In this study, we suggest that an attenuated vaccinia virus m8 strain that was licensed in 1975 in Japan as the second-generation smallpox vaccine is as efficacious as the first-generation LO vaccine that was used worldwide in the WHO smallpox eradication program.
The m8 vaccine was not used in a large population in areas of endemicity because smallpox was almost eradicated when it was developed. Today, no vaccines under development or in human trials can be tested for protective efficacy against smallpox by infection of humans with the causative virus, VAR. However, a pathogenic vaccinia virus WR strain provides an alternative small-animal model suited for evaluating protective immunization (2
). VV has rather low infectivity for mice, but WR is an exception, because it is adapted to mice by repeated passages in the mouse brain (27
). Intranasal inoculation with as little as 105
PFU of WR elicited severe illness and 50% death in BALB/c mice, although they were less susceptible to VV infection than C57BL/6 and C3H/He mice (unpublished data). Thus, BALB/c mice vaccinated with the LO and LO-derived vaccine strains failed to develop definite erythema or pustules at the inoculated skin sites, which is classified as a “take” that is indicative of viral replication and therefore successful immunization in other vaccinia virus-sensitive hosts, such as humans, cows, and rabbits. Anti-B5R, -EEV, or -IMV antibodies were certainly undetectable or at low levels in vaccinated BALB/c mice. Nevertheless, the m8, mO and LO vaccines all protected mice comparably and completely against challenge with 106
PFU of WR. Notably, a single subcutaneous vaccination with m8 primed mice to render them as protective as vaccination with mO and LO, even at a low dose (105
PFU). Furthermore, with an increased WR challenge dose (107
PFU), 100% of mice vaccinated percutaneously with m8 (105
PFU) survived, while they lost significant weight temporarily and comparably to those vaccinated with the LO or NYBH strains (unpublished data) that had been used in humans.
OPVs are known to be highly cross-reactive among themselves in immune protection. Indeed, the m8 vaccine protected monkeys against MPV challenge (unpublished data), as recently described for the MVA vaccine (9
). On the basis of these historical and experiential facts, CPV is thought to have been used in 1798 as the first human vaccine against VAR, and VV became the smallpox vaccine in the modern era. Similarly, OPVs are genetically highly conserved. Complete OPV genome sequences from VV, VAR, CPV, MPV, ectromelia virus, and camelpox virus have recently been investigated for phylogenetic analyses, with results indicating that CPV (strain GRI) is closely related to VV and that the genetic distances from VAR were lowest for camelpox virus (<0.0155), next lowest for VV (<0.0259), high for MPV (<0.0307), and highest for ectromelia virus (<0.0354) (22
). These analyses may lead to the prediction that complete genome sequence data from VVs or OPVs will provide insight into the efficacy of smallpox vaccine strains.
Therefore, we determined the complete genome sequences of the licensed m8, parental mO, and grandparental LO strains. Our data may be interpreted to mean that the LO-related vaccines have similar abilities that would induce immune protection, supporting the above-mentioned prediction. Only four missense mutations occurred among the >280 deduced ORFs of m8 during evolution from the parental mO strain. The major change was a truncating mutation of the B5R gene. It is therefore noted that B5R was the only destroyed gene in m8 compared to mO. Furthermore, m8 and mO possessed almost all ORFs corresponding to the vaccinia virus CPN strain (21
). As the grandparental LO strain has never been plaque cloned, its genome sequence exhibited huge polymorphisms, which were previously suggested by analyses of restriction enzyme fragments and pock or plaque size (46
). However, our PCR sequencing of the EEV env
-related genes indicated that they were all preserved in mO, and in LO as well, and that m8 was probably derived from a low-virulence clone of divergent LO. This genomic background of m8 suggests that it functions like LO as a smallpox vaccine, except for B5R.
B5R is the only NAb-inducing antigen of EEV so far identified (19
). EEVs are extracellular free virions released from infected cells and seem to be prevented by NAbs (12
). Destruction of B5R reduced the formation of EEV 5- to 10-fold (36
), although they comprise less than 1% of the total virus population (41
). In light of these findings, a concern has arisen that the m8 vaccine seems to contain reduced amounts of EEV that lacks the B5R antigen and might not be protective against long-range spread of VAR EEV (5
). Our study of multiple immunizations with recombinant B5R proteins adsorbed to adjuvant showed that antigenic activity was absent in the truncated B5R protein of m8 but present in the intact protein of LO. In addition, infection or vaccination with live VV induced very few anti-EEV NAbs, and repeated inoculations were required to induce moderate NAb levels (19
), probably because of the small EEV population. Alternatively, low levels of the antibodies may be due to the low sensitivity of conventional assay systems. Wyatt et al. recently reported that NAbs can be produced after a single percutaneous vaccination (56
). They recently developed and used a highly sensitive system, a semiautomated flow cytometric assay with recombinant VV expressing enhanced green fluorescent protein (8
It was therefore important to examine the levels of protection against virulent WR infection in m8-vaccinated mice, irrespective of the absence of EEV B5R-specific antibody responses. Our results confirmed that a single vaccination with m8, mO, and LO failed to induce detectable levels of anti-EEV and anti-B5R antibodies. Nevertheless, mice immunized with these vaccines were 100% protected against pathogenic WR challenge as early as 3 weeks after vaccination. Moreover, m8 with the whole B5R gene deleted protected mice from lethal WR challenge (32
). These findings suggest that many viral antigens other than B5R are also involved in protective immunity to EEV. In this regard, antibodies to the A33R Env antigen did not neutralize EEV but provided mice with 100% protection (19
). Anti-A33R might disrupt fragile EEV Env and convert to IMV, which is easily neutralized by anti-vaccinia antibodies (19
). Alternatively, A33R-specific cellular immunity may be crucial for protective immunity.
We have only limited knowledge about the protective immune mechanisms against smallpox. Experience with worldwide vaccination, however, has suggested that the protective mechanisms involve innate immunity, including interferons, natural killer cells, and complements, and also acquired immunity, including specific antibody- and T-cell-mediated immune responses (12
). Indeed, recent papers have revealed the involvement of gamma interferon-expressing CD8 and CD4 T cells, vaccinia-specific cytotoxic T cells, and T-helper type 1 memory in humans (6
) and mice (16
). Several studies conducted out of urgency in the last few years using smallpox vaccine candidates came to similar conclusions with regard to the contribution of overall immunity to smallpox protection (2
). Moreover, priming effects in vaccinated persons were recently shown to be long-lived or long-lasting, for as long as 75 years after vaccination (23
). These historical and most recent studies imply that vaccine priming for immunological memory is important so that effecter components, such as NAbs, CD4+
T cells, and various cytokines can promptly be induced or boosted to protective levels by VAR infection, regardless of whether they are above measurable levels before infection. In support of this hypothesis, we found that mice that received a single dose of LO-related vaccines could not fully develop antibody responses as early as 3 weeks after vaccination but could produce enhanced levels of antibodies and complete immune protection after pathogenic-virus infection.
The need to produce safer and more effective vaccines may increase in the future. Here, we determined the nucleotide sequences of the whole genomes from the m8, intermediate mO, and original LO vaccine strains. The accumulating information on complete genome sequences of attenuated or pathogenic VVs and other OPVs will provide a basis for producing new genetically engineered vaccines. The double-stranded DNA genomes of OPVs are known to be highly stable. However, a single nucleotide insertion just upstream of the m8 B5R mutation site has recently been reported to restore the ORF to the parental mO phenotype after repeated (10 or more) virus passages. Although the repaired viruses were a marginal population, attenuation that is achieved by a deletion of the whole B5R gene prevented the reversion of m8- to mO-type viruses (32
), which have, however, much lower virulence than LO and NYBH (24
). In turn, the genetic manipulation of m8 to replace genes related to protective immunity, but not to pathogenicity, with the counterpart genes of VAR may make m8 more efficacious. It will be necessary to study in detail the correlation between individual gene functions and antigenicity of the gene products for inducing protective immunity in the future.