). The mean age was 23 years, with a range of 18 to 29 years (Monath et al., 2004).
  • Persistence: Of the 99 subjects who experienced a major cutaneous reaction, 9% had a major cutaneous reaction by Day 3, and the rest experienced a major cutaneous reaction by Day 7. The progression of the cutaneous reaction and its size and appearance were similar to those observed in the trials of ACAM1000. The great majority (96%) developed ≥four fold increases in neutralizing antibodies. The geometric mean neutralizing antibody titer on Day 30 was 225. Four (4%) of 100 subjects did not have a four-fold increase in neutralizing antibody titer on Day 30. However, these 4 subjects all had a major cutaneous reaction by Day 7 (Monath et al., 2004).
  • Side Effects: With the diminishing threat of smallpox and increased focus on adverse events, vaccination in the United States was discontinued in 1972 for the general public and in 1989 for military personnel. The safety of ACAM2000 was assessed by documentation of adverse events, physical examination findings, lymph node assessments, measurements of vital signs, and clinical laboratory tests, including hematology, clinical chemistry, and urinalysis. Subjects in the study kept a diary of adverse events and took daily oral temperatures. There were no serious adverse events. All 70 subjects (100%) experienced at least one treatment-emergent, expected adverse event during the study. The adverse events were generally mild and did not interfere with the subjects’ daily activities. One subject experienced a serious adverse event, a single new onset seizure on Day 8; this event was considered by the investigator to be remotely related to the study vaccine. The most commonly reported treatment-emergent adverse events were related to the vaccination site and associated lymphadenitis, and the majority of adverse events reported were assessed as mild or moderate in intensity. Elevated temperature was reported as an adverse event for 9 (9%) subjects. Fortunately, cardiac adverse events appear to be self-limiting (Monath et al., 2004).
  • Efficacy: Ninety-nine percent of the subjects experienced a successful vaccination (Monath et al., 2004).
  • Description: Phase 1 clinical trials of ACAM1000 and 2000 indicate that the original goal of producing a second generation vaccine that closely matched the safety and immunogenicity of calf-skin vaccine (Dryvax®) was met. The cutaneous, antibody, and T cell responses in primary vaccinees were similar to those elicited by Dryvax®. The appearance and size of the cutaneous lesion and pattern of virus shedding from the vaccination site were also similar. Phase 2 trials in naïve and previously vaccinated subjects have been completed to define the dose response, and to extend safety and immunogenicity data. Phase 3 clinical trials are in progress (Monath et al., 2004).

Human Response

  • Host Strain: Healthy adults age 18-65 years
  • Vaccination Protocol: The study was a randomized, blind single-center comparative trial in healthy adult volunteers. Cohorts 1-4 were randomly assigned equivalent doses (2·5×105 plaque-forming units [pfu]) of either CCSV or Dryvax in a double-blind fashion. Participants were stratified by previous exposure to vaccinia (naive vs non-naive) and randomly assigned to vaccine group with a computer-generated process. In the vaccinia-naive group, a ratio of 2 to 1 (CCSV to Dryvax) was used, whereas in the non-naive population, the ratio was 1 to 1. All cohorts were enrolled consecutively with at least a 21-day delay between vaccination of successive cohorts. Cohorts 1-3 consisted of 15, 45, and 90 vaccinia-naive individuals, respectively (100 assigned CCSV and 50 Dryvax). Cohort four consisted of 100 non-naive individuals (50 CCSV and 50 Dryvax). Doses in cohort five (vaccinia-naive) were single-blind (to volunteer only) to one of the following five dilutions of CCSV (20 per group, CCSV to diluent): undiluted, 1:5, 1:10, 1:25, and 1:50. A random subset of 60 volunteers from cohort three (40 CCSV and 20 Dryvax) and 40 volunteers from cohort four (20 from each group) had blood samples taken for testing of cell-mediated immune responses (Greenberg et al., 2005).
  • Persistence: Participants kept a daily diary of symptoms and body temperature for the first 2 weeks after vaccination, and returned to the clinic for follow-up on days 3, 6, 8, 10, 14, 28, 45, 60, and 180 after vaccination. In vaccinia-naive individuals, titres peaked on day 28, whereas in non-naive people, they peaked on day 14. Although PRN50 geometric mean titres were generally higher for recipients of Dryvax rather than CCSV, their overall patterns on days 14, 28, 60, and 180 after vaccination did not differ significantly between the two vaccine groups for either vaccinia-naive or non-naive individuals (Greenberg et al., 2005).
  • Side Effects: 349 (99·7%) of 350 volunteers developed pock lesions; one vaccinia-naive individual who received a 1 in 25 dilution of CCSV did not. The rate of adverse events related to vaccine and the extent of humoral and cellular immune responses did not differ between the vaccine groups in vaccinia-naive or non-naive people. During clinic visits in the first 28 days, individuals were assessed for adverse events (vital signs, diary inspection, and concomitant medication) and formation of pock lesions (pock lesion inspection, measurements, and photographs). Intensity of adverse events was classified as mild (awareness of signs and symptoms that are easily tolerated), moderate (signs and symptoms produce discomfort sufficient to interfere with, but not prevent, normal daily activities), or severe (signs and symptoms produce sufficient discomfort to prevent normal daily activities). Differences between two proportions (eg, proportion with adverse events or proportion testing positive by an immunological assay). No serious vaccine-related adverse events were reported. Nobody withdrew from the study because of an adverse event. Other than rashes, no notable differences in frequency or severity of adverse events were recorded in the group receiving undiluted CCSV compared with those receiving diluted CCSV, and there were no notable differences in frequency of adverse events in the 1 in 50 group compared with those in other dilution groups. The adverse events reported for both vaccines were similar in severity and frequency, indicating that the manufacturing process in early human testing did not select for a more reactogenic vaccine, although it never entered larger clinical trials. As expected, vaccinia-non-naive participants tended to have fewer and milder adverse events than their vaccinia-naive counterparts. Overall, the adverse events in the diluted cohort were consistent with those of the other vaccinia-naive cohorts (Greenberg et al., 2005).
  • Efficacy: The take rate was 100% for all volunteers who received undiluted CCSV, irrespective of previous vaccinia-exposure. The 95% CI of the point estimate for vaccinia-naive individuals was 96–100% for CCSV; similarly, the 95% CI for vaccinia-non-naive individuals was 93%–100%. CCSV was immunogenic in vaccine-naive volunteers at a dose 50 times lower than that approved for Dryvax. CCSV seems to be a safe and immunogenic alternative to calf-lymph derived vaccine for both vaccinia-naive and non-naive people (Greenberg et al., 2005).
  • Description: U.S. government organizations have identified the need for a new smallpox vaccine to replenish limited stocks of the approved calf-lymph derived vaccine. Previous manufacturing methods using calf lymph are no longer acceptable because of the absence of controls in the process and the potential risk of contamination with the infectious agent associated with the prion disease bovine spongiform encephalitis. New manufacturing methods will need to eliminate the bovine intermediary. Because of ethical and safety considerations, challenge studies or field trials cannot be done to show efficacy. The strategy in designing the cell-cultured smallpox vaccine (CCSV) entailed selection of a well characterised isolate from a master vaccine seed stock used in the WHO eradication campaign. Methods of manufacture included consistency-validated processes for all aspects of manufacture, purification, storage, and distribution. Advantages of manufacture of this vaccine include the breadth of previous experience with well defined human diploid fetal lung fibroblasts in the production of other live, viral human vaccines, and the fact that the process is free from antimicrobial compounds that could produce reactions in sensitised individuals. The aims of this phase 1 clinical trial were to assess safety (frequency and severity of local and systemic adverse events), reactogenicity (frequency and characteristics of pock lesions), and immunogenicity (humoral and cellular immunity assays) of equivalent doses of CCSV and Dryvax in both vaccinia-naive and non-naive, healthy adult volunteers. Additionally, CCSV doses up to 50 times more dilute than the recommended dose for the Dryvax were assessed in a vaccinia-naive population (Greenberg et al., 2005).

Human Response

  • Host Strain: federal, state, and local potential first responders
  • Vaccination Protocol: A total of 37,901 volunteers in 55 jurisdictions received at least 1 dose of smallpox vaccine (Casey et al., 2005).
  • Persistence: Although the vaccine is effective, it is unclear how long it provides protection. Data suggest vaccine-specific memory B cells may persist for more than 50 years after vaccination, but not knowing which immunologic responses determine protection makes it difficult to define the duration of protective immunity.17 Alternative vaccine strategies designed to be safer than the presently available live virus vaccines are being pursued (Parrino et al., 2006).
  • Side Effects: A total of 590 adverse events (72%) were reported within 14 days of vaccination. Nonserious adverse events (n = 722) included multiple signs and symptoms of mild and self-limited local reactions. One hundred adverse events (12%) were designated as serious, resulting in 85 hospitalizations, 2 permanent disabilities, 10 life-threatening illnesses, and 3 deaths. Among the serious adverse events, 21 cases were classified as myocarditis and/or pericarditis and 10 as ischemic cardiac events that were not anticipated based on historical data. Two cases of generalized vaccinia and 1 case of post-vaccinial encephalitis were detected. No preventable life-threatening adverse reactions, contact transmissions, or adverse reactions that required treatment with vaccinia immune globulin were identified. Serious adverse events were more common among older revaccinees than younger first-time vaccinees (Casey et al., 2005).
  • Efficacy: A total of 38,885 smallpox vaccinations were administered, with a take rate of 92%. VAERS received 822 reports of adverse events following smallpox vaccination (overall reporting rate, 217 per 10,000 vaccinees) (Casey et al., 2005).
  • Description: The US Department of Health and Human Services (DHHS) implemented a preparedness program in which smallpox vaccine was administered to federal, state, and local volunteers who might be first responders during a bioterrorism event (Casey et al., 2005).

Human Response

  • Host Strain: US service members and DoD civilian workers eligible for smallpox vaccination
  • Vaccination Protocol: To develop vaccination policy, the US Department of Defense (DoD) drew from its own physicians, scientists, and administrators as well as colleagues in government and academia. The military vaccination program included vaccination for smallpox epidemic response teams (2000-5000 people) to assist with epidemic control and contact tracing in an outbreak, medical teams for hospitals and clinics (10,000-25,000 people) to care for smallpox cases, and operational forces (up to 500,000 people) to preserve critical capabilities. The licensed full-strength smallpox vaccine containing the NYCBH strain of vaccinia was used. First-time vaccination entailed punctures with a bifurcated needle. Previous vaccinees received 15 punctures. Those who did not respond with a major reaction as defined by the World Health Organization (WHO) were vaccinated again. Smallpox vaccinations began at 4 pilot sites: Walter Reed Army Medical Center, Washington, DC; Aberdeen Proving Ground, MD; Wilford Hall Air Force Medical Center, Lackland Air Force Base, San Antonio, TX; and the National Naval Medical Center, Bethesda, MD. For quality control, clinics tracked the vaccination response rates of the first 25 people for each vaccinator (Grabenstein et al., 2003).
  • Persistence: In addition to unacceptable side effects and problems related to production, although the vaccine is effective, it is unclear how long it provides protection. Data suggest vaccine-specific memory B cells may persist for more than 50 years after vaccination, but not knowing which immunologic responses determine protection makes it difficult to define the duration of protective immunity.17 Alternative vaccine strategies designed to be safer than the presently available live virus vaccines are being pursued (Parrino et al., 2006).
  • Side Effects: One case of encephalitis and 37 cases of acute myopericarditis developed after vaccination; all cases recovered. Among 19,461 worker-months of clinical contact, there were no cases of transmission of vaccinia from worker to patient, no cases of eczema vaccinatum or progressive vaccinia, and no attributed deaths (Grabenstein et al., 2003).
  • Efficacy: In 5.5 months, the DoD administered 450,293 smallpox vaccinations (70.5% primary vaccinees and 29.5% revaccinees). In 2 settings, 0.5% and 3.0% of vaccine recipients needed short-term sick leave. Most adverse events occurred at rates below historical rates (Grabenstein et al., 2003).
  • Description: The US implemented a program of smallpox vaccinations for approximately 500,000 military personnel. The directive came as part of a national program of preparedness against biological attack. Pre-attack vaccination was determined to be the best way to personally protect troops so that they could continue their missions. The program was therefore mandatory for designated service members and employees except those with contraindications (Grabenstein et al., 2003).

Human Response

  • Host Strain: Adult males
  • Vaccination Protocol: Healthy male subjects aged 20–55 years were eligible for recruitment. The study design was divided into two parts: Part I subjects (n = 68) had no prior history of smallpox vaccination, while Part II (n = 18) subjects had a prior history of smallpox vaccination, documented by a vaccination certificate or a typical vaccination scar. Part I subjects were randomly assigned to receive a dose of either 106 (Group 1, n = 18), 107 (Group 2, n = 16), 108 (Group 3, n = 16) TCID50 MVA-BN in a double-blind manner, or 108 TCID50 (Group 4, n = 18) open-label, on day 0 and d28. Part II participants received a single dose of 108 TCID50 (Group 5, n = 18), open-label, to evaluate a boost response in previously vaccinated subjects. Study-specific assessments were conducted at screening and on d 0, 7, 28, 35, 42, and 126 (Vollmar et al., 2006).
  • Persistence: T-cell immunity can persist for up to 50 years after immunization (Vollmar et al., 2006).
  • Side Effects: 15 of the 64 general adverse events were assessed as possibly related to the study vaccine. 2 of these each occurred in Groups 1, 3, and 4, respectively, and 9 (28%) in the pre-immunized subjects. Only 1 serious adverse event was reported during the study: a subject in Group 4 was hospitalized due to an infected epidermal cyst on the face, 12 days after the second injection; however, this event was judged to be unrelated to the study vaccine, and the subject recovered without sequelae (Vollmar et al., 2006).
  • Efficacy: The immune responses achieved after administration of MVA-BN were highly dose-dependent. Total IgG seropositivity rates, as determined by the ELISA, reached a maximum of 81% and 88% following a single vaccination using the highest dose (108 TCID50) via the s.c. or i.m. routes, respectively. They reached 100% following the second vaccination. On the other hand, the pre-immunized subjects attained 100% seropositivity after a single vaccination with MVA-BN although only 4 of these subjects had detectable antibody titers prior to inclusion, implying a pre-existing boostable immunity more than 20 years post-vaccination (Vollmar et al., 2006).
  • Description: The primary objective of the study was to demonstrate safety and tolerability of MVA-BN at different doses administered to healthy subjects with or without a history of smallpox vaccination. Immunogenicity was assessed in all subjects as a secondary endpoint and was also used to evaluate dose-related responses and optimal route of application (Vollmar et al., 2006).

Human Response

  • Host Strain: rhesus macaque (Macaca mulata)
  • Vaccination Protocol: The challenge experiment included 4 groups: group 1 consisted of 3 monkeys vaccinated with the 4pox DNA vaccine, group 2 consisted of 2 monkeys vaccinated with the L1R DNA vaccine, group 3 (negative controls) consisted of 3 monkeys vaccinated with a Hantaan virus DNA vaccine, and group 4 (positive controls) consisted of 2 monkeys vaccinated with the human smallpox vaccine (Dryvax). The L1R DNA vaccine was tested to determine the degree to which vaccination with a single immunogen eliciting IMV-neutralizing antibodies could confer protection. The DNA vaccines were administered by gene gun. Five weeks before challenge, all monkeys, except the monkeys vaccinated with Dryvax and one of the negative controls, were vaccinated with new preparations of the same DNA vaccine they had received 1-2 years earlier. This booster vaccination was administered to affirm that immunological memory had been elicited by the initial vaccination series and to ensure robust responses to the DNA vaccines with the intent to prove concepts rather than explore minimal requirements for protection. Based on the dosing experiments, a dose of 2 x 107 PFU was chosen for the vaccine evaluation experiment. Vaccinated monkeys were challenged with MPOV-Z79 by i.v. injection into the right or left saphenous vein. At 2-day intervals, whole-blood, serum, and throat swab samples were collected, and rectal temperature, pulse, and blood oxygen saturation were measured (Hooper et al., 2004).
  • Persistence: Gene gun vaccination with the 4pox DNA vaccine or the L1R DNA vaccine elicited a memory response that was maintained for at least a year and up to 2 years (Hooper et al., 2004).
  • Side Effects: Although VACV is highly immunogenic and is known to confer long-lasting protective immunity to smallpox, the adverse events associated with the present smallpox vaccine (i.e., Dryvax) pose a significant obstacle to successful vaccination campaigns. Adverse events historically associated
    with VACV range from the nonserious (e.g., fever, rash, headache, pain, and fatigue) to life threatening (e.g., eczema vaccinatum, encephalitis, and progressive vaccinia). Serious adverse events that are not necessarily causally associated with vaccination, including myocarditis and/or myopericarditis, have been reported during past and present smallpox vaccination programs. Several adverse cardiac events reported in the first 4 months of the 2003 civilian and military vaccination campaigns prompted the CDC to revise their recommendations for exclusion of potential smallpox recipients to include those persons with heart disease or several other conditions. In addition, identifying protective immunogens might allow the development of a subunit smallpox vaccine that affords protection with negligible adverse events (Hooper et al., 2004).
  • Efficacy: Monkeys vaccinated with the 4pox DNA vaccine were protected not only from lethal monkeypox but also from severe disease. This is the first demonstration that vaccination with a combination of VACV immunogens, rather than the whole infectious virus, is sufficient to protect NHPs against any poxvirus disease (Hooper et al., 2004).
  • Description: Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine administered to the skin, the vaccine itself can pose a serious health risk. The present study demonstrates that monkeys vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R), which encodes a target of neutralizing antibodies, developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible (Hooper et al., 2004).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: The following lethal intranasal vaccinia (strain WR) challenge model was used to evaluate protective immunization by vaccinia clones in mice. Mice were immunized by scarification with graded doses of ACAM1000 and then challenged by intranasal inoculation with 100 times the median lethal dose (LD50) of vaccinia-WR (Weltzin et al., 2003).
  • Persistence: In one mouse model, all sham-immunized animals died (average survival time [AST] = 5.2 d), whereas immunized mice all survived (3-5 weeks). Another mouse model involving IN challenge showed protection with minimal transient weight loss, while sham-immunized mice had severre weight loss and all died (AST = 12.6 d) (Weltzin et al., 2003).
  • Side Effects: transient weight loss (Weltzin et al., 2003)
  • Efficacy: All mice immunized with 7 or 8 log10 PFU/ml survived. At lower vaccine doses, survival was reduced in a dose-dependent manner. Body weight decreased 1−2 d after challenge but increased subsequently in mice receiving the highest doses of vaccine viruses. Protection by all clones was similar to that of Dryvax. The dose that protected 50% of mice from death (PD50) was 5.5 log10 PFU/ml for Dryvax (Weltzin et al., 2003).
  • Description: Vaccine candidates were purified from Dryvax either by sequential plaque selection to isolate clones or by passage at low multiplicity of infection (MOI) to isolate a polyclonal virus. The starting material was a pool of 30 vials (3,000 doses) of Dryvax from three different production lots. Six clones were isolated by three sequential rounds of plaque purification in MRC-5 cells (human lung fibroblast cell line). The clones were then amplified in fluid cultures to produce vaccine candidates. The polyclonal strain was produced by passage three times in MRC-5 cells at MOI 0.001 plaque-forming units (PFU)/cell. HindIII restriction endonuclease analysis was carried out on viral DNA isolated from the seven vaccine candidates and Dryvax. All DNA samples yielded digestion products corresponding to those of Dryvax, indicating that there were no major genetic rearrangements. Minor variations in the molecular weights of individual bands, such as band K of clone 3 and possibly the higher-molecular-weight bands of clone 2, were observed. Based on its attenuated phenotype in mice and similarity to Dryvax in other characteristics, clone 2 was selected as the best candidate for further development and was renamed ACAM1000 (Weltzin et al., 2003).

Mouse Response

  • Host Strain: 3-4 day-old outbred ICR mice
  • Vaccination Protocol: Groups of mice were inoculated with graded doses (0.3 to 3.0 log10 pfu) (Monath et al., 2004).
  • Persistence: Survival analysis showed that ACAM1000 and ACAM2000 did not differ from one another but had significantly longer survival than Dryvax (Monath et al., 2004).
  • Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
    encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
  • Efficacy: The median lethal dose (LD50) and 90% lethal dose (LD90) were higher for mice receiving ACAM2000 and ACAM1000 compared to Dryvax (Monath et al., 2004).
  • Description: The neurovirulence profiles of ACAM2000 and ACAM1000 vaccines were compared to Dryvax in a lethal dose assay (Monath et al., 2004).

Mouse Response

  • Host Strain: Young adult BALB/c mice.
  • Vaccination Protocol: Groups of 5 mice were immunized with graded doses (4 to 7 log10 PFU/mL) of ACAM 2000 and then challenged 3 weeks later with 100 LD50 of vaccinia WR virus. Survival and body weight were recorded daily for 14 days after challenge (Monath et al., 2004).
  • Persistence: The survival times were not statistically different between treatment groups (Monath et al., 2004).
  • Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
    encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
  • Efficacy: Protective efficacy of the 3 viruses tested was similar (Monath et al., 2004).
  • Description: Mice were used to compare the protective efficacy of immunization with ACAM2000, ACAM1000, and Dryvax (Monath et al., 2004).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: Adult (16–23 g) female BALB/c mice were vaccinated in the skin of the thigh using an Easy Vax™ DNA vaccine delivery system to deliver the vaccine plasmids on weeks 0, 3, and 8. Anesthetized mice were scarified by placing 10 μl of PBS containing live VACV on the tail. A 26 gauge 5/8" needle was used to scratch the tail to facilitate infection/vaccination. A lesion (pock) at the site of scarification on d 10 indicated successful vaccination. Mice were anesthetized and weighed before i.n. injection of 50 μl of PBS containing 2 × 106 pfu of VACV strain IHD-J using a plastic pipette tip. After challenge, mice were observed and weighed daily for 3 weeks (Hooper et al., 2006).
  • Persistence: (Hooper et al., 2006)
  • Side Effects: There are several drawbacks to the current anthrax vaccines including nonserious and serious adverse events that make the vaccine contraindicated in large segments of the population (e.g., persons who are immunodeficient, immunosuppressed, pregnant, breastfeeding, or have history of cardiac disease), and because this vaccine results in a localized skin infection containing infectious virus (i.e. pock), the infection can spread to other sites on the body (e.g. ocular autoinoculation) or to persons who come in close contact with the vaccinee. Identification of the genes associated with protective immunity and, conversely, the genes associated with adverse events unrelated to dissemination or transmission will be important for characterizing the next-generation smallpox vaccines and for engineering future smallpox vaccines (Hooper et al., 2006).
  • Efficacy: Mice vaccinated with the 4pox DNA vaccine using the Easy Vax™ device were completely protected from i.n. challenge with >10 LD50 of VACV, strain IHD-J (Hooper et al., 2006).
  • Description: The enhanced immunogenicity of DNA vaccines delivered by gene gun likely involves the direct introduction of plasmid DNA to cells in the skin, including specialized antigen-presenting cells (APCs). While the gene gun has yielded among the most promising immune responses for a DNA vaccine thus far, there is the possibly that all of the criteria required for successful product development will not be satisfied. Hence, it is important to continue to evaluate alternative technologies that might better facilitate the development of licensed human vaccines. Alternative means of delivering DNA vaccines under investigation include the use of electric field technologies. Electroporation is a process whereby cells are transiently permeabilized by high-intensity electric field pulses. The present study tests a novel device capable of targeting electroporation to the dermis using a microneedle array. The plasmid DNA is dried onto the tips of the microneedles, which are inserted into the skin where the DNA dissolves in interstial fluid and is then transfected into the surrounding cells by electroporation (Hooper et al., 2006).

Monkey Response

  • Host Strain: Young adult rhesus monkeys.
  • Vaccination Protocol: Monkeys (six per group) were vaccinated by scarification using a bifurcated needle. All 18 monkeys developed typical primary cutaneous reactions. Neutralizing antibodies against both variola and vaccinia viruses were measured 30 d after vaccination (Weltzin et al., 2003).
  • Persistence: Neutralizing antibodies against both variola and vaccinia viruses were present at >30 d post-vaccination (Weltzin et al., 2003).
  • Side Effects: Dryvax can causes severe neurobiological illness and mortality via nonpurulent meningitis. ACAM 1000 can lead to mild edema and small areas of lymphoid infiltration (Weltzin et al., 2003).
  • Efficacy: Antibodies to variola at titers 1:40 were present in two of six monkeys inoculated with ACAM1000. Neutralizing antibodies to vaccinia virus appeared in five of six inoculated with ACAM1000 (titers, 1:10−40) (Weltzin et al., 2003).
  • Description: Vaccine candidates were purified from Dryvax either by sequential plaque selection to isolate clones or by passage at low multiplicity of infection (MOI) to isolate a polyclonal virus. The starting material was a pool of 30 vials (3,000 doses) of Dryvax from three different production lots. Six clones were isolated by three sequential rounds of plaque purification in MRC-5 cells. The clones were then amplified in fluid cultures to produce vaccine candidates at MRC-5 passage. The polyclonal strain was produced by passage three times in MRC-5 cells at MOI 0.001 plaque-forming units (PFU)/cell. HindIII restriction endonuclease analysis was carried out on viral DNA isolated from the seven vaccine candidates and Dryvax. All DNA samples yielded digestion products corresponding to those of Dryvax, indicating that there were no major genetic rearrangements. Minor variations in the molecular weights of individual bands, such as band K of clone 3 and possibly the higher-molecular-weight bands of clone 2, were observed. Based on its attenuated phenotype in mice and similarity to Dryvax in other characteristics, clone 2 was selected as the best candidate for further development and was renamed ACAM1000. This model was performed to confirm the immunogenicity of ACAM1000 that was observed in mice (Weltzin et al., 2003).

Monkey Response

  • Host Strain: cynomolgus macaques (Macaca fascicularis)
  • Vaccination Protocol: Four groups of six captive-bred sub-adult healthy monkeys each were vaccinated: the first group was vaccinated twice with a high dose of 108 TCID50 MVA-BN at an interval of 4 weeks, the second group was vaccinated with a low dose of 2 x 106 TCID50 MVA-BN followed 10 days later by a s.c. vaccination with Elstree-RIVM, the third group was vaccinated with one s.c. standard dose of Elstree-RIVM, and the fourth group was vaccinated s.c. with one standard dose of Elstree-BN. Group V was sham vaccinated. 15 weeks after the last vaccination, all of the animals were challenged intratracheal (i.t.) with either 106 PFU (3 animals per group) or 107 PFU (3 animals per group) of MPXV, which were chosen as sub-lethal and lethal challenges, respectively (Stittelaar et al., 2005).
  • Persistence: (Stittelaar et al., 2005)
  • Side Effects: Elevated body temperatures were observed (Stittelaar et al., 2005).
  • Efficacy: All vaccinated animals that were challenged showed an episode of elevated body temperature (>1°C; ~2.65%) that occurred between days 5 and 8 post-challenge which returned to normal by d 12. Only one vaccinated animal developed pocks upon MPXV challenge, while all others showed no clinical signs of the disease apart from an elevated body temperature. This animal, which was vaccinated with MVA-BN (group I), initially developed pocks (>70) on d 11 after the challenge with MPXV (Stittelaar et al., 2005).
  • Description: The present study investigated different combinations of candidate and traditional vaccines, followed by MPXV challenge i.t. The MVA strain (MVA-BN, or IMVAMUNE) is currently being tested in >300 human subjects in on-going phase I and II clinical studies, including individuals for whom vaccination with traditional smallpox vaccines is traditionally contraindicated. For the present study, the immune response and efficacy of MVA-BN vaccination were compared to those of a primary vaccination with MVA-BN followed by vaccination with a first-generation smallpox vaccine produced on calf skins (Elstree-RIVM). For this purpose, a low dose of MVA was chosen to prime the immune system, thus reducing the side effects of vaccination with a traditional vaccine shortly thereafter without changing the take rate of the traditional vaccine. In addition, vaccination protocols with Elstree-RIVM alone and vaccination with a second-generation vaccine (Elstree-BN) were evaluated. Elstree-BN is based on the same vaccinia virus strain as Elstree-RIVM, but the former was passaged and produced on chicken embryo fibroblasts to further attenuate the virus and to make a better defined vaccine preparation that does not depend on the use of calves (Stittelaar et al., 2005).
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