• 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
• Allergen: Neomycin, Polymyxin B
• 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 × 10^8 pfu/mL, filled into vials containing 0.3 mL (Monath et al., 2004).
• Immunization Route: Percutaneous
• 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).
• Approved Age for Licensed Use: 1 year of age and older
• 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 1×10^8 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).

• 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 × 10^6, 2 × 10^7, 2 × 10^8 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 10^6, 10^7, 10^8 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:
  • Type: Protein
  • Description: (Hooper et al., 2004)
  • Detailed Gene Information: Click Here.
• 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).

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: 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 10^6 (Group 1, n = 18), 10^7 (Group 2, n = 16), 10^8 (Group 3, n = 16) TCID50 MVA-BN in a double-blind manner, or 10^8 TCID50 (Group 4, n = 18) open-label, on day 0 and d28. Part II participants received a single dose of 10^8 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 (10^8 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 10^7 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: 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 × 10^6 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: 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 10^8 TCID50 MVA-BN at an interval of 4 weeks, the second group was vaccinated with a low dose of 2 x 10^6 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 10^6 PFU (3 animals per group) or 10^7 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).