• Vaccine Name: ACAM2000
• Target Pathogen: Variola virus
• Target Disease: Smallpox
• 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).

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).

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).