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

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).