Studies using VacV have led to a comprehensive understanding of orthopoxvirus replication, dissemination, and pathogenesis. Additionally, VacV, VarV, and MPX share 98% sequence homology. However, some variance exists among poxvirus strains and clades with respect to the precise mechanisms of dissemination. For example, different strains of VarV exhibit distinct plaque phenotypes in vitro
and different mortality profiles in vivo
). Given the potential clinical significance of VarV and MPX, we assessed whether the mode of dissemination was conserved between these viruses and VacV. Our data demonstrate that VarV and MPX are capable of inducing actin tails in a manner analogous to that of VacV. All of these viruses localize host factors known to regulate actin polymerization, such as Grb-2 and Nck. Like VacV, VarV and MPX also appear to utilize Src and Abl family tyrosine kinases in a redundant fashion (24
). Of potential importance from a clinical perspective, actin tails formed by VacV, MPX, and VarV are similarly sensitive to Src and Abl family tyrosine kinase inhibitors. In plaque assays, dasatinib and PD-166326 reduced the sizes of plaques and comets, whereas imatinib mesylate reduced comet size without diminishing plaque size. The findings of EEV assays were generally consistent with those of the comet assay, with one exception. Although imatinib mesylate inhibited comet formation by VarV-BSH, VarV-SLN, MPX, and VacV, the drug appeared to have less dramatic effects in EEV assays with MPX. Because PD-166326 and dasatinib were effective in both the comet and EEV assays with MPX and because the comet assay was consistent across all strains tested, we cannot rule out that adsorption of EEV to infected cells or incomplete neutralization of IMV may contribute to apparent quantitative differences in EEV assays.
Drugs that affect poxvirus replication or spread are important to mollify symptoms associated with vaccination or for smallpox or monkeypox virus infections in individuals for whom vaccination poses a significant risk or would prove ineffective. The therapies currently approved or used on the investigational level for poxvirus infections are vaccinia immune globulin (VIG) (5
) and cidofovir, a DNA polymerase inhibitor (3
). However, the efficacy of VIG in late-stage infections is limited, and while effective, cidofovir causes severe renal toxicity at the doses required and must be administered with intravenous hydration and in conjunction with probenecid, a renal tubular blocker that is also not without complications (14
). It is unlikely that this regimen could be implemented to successfully treat a significant number of infected individuals. Another drug, ST-246, blocks formation of CEV and EEV and has shown efficacy in mouse and nonhuman primate models of poxvirus infection (12
), though it apparently engenders resistance. ST-246 is currently in human trials.
Would tyrosine kinase inhibitors such as dasatinib and imatinib mesylate prove efficacious in vivo
? The in vivo
shortcomings of dasatinib stand in stark contrast to its apparent promise based on in vitro
assays. Despite robust in vitro
effects on plaque size and comets, dasatinib neither reduces viral loads nor protects mice from lethal challenge. During the course of our experiments, the European Medicines Agency reported immunotoxicity for dasatinib (10
). Specifically, treatment with a dose of 25 mg/kg, but not 15 mg/kg, delivered once daily prevents graft rejection in a murine cardiac transplant model. Furthermore, dasatinib inhibits murine splenic T-cell proliferation and induces lymphoid depletion of the thymus and spleen. These data are in accordance with our observation that dasatinib induces splenopenia and suppresses the effects of imatinib mesylate on dissemination of VacV. Taken together, these data indicate that immunotoxicity of dasatinib likely accounts for its failure to provide benefit for poxvirus infections. Unfortunately, we were unable to define a concentration or dosing regimen that would minimize immunosuppressive effects yet still abrogate viral dissemination.
The most likely explanation for the immunosuppressive effects of dasatinib is the inhibition of Src family kinases rather than Abl family kinases. In particular, Fyn and other Src family tyrosine kinases have been implicated in various aspects of the immune response, including innate and antigen signaling, phagocytosis, and T- and B-cell development (15
). Dasatinib also inhibits Abl family kinases more potently than imatinib mesylate does. However, our data with the latter suggest that inhibition of Abl family kinases per se
likely does not contribute to significant immunosuppression: imatinib mesylate did not prevent acquisition of protective immunity to poxviruses, and the drug is well tolerated in human patients, who show little increased incidence of infection (35
). Furthermore, we demonstrated the ability of imatinib mesylate to limit dissemination of virus in vivo
, a finding consistent with our in vitro
data (Fig. ). Together, these data suggest that dual Src/Abl inhibitors provide little in vivo
benefit against microbial infections, despite their apparent efficacy in vitro
In contrast to the case for dasatinib, the data presented here suggest that imatinib mesylate can provide significant protection when administered postinfection, in addition to its prophylactic effects reported previously (24
). Nevertheless, the benefits decrease in a time-dependent manner following inoculation. The absence of drug during the period following inoculation may permit virus to establish infection and to spread to distal tissues. Once established in distal tissues, viral replication and actin tails may contribute to further expansion of virus, despite the addition of the drug. A similar argument may account for our observation that increasing the inoculum to 2 × 105
PFU, or 10 times the LD100
, overcame the protective benefit of imatinib mesylate (data not shown). Notably, the effects of imatinib mesylate on limiting dissemination were particularly evident at low viral titers (Fig. ), akin to the infectious dose for VarV in humans, in luciferase assays. Another factor contributing to the efficacy of imatinib mesylate following inoculation may be that drug delivered via an osmotic pump reaches therapeutic levels only after 16 to 18 h. Despite these caveats on the precise timing of its delivery, imatinib mesylate provides a significant level of protection pre- or postinfection, perhaps by allowing time for an effective immune response to develop, in a manner that does not interfere with acquisition of protective immune memory. Collectively, these data suggest that the potential utility of imatinib mesylate for treatment of poxvirus infections should be evaluated further.
In this regard, prairie dogs may offer a means to assess the therapeutic value of imatinib mesylate for MPX infections (12a
). Similar to the case in the murine model, an inoculum of 5 × 104
PFU i.n. is used. However, this model is distinguished by the appearance of disseminated lesions or “pox” at 9 to 12 days postinfection, a phenotype previously observed only in primate models. In humans, pox lesions generally appear 7 to 19 days following infection and have been attributed to migration of EEV through the lymphatic system to the skin. Thus, presentation of pox in the prairie dog model recapitulates an important aspect of disease progression seen in humans but not in other small-animal models. Our data demonstrating that imatinib mesylate limits EEV release in vitro
and dissemination in vivo
, especially at low inoculums (Fig. ), suggest that this drug may have efficacy against MPX in prairie dogs and possibly primates, using rash illness progression as a disease marker, a prospect we are now testing.
Imatinib mesylate may also have utility when coadministered with other compounds under consideration as poxvirus therapeutics, such as ST-246 and cidofovir. ST-246 protects mice from lethal challenge when administered by up to 3 days postinfection (2
). ST-246 acts more distally than imatinib mesylate by inhibiting F13 and interfering with IEV production and viral dissemination (36
). Notably, however, variants resistant to ST-246 have been described that result from a single base change in F13L (36
). Similarly, resistance to cidofovir is conferred by point mutations in E9L, the DNA polymerase gene (1
). In contrast, imatinib mesylate is less likely to engender resistant mutants because it targets host kinases. Moreover, when coadministered, imatinib mesylate may reduce viral loads and decrease the probability of developing mutants resistant to ST-246 or cidofovir.
In summary, we describe a conserved mode of dissemination within the orthopoxvirus family and the mechanism of actin tail formation and EEV release by MPX and VarV. In addition, we show that dual Src/Abl inhibitors effectively limit both actin tail-based motility and EEV release in vitro. However, their utility against poxvirus infections in vivo is precluded by their immunosuppressive activity. In contrast, we show that imatinib mesylate can be used in a therapeutic context and does not interfere with the acquisition of immune memory, which may warrant further testing of this or related drugs in animal models of poxvirus infection.