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Background To evaluate the potential to increase the supply of smallpox vaccine (vaccinia virus), we compared the response to vaccination with 108.1, 107.2, and 107.0 plaque-forming units (pfu) of vaccinia virus per milliliter.
Methods In this randomized, single-blind, prospective study, 680 adults who had not been previously immunized were inoculated intradermally with undiluted vaccine (mean titer, 108.1 pfu per milliliter), a 1:5 dilution, or a 1:10 dilution of vaccinia virus with use of a bifurcated needle, and the site was covered with a semipermeable dressing. Subjects were monitored for vesicle formation (an indicator of the success of vaccination) and adverse events for 56 days after immunization.
Results Success rates did not differ significantly among the groups and ranged from 97.1 to 99.1 percent after the first vaccination. Both the undiluted and diluted vaccines were reactogenic. In addition to the formation of pustules, common adverse events included the formation of satellite lesions, regional lymphadenopathy, fever, headache, nausea, muscle aches, fatigue, and chills consistent with the presence of an acute viral illness. Generalized and localized rashes, including two cases of erythema multiforme, were also observed.
Conclusions When given by a bifurcated needle, vaccinia virus vaccine can be diluted to a titer as low as 107.0 pfu per milliliter (approximately 10,000 pfu per dose) and induce local viral replication and vesicle formation in more than 97 percent of persons.
On the basis of the results of a pilot dose-ranging study reported elsewhere in this issue of the Journal,2 we sought to determine with greater precision (within 5 percent) the rate of success of inoculation with dilutions expected to have a moderate-to-high success rate: undiluted vaccine (geometric mean titer, 108.1 plaque-forming units [pfu] per milliliter), a dilution of 1:5 (geometric mean titer, 107.2 pfu per milliliter), and a dilution of 1:10 (geometric mean titer, 107.0 pfu per milliliter). Given the reactogenicity consequent to viral replication in previously unexposed persons, we also sought to determine the frequency and range of adverse events associated with the administration of the vaccine with a bifurcated needle at these dilutions.
Methods
Vaccine and Diluent
The smallpox vaccine (Dryvax, Wyeth Laboratories, Marietta, Pa.) used in this study was produced in 1982 or earlier (lot no. 4008284) and was provided by the Centers for Disease Control and Prevention in Atlanta. The vaccine lot and diluent differed from the preparations used in the pilot study, and the vaccine contained a larger quantity of virus (108.1 vs. 107.8 pfu per milliliter).2 Lyophilized vaccine was reconstituted with a new formulation of diluent (lot no. 1468-1A, Chesapeake Biological Laboratories, Baltimore) consisting of 50 percent glycerin and 0.21 percent phenol in sterile water. Unlike the original vaccine diluent, this formulation did not contain brilliant green dye.
The vaccine was prepared at each site by adding 0.25 ml, 1.25 ml, or 2.5 ml of diluent directly to the vials, which corresponded to undiluted vaccine, a 1:5 dilution, and a 1:10 dilution, respectively. Vaccine was used for up to seven days after reconstitution and stored at 2 to 8°C between uses in accordance with the results of analyses of the stability of vaccinia virus. An aliquot from each dilution of vaccine used at each site was collected at the time of preparation and frozen for subsequent evaluations of the viral titer. Titers of every vaccine preparation were determined by plaque assay in the central laboratory (Saint Louis University, St. Louis).3 The quantity of virus on three representative bifurcated needles was also measured by plaque assay.
Study Design and Subjects
The study was a randomized, single-blind trial conducted at the National Institute of Allergy and Infectious Diseases Vaccine and Treatment Evaluation units in St. Louis, Baltimore, and Rochester, N.Y., and at the Respiratory Pathogens Research Unit in Houston. The study was approved by the institutional review board of each participating facility. Subjects were enrolled from November 2 to November 28, 2001, after providing written informed consent.
Healthy adults 18 to 32 years of age were eligible if they had no vaccination scar, no history of vaccinia virus vaccination, and no antibodies against human immunodeficiency virus. In the wake of the September 11, 2001, terrorist attacks, public announcement of the study resulted in a vigorous public response among young adult volunteers. Many were students at the participating trial sites. Exclusion criteria included the contraindications against vaccination noted in the package insert (pregnancy, immunosuppression, and eczema), a history of vaccination with any type of live attenuated virus within 60 days before the study, the receipt of blood products or immune globulin within 6 months before the study, and household contact, sexual contact, or occupational exposure to pregnant women, immunosuppressed persons, persons with eczema, or infants less than 12 months of age.
A total of 680 subjects were randomly assigned to receive undiluted vaccine, a 1:5 dilution of vaccine, or a 1:10 dilution of vaccine. The subjects were inoculated with a bifurcated needle that held a drop of vaccine and that was pressed 15 times into the skin of the upper arm. The vaccination sites were covered with folded gauze and a semipermeable adhesive membrane to avoid autoinoculation or exposure of personal contacts to vaccinia virus (Figure 1).2 Dressings were changed and the vaccination sites were assessed every three to five days until the lesions dried and an eschar formed. Subjects were evaluated for adverse events for 56 days after immunization.
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Neutralizing Antibody and Vaccine-Stability Assays
Serum samples were collected before vaccination (day 0) from all subjects. Serum neutralizing antibody titers were determined in prevaccination samples from all 15 subjects in whom the initial vaccination failed, as well as in 28 subjects in whom vaccination was successful. The end point was a 60 percent reduction in the number of plaque-forming units, as described previously.3 In addition, plaque assay was used to determine the stability of the vaccine reconstituted with the new diluent after storage at 2 to 8°C.5
Statistical Analysis
The goal of the study was to establish precise estimates of the rates of response to a 1:5 dilution and a 1:10 dilution and secondarily to test the hypothesis of the noninferiority of these dilutions as compared with undiluted vaccine. Noninferiority was defined by an alpha level of 0.05 and a difference in success rates (the rate for undiluted vaccine minus the rate for diluted vaccine) of no more than 5 percent. The sample size was based on the results of the pilot study.2 We estimated that 684 subjects would need to be enrolled — 107 in the group given undiluted vaccine, 241 in the group given a 1:5 dilution, and 336 in the group given a 1:10 dilution. We used exact methods to calculate the 95 percent confidence intervals for the success rates in each group and for the differences in these rates among the groups.6 We used a one-sided t-test to calculate the confidence intervals for the difference in rates, in keeping with the nature of the study as an evaluation of noninferiority.
The groups were compared with the use of analysis of variance or the Kruskal–Wallis test7 for continuous outcomes. Categorical outcomes were compared with the use of exact or asymptotic contingency-table tests and logistic regression. Unless otherwise noted, longitudinal outcomes, such as the reactogenicity of the vaccine, reflect the worst possible (most severe) outcome, rather than the best possible outcome. The statistical significance of these results was confirmed with the use of generalized estimating equations. According to the study protocol, the vaccination had to be evaluated at least once on day 7, 8, or 9 and again on day 13, 14, or 15 and could be evaluated at other follow-up visits at the discretion of the evaluators. In addition, one clinical center evaluated all vaccination sites on day 10, 11, or 12. To allow for the possibility that a small number of subjects had been exposed to vaccinia virus before the study, we analyzed the data according to the intention-to-treat principle.
No subjects were lost to follow-up. One subject who had no response to the initial vaccination was not revaccinated because of logistic constraints.
Results
Characteristics of the Subjects
A total of 680 subjects were enrolled: 106 received undiluted vaccine, 234 received a 1:5 dilution, and 340 received a 1:10 dilution. Five hundred eighty-four subjects were white (85.9 percent), 39 were black (5.7 percent), 26 were Asian (3.8 percent), 16 were Hispanic (2.4 percent), 3 were Native American (0.4 percent), 1 was a Pacific Islander (0.1 percent), and the racial or ethnic background of 11 subjects (1.6 percent) was unknown. There were 346 women (50.9 percent), and the mean (±SD) age of the subjects was 24.8±3.7 years. There were no significant differences (P=0.51 by Pearson's exact chi-square test6) in these characteristics among the groups.
Titers and Stability of Vaccine
The geometric mean titers of vaccinia virus were 108.1 pfu per milliliter in the case of undiluted vaccine (range, 107.8 to 108.4), 107.2 pfu per milliliter in the case of the 1:5 dilution (range, 106.9 to 107.5), and 106.8 pfu per milliliter in the case of the 1:10 dilution (range, 106.0 to 107.2). At 1 site two of the three vaccine preparations of the 1:10 dilution had lower titers (106.0 and 106.2 pfu per milliliter, as compared with a mean titer of 107.0 pfu per milliliter at the 12 other sites). The reason for the lower titers among 2 of 14 preparations of the 1:10 dilution of vaccine is unknown, but vaccination was successful in all the subjects who received these preparations; handling and shipping of the vaccine to the central laboratory may have led to the decrease in titer in these 2 instances. Therefore, throughout this article we use 107.0 pfu per milliliter as the mean titer for the 1:10 dilution.
To understand the amount of virus potentially delivered, we measured the number of plaque-forming units of virus on three representative bifurcated needles. The needles with undiluted vaccine had a mean of 105.0 pfu (range, 104.8 to 105.2), the needles with the 1:5 dilution of vaccine had a mean of 104.3 pfu (range, 104.1 to 104.5), and the needles with the 1:10 dilution of vaccine had a mean of 103.9 pfu (range, 103.8 to 104.1).
Assessments of the stability of the vaccinia virus demonstrated that when reconstituted in the new diluent, it remained viable, with consistent titers (within 100.3 pfu per milliliter), for seven days at 2 to 8°C. The titers of the undiluted vaccine were 107.8, 107.9, and 107.4 pfu per milliliter on day 0, on day 7, and at four months, respectively. The titers of the 1:5 dilution were 107.5, 107.2, and 106.6 pfu per milliliter on day 0, on day 7, and at four months, respectively. The titers of the 1:10 dilution were 106.9, 106.9, and 106.5 pfu per milliliter on day 0, on day 7, and at four months, respectively.
Success Rate
The initial vaccination was successful in 665 of the 680 subjects (97.8 percent). There was no significant difference in the rate of vesicle formation over the range of titers tested — 108.1, 107.2, and 107.0 pfu per milliliter (Table 1). When the results of the initial vaccination were combined with those of revaccination, the upper limit of the one-sided 95 percent confidence interval for the difference in values was 0.5 percentage point for the 1:5 dilution and 1.7 percentage points for the 1:10 dilution; thus, the noninferiority of the diluted vaccines as compared with the undiluted vaccine was established. This criterion was also achieved when the analysis included only the results of the initial vaccination (the upper limit of the one-sided confidence interval for the difference in values was 1.8 percentage points for the 1:5 dilution and 4.6 percentage points for the 1:10 dilution). The criterion was essentially met with the use of a more conservative two-sided confidence interval (data not shown), except that the upper limit of the 95 percent confidence interval for the difference in values was 5.1 percentage points for the comparison of a single vaccination of a 1:10 dilution with a single vaccination of undiluted vaccine.
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Reactogenicity
Local signs and symptoms of vaccinia virus replication among all 665 subjects in whom the initial vaccination was successful are summarized in Table 2. The diameter of the pustule was maximal on day 13 or 14 (mean, 12.4 mm) (Figure 2A, Figure 2B, and Figure 2K). Erythema and induration were maximal on day 10, 11, or 12 (mean, 51.4 and 48.1 mm, respectively), then decreased rapidly (Figure 2A, Figure 2B, Figure 2I, and Figure 2J). However, some subjects had very large areas of redness and swelling. Ten percent had diameters of redness exceeding 10 cm on day 10, 11, or 12, and 50 percent had diameters of redness exceeding 4 cm. Local satellite lesions near the site of inoculation were observed in 5.8 percent of subjects on day 13 or 14 (Figure 2B and Figure 2C) and were more common among subjects who had received diluted vaccine. Regional lymphadenopathy was common, occurring in 30.5 percent of subjects on day 7, 8, or 9.
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Pain at the vaccination site was common and was moderate or severe in 225 subjects (33.8 percent) on day 7, 8, or 9 and in 202 subjects (30.4 percent) on day 10, 11, or 12. More than one third of the subjects (36.4 percent) were sufficiently ill to miss school, work, or recreational activities or to have trouble sleeping.
The differences in local signs of vaccinia virus replication after vaccination with any of the three titers of vaccine are summarized in Table 4. There was no significant difference in the mean diameter of the pustule on day 13 or 14 among the three groups (P=0.10). However, subjects given undiluted vaccine had significantly larger areas of erythema (P<0.001) and induration (P=0.004) on day 7, 8, or 9 and a significantly higher incidence of regional lymphadenopathy on day 7, 8, or 9 (P=0.003) but a significantly lower incidence of satellite lesions on day 13 or 14 (P=0.003).
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Discussion
Our results suggest that the current stocks of vaccinia virus can be diluted to a titer of approximately 107.0 pfu per milliliter and still induce local viral replication and vesicle formation in a high proportion of persons who had never before been vaccinated. The lot of vaccine we used in our pilot study had a lower titer (107.8 pfu per milliliter), and a 1:10 dilution yielded a titer of 106.5 pfu per milliliter.2 The higher rate of response to the 1:10 dilution in the current study indicated that a titer of approximately 107.0 pfu per milliliter is needed to obtain a success rate of 95 percent or more.
Only eight subjects (1.2 percent) did not have a response after two vaccinations. Five of these eight were among the subjects who had preexisting neutralizing antibodies, suggesting that they had been vaccinated as infants. The use of a 1:10 dilution of vaccine in persons who have never been vaccinated, followed by a second vaccination in those with no response after seven days, could potentially protect nearly 10 times as many persons as would be protected by the administration of undiluted vaccine.
The rate of adverse reactions to vaccinia vaccine is dependent on age and immune status.4,5,8,9,10,11,12,13,14,15,16 Although there were no life-threatening adverse events, such as encephalitis or progressive vaccinia, in our study, there was a substantial degree of reactogenicity. The high frequency of local pain and erythema at the inoculation site, regional lymphadenopathy, and fever reflect active replication of vaccinia virus, with resultant acute viral illness. For the most part, the symptoms were mild to moderate. Although these adverse events were not serious, in more than one third of subjects they were the cause of missed work, school, sleep, or recreational activities. Autoinoculation of the eyes or the genitalia may occur in vaccinated children, and vaccinated persons may also inadvertently inoculate unvaccinated persons; we avoided these potentially serious events by applying semipermeable-membrane bandages to the inoculation sites.
There were significant differences in the incidence of local reactions between the group given undiluted vaccine and the groups given diluted vaccine. Undiluted vaccine resulted in larger areas of inflammation and a higher incidence of regional lymphadenopathy, whereas diluted vaccine resulted in a higher incidence of satellite lesions. These two observations may be related; increased inflammation may limit viral replication and therefore reduce the formation of local satellite lesions in persons who receive a high-titer vaccine. These clinical observations are also consistent with the observation in the pilot study that, although all subjects in whom vaccination was successful had cellular immune responses, the responses were more vigorous in the group that received undiluted vaccine. Whether or not the increased local inflammation associated with undiluted vaccine is due to a more rapid rate of replication soon after vaccination or to the inoculation of a minor subpopulation of more virulent vaccinia is not known. The morphology of vaccinia virus plaques in tissue culture is highly variable, suggesting that a mixed population of virus is present in the vaccine.
Whether or not persons vaccinated more than 30 years previously can be successfully revaccinated with diluted vaccine (e.g., doses as low as 107.0 pfu per milliliter) remains to be determined. Our observation that 6 of 14 subjects with no response after the initial vaccination had preexisting neutralizing antibodies suggests that the success rates in this population may be lower. Studies are being designed to determine whether protection is associated with preexisting antibody levels, cellular immunity, or both and whether a significant boost in immunity can occur in the absence of a primary response to vaccination.
Supported by contracts with the National Institute of Allergy and Infectious Diseases (N01-AI-45150, N01-AI-45248, N01-AI-45257, N01-AI-85342, N01-AI-65298, and N01-AI-15448) and by a contract from the National Center for Research Resources (M01-RR00188).
We are indebted to John Becher and Lisa Rotz at the Centers for Disease Control and Prevention for their assistance and thoughtful discussions; to Elizabeth Beim and the entire Emmes staff for their support of the protocol; to Edwin Anderson, M.D., Dennis Cunningham, M.D., James D. Campbell, M.D., Karen Kotloff, M.D., Genevieve Losonsky, M.D., Steven S. Wasserman, Ph.D., Michele Trucksis, Ph.D., M.D., Paul S. Sehdev, M.D., Michael Keefer, M.D., Robert Betts, M.D., Jeff Laduca, M.D., Thomas R. Cate, M.D., Ramona Simionescu, M.D., and Deep Ajmani, M.D., for their clinical assistance; and to the staff at the clinical sites.
Source Information
From the Department of Medicine, National Institute of Allergy and Infectious Diseases Vaccine and Treatment Evaluation Unit, Saint Louis University School of Medicine, St. Louis (S.E.F., F.K.N., R.B.B.); the Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston (R.B.C., R.L.A.); the Center for Vaccine Development, University of Maryland School of Medicine, Baltimore (C.O.T., R.E.); the University of Rochester School of Medicine and Dentistry, Rochester, N.Y. (J.J.T., C.M.N.); and the Emmes Corporation, Rockville, Md. (M.W.).
This article was published at www.nejm.org on March 28, 2002.
Address reprint requests to Dr. Belshe at the Division of Infectious Diseases and Immunology, Saint Louis University Health Sciences Center, 3635 Vista Ave. (FDT-8N), St. Louis, MO 63110.
References
In addition to the authors, the National Institute of Allergy and Infectious Diseases Smallpox Vaccine Study Group includes Stephen P. Heyse, M.D., M.P.H., Holli Hamilton, M.D., M.P.H., Pamela McInnes, D.D.S., Walla Dempsey, Ph.D., Lydia Falk, Ph.D., and Wendy Fanaroff-Ravick, R.N., M.S.N., all of the National Institute of Allergy and Infectious Diseases, Bethesda, Md.
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Related Letters:
Responses to Smallpox Vaccine
Sauri M. A., Frelinger J. A., Garba M. L., Belshe R. B., Frey S. E.
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Full Text |
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N Engl J Med 2002;
347:689-690, Aug 29, 2002.
Correspondence
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