There is a need for a safer smallpox vaccine. The current U.S.-approved smallpox vaccine has the highest adverse event rate of any vaccine being used [29
]. Historically 1000 out of a million vaccinees suffer complications from smallpox vaccination. More than 50 out of a million events will be life-threatening and 1-2 out of a million will result in death [5
]. Life-threatening vaccine-related complications most often arise in individuals with a disease, condition, or treatment resulting in a disrupted skin barrier or compromised immunity. Approximately, 25% of the U.S. population is at risk or would place a close contact at risk of complication from the vaccine [29
Current efforts to generate safer smallpox vaccines include the use of live but attenuated strains of vaccinia such as Modified Vaccinia Ankara (MVA) or defective Lister LC16m8 [21
]. Alternatives to the live virus approach to smallpox vaccine development include the use of subunit DNA [38
], peptide [25
], or protein vaccines [41
] and bacterial vectors [43
]. These alternatives are in the early stages of development and significant developmental, clinical and regulatory hurdles are likely to be encountered prior to approval for wide-spread clinical use. It should be noted that in the U.S., the Dryvax vaccine will very likely be replaced by ACAM2000 (Acambis plc, Cambridge, UK), a new live virus vaccine derived from Dryvax. While this vaccine has been demonstrated to be as effective as Dryvax, the risk of adverse events due to vaccination apparently remains the same. Immune responses to this vaccine with and without ST-246 as an adjunct should also be tested also.
We propose an alternative approach to the new vaccines in development; one in which the Dryvax vaccine (or ACAM2000), which is proven to be effective but not entirely safe, is used in combination with ST-246, a novel antiviral drug in development as a smallpox therapeutic, to improve its safety. In previous animal studies [13
], it has been demonstrated that ST-246 protects animals from lethal poxvirus challenge and suggests that protective immunity is elicited even in the presence of the drug. Previous to this report, a detailed comparative analysis of immune responses elicited by smallpox vaccination in the presence of ST-246 has not been performed.
The data presented in this study demonstrate that protective immunity elicited by the smallpox vaccine is not compromised by concurrent treatment with ST-246. ST-246 reduces the severity of lesion formation due to vaccination with VV-WR but has no effect on the less severe lesion formation due to Dryvax vaccination (Figs. and ). This suggests that ST-246, if used as an adjunct to the smallpox vaccine in humans, will not inhibit the vaccine “take”—the lesion formed as a result of vaccination that is often used as evidence of vaccine-induced protective immunity. We consider this to be significant considering that in the event of a smallpox outbreak the vaccine will be administered to all suspected of exposure. In the absence of a lesion, serological testing would be necessary to confirm vaccine-induced protective immunity.
The IgG responses to whole virus or individual virion proteins as determined by ELISA are slightly reduced by ST-246 (), but the virus-neutralizing serum responses are unaffected by the drug ( and ). Cellular immune responses were not significantly affected by ST246: the cytokine release assays demonstrate that TNF-α, IFN-γ, and IL-2 responses are slightly enhanced by ST-246 (Figs. and ) at early times post-vaccination, while the proliferative responses to vaccinia antigens are unaffected (). Our observations that early cellular responses may be slightly enhanced by ST-246 were initially considered to be the result of normal experimental variability, but in all repeats of the experiment, the trend remained consistent. Additional studies will be necessary to further confirm the reproducibility of this phenomenon and understand the mechanism. Considering our results, it is possible that vaccination in combination with ST-246 enhances natural killer cell activity, or NK1.1 or γδ T cell activation at early times post-infection since cytokine production did not appear to be due to detectable CD4+ or CD8+ T cell activation although the response was virus-specific. We will address these possibilities in future studies. It was intriguing that the IFN-γ response on day 180 post-vaccination as determined by the CBA assay was elevated relative to earlier time-points. To our knowledge there is no precedent in the literature for this observation. These assays were necessarily performed at different times (although the assay conditions were identical) so it is possible that the results reflect inter-assay variability. We consider this unlikely in light of the fact that the TNF-α and IL-2 responses, measured from the same sample, as well as our negative control samples, are not correspondingly elevated. Analysis of the IFN-γ response by ICCS at these same time-points using the same preparation of splenocytes and stimulator cells demonstrates that the number of cells producing IFN-γ in response to restimulation is very similar at days 33 and 180 post-vaccination. It is possible that at day 180 post-vaccination, T-cell avidity is higher, thus enabling a more robust recall response to restimulation or, alternatively, non-CD4+ or -CD8+ antigen-specific responses are contributing to the overall response similar to what was observed on day 7 post-vaccination.
ST-246 is in development as a post-exposure therapeutic for smallpox. It has been proven to be extremely efficacious in preventing poxvirus disease in a post-exposure setting. Considering this, it is reasonable to think that ST-246 may be used to prevent or treat smallpox vaccine-related adverse events. It is debatable whether a 14-day treatment regimen is feasible under the conditions in which smallpox vaccination would be given (i.e., prior to deployment for military personnel, prophylactic vaccination of “first responders”, or emergency vaccination of the civilian population in the event of a smallpox outbreak). This issue cannot be specifically addressed at this time considering that ST-246 is still under development as a drug and that final formulations and dosing requirements are unknown.
In this study, we demonstrate that protective immune responses to the vaccine are not inhibited by ST-246. This study was performed in immunocompetent mice so that a direct comparison of robust immune responses could be made. The risk of adverse events in humans is elevated in immunocompromised individuals or those with a history of eczema (atopic dermatitis) or other exfoliative skin disorders amongst other contraindications. Murine models for these situations exist. Future studies will be focused on testing the pathogenicity of the smallpox vaccine and prophylactic and therapeutic efficacy of ST-246 in these models Additionally, we will begin to address the in vivo mechanism of action of ST-246 on poxvirus replication and dissemination both at the site of inoculation (the “pock”) and at in distal tissues as well as dissemination between hosts. Based on our previous data [13
], the observations of others [14
] and findings presented here, we hypothesize that ST-246 will be effective in preventing or treating smallpox vaccine-related adverse events in these models without compromising immune responses. This raises the possibility that ST-246 may be used as an adjunct to the smallpox vaccine in individuals, for whom the vaccine is currently contraindicated, protecting them from serious adverse events, without inhibiting the generation of protective immunity.