Dilution of Smallpox Vaccine, Revisited
By Luciana Borio, M.D., March 1, 2007
There are currently three types of smallpox vaccine available for use in the U.S. in the event of a bioterrorism attack. The vaccines are derived from the New York City Board of Health (NYCBH) strain of vaccinia, and include the freeze-dried Dryvax, produced in 1978 by Wyeth, and the liquid Wetvax, produced in 1958 by Aventis, both of which were grown on calf-skin, and the recently developed freeze-dried ACAM2000, produced by Acambis and grown in Vero cells. All three are made from live, replicating vaccinia virus that produces antibodies that are protective against the smallpox virus. Each requires that a small quantity of the vaccine virus be inoculated into the superficial layers of the skin where it replicates producing a vaccinial infection. Of these, only Dryvax is licensed. The other two are available under an IND, although it is anticipated that ACAM2000 will be licensed soon.
Previous trials have shown that, under emergency circumstances, Dryvax and Wetvax could be diluted 1:5, if not 1:10, with minimal loss of efficacy [1, 2]. ACAM2000 may not be diluted . A study using ACAM2000 showed a steep reduction in takes with dilution: 86% for a 1:5 dilution and 59% for a 1:20 dilution.
Now, Robert Couch and colleagues have conducted additional dilution trials with Dryvax and Wetvax, and have shown that diluted vaccine not only remains effective, but is associated with reduced vaccine-associated morbidity . In this study, 330 vaccinia naïve persons were randomly assigned to 1 of 11 vaccine-dose groups (see table below) in a double-blinded fashion. Virus titers in the undiluted vaccine ranged from 107.46 to 108.27 pfu/mL (mean 108.2 pfu/mL) as measured via chicken chorioallantoic membrane assay by the CDC. Vaccine dilutions were made at each study site.
“Take” rates, defined by the occurrence of a vesicle and best detected 6 to 8 days after vaccination, are shown in the table below. The combined take rates for recipients of 1:100 diluted Dryvax was 83.3%, mainly due to 4 of 6 subjects without a take at one of the sites. It was found that the vaccine virus titers were lower for these 4 subjects than for the 2 remaining subjects with a take; however, they were similar to subjects with takes at the other sites. Differences in take rates for each group were not statistically significant, other than a linear trend toward reduced take with reduced dose of Dryvax.
Table: Percent of subjects experiencing a “take” in each group
Wetvax (lot 1)
Wetvax (lot 2)
There was a high correlation between take and the development of serum neutralizing antibodies, as 97.5% of subjects with a take developed an increase in serum neutralizing antibody levels. No responses were detected in subjects who did not develop a take. Further, there were no dose-related trends in the frequency of serologic responses for any of the vaccine groups.
There were fewer instances of fever and systemic reactions (i.e. chills, headache, nausea, fatigue) in the lower-dose groups. Fever occurred earlier in the higher-dose groups and later in the lower-dose groups. In recent trials, 25% to 36% of recipients with a vaccine take missed normal daily activities due to adverse effects [1, 2]. In this trial, 29% of subjects with a take missed school, work, or social activities for at least 1 day, 9% for 2 days, and 4% for 3 days or more. However, there was a significant decrease in missed daily activities and mean symptom scores in the lower-dose groups.
Of interest, the classic description of vaccinia take lesion progressing from vesicle to pustule to eschar was not observed in most subjects. Instead, vesicles/pustules ruptured spontaneously under the occlusive dressing, resulting in draining shallow ulcers until 21 to 28 days after vaccination. In this study, the vaccinated area was covered with gauze and two overlapping occlusive dressings. In a previous study , vaccination sites were covered with folded gauze and one semipermeable adhesive membrane. The choice of dressing may contribute to the progression and healing of vaccinia lesions .
There were no differences in take rates or serum neutralizing-antibody responses between groups given higher-doses versus lower-doses.
Systemic reactions and symptom scores were greater for the higher-dose groups.
Vaccine dilution is safe and effective and could create a large stockpile of vaccine, which is of particular importance to the developing world where vaccine is no longer produced. The authors conclude that a 1:5 dilution would make hundreds of millions of vaccine doses available, and that even a 1:10 dilution can be used without a significant reduction in take rates.
However, even in this trial, conducted at experienced centers, two subjects were inadvertently given a 1:3.2 dilution vaccine. Thus, if vaccine is to be diluted in an emergency, where global demand exceeds vaccine capacity, careful planning must be conducted in advance. Provisions to have an adequate amount of diluent and bifurcated needles on hand need to be made in advance.
In addition, the authors believe that vaccination techniques may have influenced the lower take rates for the 1:100 dilution of Dryvax in one of the study sites. This reinforces the concept that high take rates can only be achieved through the use of adequate vaccination technique.
One important note: The ACAM2000, the new smallpox Vero cell-culture vaccine purchased by the U.S. government for the SNS, cannot be diluted. In a previous study, virus titers in the undiluted ACAM2000 vaccine were only approximately 106.5 pfu/mL, considerably lower than titers present in the stored Dryvax and Wetvax vaccines .
Couch RB, Winokur PL, Edwards KM, et al. Reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses. The Journal of Infectious Diseases 2007;195:826-32.