INTRODUCTION
Smallpox, caused by variola virus (genus
Orthopoxvirus, family
Poxviridae), accounted for millions of deaths throughout human history. The disease was declared eradicated in 1980 after a worldwide program implemented by the WHO involving strict surveillance and intensive vaccination using vaccinia virus (VACV;
Poxviridae). After disease eradication, smallpox vaccination was discontinued on all continents (
1,
2), and variola virus stocks were transferred to two maximum security laboratories at the CDC, Atlanta, GA, and Vector, Koltsovo, Novosibirsk region, Russia (
3).
Since the 1990s, and mainly after the terrorist attacks on the United States in 2001, there has been a general concern that smallpox could reemerge as a biological weapon (
3). Therefore, different countries and the WHO have increased their stockpile of smallpox vaccine over the years (
4,
5). However, the first-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current health care standards (
6,
7). Therefore, the search for new-generation vaccines that combine low pathogenicity and immune protection is important.
Recent studies using different sequencing technologies demonstrate that first-generation vaccines are genetically heterogeneous and consist of a pool of quasispecies related to the uncontrolled passage in calf/sheep skin or embryonated chicken eggs for several decades. Viral clones isolated from these vaccines may differ in virulence, efficacy of immune protection, and genetic content (
8–12). In fact, the second-generation smallpox vaccine currently produced in the United States, ACAM2000, is a clone of the first-generation vaccine used in the country, Dryvax. ACAM2000 induces immune responses and protection similar to that of the parental strain and was selected in parallel to the neurovirulent clone 3 (CL-3), also isolated from the Dryvax vaccine (
4,
9). Work involving additional clonal selection further confirmed the genotypic diversity of Dryvax (
11,
13).
In addition to the search for safer vaccines, recent genetic studies of clonal variants of first-generation vaccines have improved our understanding of the phylogenetic relationships and obscure origins of different VACV strains (
8,
11,
12). Nevertheless, numerous questions remain unanswered.
The major smallpox vaccine producer in Brazil, among three others, was Instituto Oswaldo Cruz in Rio de Janeiro (
1,
14). VACV strain IOC (VACV-IOC) was exclusively used by the Institute to manufacture the vaccine that was widely distributed in Brazil during the eradication campaign (
1). The vaccine was produced in calf skin or chicken eggs (
14) and usually had take rates of >98% (
1) and low rates of adverse effects (H. Schatzmayr, personal communication). Unfortunately, poor documentation resulted in scant existing information about the origins of VACV-IOC. However, it is suggested that the VACV-IOC used in the 1970s originated from the Beaugency strain (J. A. Espmark to D. A. Henderson, correspondence on March 31, 1969, file 88-001-10, Sanofi Pasteur Archives [Connaught Campus]) that probably constituted the first animal-based vaccine samples imported to Brazil in 1887 from Chambon Institute, France (
15,
16).
Damaso et al. (
17) named the vaccine VACV-IOC, after the Instituto Oswaldo Cruz, during comparative studies with Cantagalo virus (CTGV). This virus is a VACV strain that infects dairy cattle and milkers in Brazil, and it constitutes a rare example of the occurrence of VACV in nature. Studies have suggested that VACV-IOC is phylogenetically related to CTGV and to other field variants related to CTGV (CTGV-like viruses), which were isolated in subsequent outbreaks after 1999 (
18–21). Nevertheless, the analyses were based on a few sequenced genes and deletion patterns (
17,
22). Hence, full-genome sequencing would be necessary to place VACV-IOC correctly in relation to other VACV strains, particularly Cantagalo virus.
In addition to the lack of genetic information regarding VACV-IOC, little is known about the biological and immunological features of this vaccine strain. Taking into account the genetic diversity of first-generation vaccines, we isolated two viral clones at random to characterize genetically homogenous populations of VACV-IOC. In this work, we show that the selected clones combined low virulence to mice with effective immune protection from lethal challenge and shared a common fragmentation pattern of several virulence genes. More importantly, based on the full-genome sequencing, we provide evidence that VACV-IOC branches as a novel independent cluster in VACV phylogeny together with the Brazilian field strains CTGV and Serro 2 virus as well as horsepox virus (HSPV). Our data strongly support the hypothesis that CTGV represents a feral VACV that evolved in parallel with the VACV-IOC used in the 1970s after splitting from a most recent common ancestor, probably an ancient smallpox vaccine sample related to horsepox virus. This work brings new insights into the origins and evolutionary relationships of VACV lineages.
DISCUSSION
VACV-IOC was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication program (
1). However, little is known about the biological and immunological features, as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. We show evidence that both clones could induce a protective immune response against a lethal infection and had low virulence in infected mice. Most notably, this study provides genetic evidence of a novel, independent cluster of VACV phylogeny formed by VACV-IOC, the Brazilian field strains CTGV and Serro 2 virus, and HSPV. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved independently of the VACV-IOC used in the 1970s after splitting from a most recent common ancestor, probably an ancient smallpox vaccine sample related to HSPV. Our data revisit the origins of VACV and propose new evolutionary relationships between ancient and extant strains.
The immunization with clone B141 or B388 conferred protection following a lethal challenge, and the profile of protection was comparable to those of the licensed vaccine ACAM2000 and the parental vaccine strain VACV-IOC. It has been reported that ACAM2000 induces immune responses similar to those of its parental strain, Dryvax, which consequently leads to protection (
4). We did not investigate the immunogenicity of the parental VACV-IOC in detail; however, our data suggest that B141 and B388 immunogenicity is comparable to that of VACV-IOC, which was successfully used during the Brazilian smallpox vaccination campaign.
An immune response profile similar to that elicited by B141 and B388 generally is observed following infections with VACV and is well established for other vaccine strains (
40–43). The humoral immune response induced by smallpox vaccines has been identified as the main arm of the immune response in the protection against orthopoxvirus reinfections (
44,
45). Sera of mice immunized with B141 and B388 showed neutralizing titers (1:29) comparable to those obtained for the first-generation vaccine Dryvax (1:22) (
43). Nevertheless, MV neutralizing antibody levels of 1:32 or higher are correlated with protection against smallpox in vaccinees (
46). Despite the somewhat low levels of MV neutralizing antibodies, both IOC clones induced a robust neutralization response against extracellular virus and protected mice against a lethal challenge, similar to other VACV strains (
40,
47).
Whether cell-mediated immune responses to VACV are essential for protection against a lethal challenge is still controversial (
6,
44,
45). Nevertheless, they are key players in controlling primary infection (e.g., vaccination) (
48,
49). In this context, immunization with B141 and B388 efficiently stimulated the production of IFN-γ, TNF-α, or IL-2 by CD4
+ and CD8
+ T cells in response to VACV. T-cell priming also has been reported following immunization with vaccine strains Dryvax, LC18m6, ACAM2000, and Lister (
40,
42,
50,
51). More importantly, a percentage of B141- and B388-primed CD4
+ or CD8
+ T cells exhibited a polyfunctional phenotype and simultaneously expressed IFN-γ in combination with either TNF-α or IL-2. Interestingly, polyfunctional CD4
+ T cells, but not CD8
+ T cells, were correlated with smaller lesions in vaccinated individuals following revaccination with VACV-Lister (
52). On the other hand, MVA primed highly polyfunctional CD8
+ T cells in vaccinees after challenge with Dryvax (
53). Based on these findings, we suggest that immunization with B141 and B388 induce a protective T-cell response.
Although equally immunogenic, B388 was, to some extent, more virulent to mice than B141, whereas the parental VACV-IOC showed intermediate virulence compared to that of the clones. In this context, it is interesting that the second-generation vaccine ACAM2000 is less virulent than its parental strain, Dryvax, in intrathalamic infection (
4). Therefore, our data suggest that, between the two VACV-IOC clones, B141 is a better alternative candidate for a second-generation vaccine that combines low virulence and immunogenicity.
B141 and B388 had similar spread rates and small-plaque phenotypes in cell culture. Therefore, differences in virulence between B141 and B388 might rely on other characteristics, such as the differential expression of virulence genes. Deletions and truncations of virulence genes usually result in VACV attenuation in animal models (
54). Interestingly, clones B141 and B388 have several fragmented virulence genes. Among these are orthologs of the VACV-Cop genes C10L, C9L, C6L, C4L, C2L, M1L, A55R, and F1L and VACV-WR gene B13R.
VACV-Cop C10L encodes a protein recently shown to bind Ku subunits of DNA-PK, leading to the inhibition of cytoplasmic DNA sensing. This protein is intact and predicted to be functional in all extant VACV strains except for MVA (
55). The truncated C10 protein encoded by IOC_019/020 (and their ITR paralogs) of B141 and B388 probably is not functional because it lacks the C terminus, which is essential for Ku-binding activity (
55).
Remarkably, clones B141 and B388 have two fragmented apoptosis-related genes: the orthologs of VACV-WR B13R and VACV-Cop F1L. B13R (IOC_227/228/229) encodes SPI-2 and also is truncated in other VACV strains (VACV-Cop B13R/B14R). On the other hand, F1L (IOC_063/064) is intact in all VACV strains, except for the IOC clones that retain 30% of the N terminus. F1L encodes a Bcl2 protein that binds to and inhibits caspase 9 activation (
56) and Bak/Bax-mediated apoptosis (
57). The absence of intact orthologs of B13R and F1L indicates that clones B141 and B388 are devoid of essential mechanisms to inhibit apoptosis. Preliminary results show the presence of cleaved poly(ADP-ribose) polymerase during infection of cells with clones B141 and B388 (L. C. Schnellrath and C. R. Damaso, unpublished results).
Despite these genotypic similarities, B141 and B388 differ by a 4.3-kb deletion in the 3′ end of the B388 genome, which is probably not related to the enhanced virulence of B388. On the other hand, clone B388 has full copies of orthologs of K3L and C3L, whereas these genes are fragmented in B141 (IOC_056/057 and IOC_042/043, respectively). K3L is conserved in all other VACV strains and is an important inhibitor of the type I IFN pathway (
58). Nevertheless, the deletion of K3L did not lead to VACV attenuation in the mouse model (
59). Therefore, the fragmentation of K3L probably does not account for the lower virulence of B141 compared to that of B388. In contrast, the deletion of C3L caused VACV attenuation in infected mice (
60). It encodes a secreted complement control protein (VCP) that binds cellular C3b and C4b through SCR domains and inhibits complement-mediated neutralization of the virus particles (
61). The truncated VCP encoded by B141 has one SCR domain, in contrast to four domains present in the full VCP of B388, and this difference probably affects the efficacy of B141 to neutralize the complement during infection. Therefore, C3L will be further investigated as a potential promoter of enhanced virulence in B388, in contrast to B141.
The evolutionary relationships and origins of VACV strains are quite nebulous. In this study, phylogenetic inferences indicated that VACV-IOC branches as a new independent VACV cluster together with CTGV-like viruses and, interestingly, with horsepox virus. The IOC clones and CTGV-like viruses map as subgroups that split from a most recent common ancestor. Most notably, HSPV branches into this novel VACV cluster as an independent subgroup. Sequence alignments and analysis of estimated maximum-likelihood distances indicated that HSPV is more closely related to VACV-IOC than to CTGV-like viruses. Therefore, these data suggest that the common ancestor to VACV-IOC and CTGV-like viruses was an ancient smallpox vaccine strain closely related to horsepox virus. We hypothesize that this ancient vaccine was the sample imported to Rio de Janeiro, Brazil, in 1887 and originated the modern VACV-IOC, but it also escaped to nature and originated feral VACV (CTGV-like viruses). In support of this hypothesis, the transit of infected cows from Rio de Janeiro to other states was reported in the late 1890s as an effort to establish vaccine-producing centers in other states (
15,
62). Moreover, reports of poxvirus-related infection in dairy cattle have been described since the beginning of the 20th century, and on several occasions they were related to the transmission of smallpox vaccine from vaccinees (
22). In some farms, dairy cows were vaccinated with the smallpox vaccine to prevent further spread of the so-called cow-pox to the entire herd (
63). An alternative hypothesis to explain the presence of CTGV-like viruses in the Brazilian wilderness has been recently proposed, suggesting the accidental entry of horses or rodents infected with VACV/HSPV in Brazil and the subsequent establishment of the virus in nature (
64). However, our work introduces new evidence based on full-genome sequence and bioinformatics analyses that favor the former hypothesis of an ancient vaccine origin for CTGV-like viruses.
The phylogenetic relationship of VACV-IOC and CTGV was first hypothesized when CTGV was isolated during an outbreak of pustular disease in cows in 1999 (
17). Previous studies have shown that VACV-IOC and CTGV-like viruses share unique genetic features, such as the 18-nt and 15-nt deletions in the orthologs of the A56R gene and K2L gene, respectively (
17,
21,
22). Moreover, these viruses share phenotypic features, such as low spreading in cell culture, low virulence in mice (
25,
65), and apoptosis induction (S. Reis, L. C. Schnellrath, N. Moussatche, and C. R. Damaso, unpublished data). Nevertheless, we show here that the panel of fragmented virulence genes differs between the IOC clones and CTGV-like viruses, and some sequences present in CTGV-like viruses are missing from the IOC clones. Despite that, both CTGV and Serro 2 virus have smaller genomes than the IOC clones, indicating a greater loss of genetic information during their divergence and coevolution over time. On the other hand, and more importantly, IOC-B141, CTGV, and Serro 2 virus share the same junction site of the 3′ ITR, which has been suggested to be a genetic feature of VACV lineages (
13). Together, these phenotypic and genotypic features support the close phylogenetic relatedness of VACV-IOC and CTGV-like viruses.
It is generally accepted that HSPV is closely related to an ancestor of the modern VACV strains (
37). Hence, the phylogenetic relatedness of HSPV and VACV-IOC is intriguing and suggests an ancient evolutionary path for the Brazilian smallpox vaccine. Based on this assumption, it would be expected that the novel cluster would branch off the base of the VACV phylogenetic tree. Nevertheless, this topology is observed only when the simplest hierarchical clustering method (unweighted pair group method with arithmetic means [UPGMA]) was used (data not shown), as also noted by others (
10,
37). The analysis of more and older strains of HSPV would certainly contribute to this discussion. Nevertheless, the use of equine pustules as smallpox vaccine is an old practice. Jenner and colleagues reported the production of smallpox vaccine using fluid from vesicular eruptions in horses with “horse-pox” in the early 1800s (
38,
66). Therefore, this novel cluster raises interesting issues regarding the origins of VACV strains in general and particularly of the Brazilian VACVs.
Historical records indicate that the establishment of an animal-based vaccine in Brazil was accomplished only in 1887, when samples of calf-lymph vaccine were brought to Rio de Janeiro from the Chambon Institute in Paris, France (
15,
16). The French Institute used the Beaugency strain (
67), which is suggested to be the origin of the VACV-IOC used in the Brazilian smallpox eradication campaign (J. A. Espmark to D. A. Henderson, correspondence on March 31, 1969, file 88-001-10, Sanofi Pasteur Archives [Connaught Campus]). Interestingly, the VACV-IOC cluster always mapped as a sister group to the American Dryvax cluster but not to the cluster of extant European VACV strains. In support of the relatedness of Dryvax and IOC/CTGV strains, HSPV occasionally mapped with the Dryvax cluster when fewer Dryvax genomes were included in the tree reconstructions (data not shown) or in the absence of IOC/CTGV-like virus genomes (
10–13). The Dryvax vaccine produced by Wyeth laboratories supposedly was derived from the New York City Board of Health (NYCBH) strain that was brought from England to the United States in 1856 (
1).
In an attempt to reconcile our phylogenetic findings, we searched historical records to gain an understanding of the evolutionary connections between VACV-IOC, HSPV, and Dryvax. In 1866, Ernst Chambon and Gustave Lanoix obtained material from spontaneous cases of “cow-pox” in Beaugency, Loire Valley, France, and used it as the seed strain to produce calf-based smallpox vaccine (animal vaccine) at the recently created Institute de Vaccine Animale in Paris, France. A second natural case of “cow-pox” occurred later in 1866 in St. Mandé, suburbs of Paris, and the samples were mixed without changing the “Beaugency” designation. The success of the Beaugency stock led to its distribution to Belgium, Germany, Sweden, Switzerland, French colonies, and the Americas in the next decades (
67). In the meantime, spontaneous cases of “horse-pox” were also documented in France from 1860 to the 1880s, and vesicular pustules from infected horses were used as seed material for the production of smallpox vaccine by different French producers (
68). The Beaugency strain was introduced into the United States in 1870 by Henry Martin of Boston, who established a private animal vaccine farm. The Beaugency lymph then was widely distributed to several cities, including New York (obtained by Frank Foster, director of the New York Dispensary) (
69–71) and Philadelphia, where the Wyeth brothers started an animal vaccine farm in 1885. In the same year, the Wyeth brothers obtained a fresh seed of Beaugency lymph from the Vaccinal Institute in Belgium (
71). Interestingly, the NYCBH apparently used different sources of vaccine virus over the years. The annual report of the NYCBH in 1872 indicates the establishment of the animal vaccine in 1871, using in most animals a bovine sample obtained from the Practical Institute for Animal Vaccine in Cuba and Puerto Rico. A few animals were double infected with more scarification points of this Cuban sample and a few points of the Beaugency strain provided by Frank Foster (
72). However, in 1874 the NYCBH reported the simultaneous use of humanized (Jennerian) and calf-based vaccines. The former derived from the vaccine stocks of J. P. Loines, who obtained the samples from England in 1856, and the latter were derived from the bovine stocks supposedly produced since 1871 (
73). If all these historical records are accurate regarding the strain names and sources, it is possible that the Dryvax vaccine is derived from the Beaugency strain, similar to VACV-IOC, and not (or at least not only) from the NYCBH strain as widely assumed. This hypothesis would account for the relatedness of VACV-IOC and Dryvax and also the mapping of the NYCBH-derived strains WR and IHD-W in a different cluster apart from the Dryvax viruses.
However, it is important to note that the second half of the 19th century was a period of intense exchange and distribution of European smallpox vaccine lymph of different origins among European countries, the United States, and throughout the world (
1,
66,
67,
69). Therefore, our data favor the interesting hypothesis proposed by Evans's group that a heterogeneous ancestor stock of VACV would be the origin of the genetic diversity found in the extant VACV strains (
13). Furthermore, our work adds new information to this hypothesis, suggesting that natural cases of so-called horse-pox and cow-pox, as well as the indiscriminate mixture of samples, probably contributed to increased heterogeneity of the subsequent VACV stocks that were randomly sampled during the 19th century.