Smallpox was eradicated in the 1970s through the use of intensive vaccination in combination with campaigns designed to discover and isolate residual pockets of disease (10
). The vaccines used in many of these campaigns were composed of live vaccinia virus (VACV) cultured in large quantities on the skins of animals, usually cows. Many different vaccinia virus strains were used as vaccines toward the end of this era, including a strain that was distributed in a lyophilized formulation called Dryvax (DVX) produced by Wyeth Laboratories (24
). This calf lymph vaccine is derived from the New York City Board of Health VACV strain and shares this origin with the most commonly studied VACV research strain, Western Reserve (WR). However, the two viruses have long been propagated in isolation. The last stocks of Dryvax were produced after passaging the virus 22 to 28 times in cows (24
), while the sequenced strain of WR (NC_006998) has a complex 70-year history of passage, first in rabbits, followed by mice and, in more recent decades, by extensive passage in cell culture (26
; R. Condit, personal communication).
These old smallpox vaccines were rarely subjected to clonal purification; in fact, the methods used to propagate them would have readily produced mixtures of viruses that are commonly called quasispecies. They were also contaminated with adventitious agents, including bacteria and bacterial debris (10
). This situation is considered intolerable for modern licensure requirements and created problems when the need arose to produce new smallpox vaccine supplies in the late 1990s. This led to the development of ACAM-2K (Acambis clone 2000), a licensed vaccine comprising a VACV strain cloned from Dryvax and cultured on Vero cells (11
). ACAM-2K was one of seven viruses originally cloned from a pool of Dryvax production lots and shown to replicate the immunogenicity of Dryvax in humans while exhibiting a seemingly comparable (albeit still not ideal) safety profile (11
Genome sequencing has suggested that vaccines like Dryvax are comprised of a complex mixture of viruses. The Esposito laboratory reported that there are 573 single-nucleotide polymorphisms (SNPs) and 53 insertions-deletions (indels) of various sizes that differentiate ACAM-2K (originally called clone 2, or CL2) from a more neurovirulent sister clone, clone 3 (CL3) (24
). Similar degrees of sequence difference are observed when these viruses are compared in a pairwise manner with other independently isolated Dryvax subclones, including VACV-3737 and VACV-Duke. VACV-Duke is of special interest because it was isolated from a patient who developed progressive vaccinia after being vaccinated with Dryvax (18
). Thus, it may illustrate an example of clonal selection operating in vivo
upon a virus presumed to have been a component of the original inoculum. The fact that old smallpox vaccines comprise a quasispecies is not restricted to Dryvax, of course. Garcel et al. have documented a diversity of phenotypes exhibited by clones isolated from a stock of the VACV strain Lister (13
), and shotgun sequencing of unpurified stocks identified >1,200 polymorphic sites distributed across a mix of Lister genomes (23
These observations raise intriguing questions about the degree of genome diversity that can be found in old smallpox vaccines. In this communication, we have taken advantage of recent advances in DNA-sequencing technologies to explore this question in greater detail. Our results illustrate the remarkable complexity of the quasispecies that characterize stocks of old, unpurified smallpox vaccines and suggest that the viruses that have been isolated to date represent only a small fraction of the diversity of viruses in these preparations. These genomic studies also provide insights into the origin of viruses like VACV-Duke and of orthopoxvirus evolution under the selection processes associated with classical VACV propagation methods.