Orthopoxviruses remain an emerging public health threat. Although smallpox was eradicated in 1980, it remains a potential bioterrorism threat. Other orthopoxviruses, including monkeypox and cowpox viruses, are zoonotic disease threats to humans. The methodology used to assess prior orthopoxvirus exposure or response to vaccination is primarily through enzyme-linked immunosorbent assay (ELISA). This assay requires four to five hours of hands-on time and nearly two days to obtain results. In this study, we utilized Simple WES^TM, an automated capillary immunoblotting platform, to develop a less labor intensive and high-sensitivity assay through identification and incorporation of immunodominant Vaccinia virus proteins to detect antibody response to smallpox vaccination.

Naïve and post-vaccination serum samples (n=142) were obtained from an IRB-approved CDC smallpox vaccination study. Sera from vaccinia-naïve individuals were used as negative controls and vaccinia immunoglobulin (VIG) was used as positive control. Samples and reagents were prepared according to the ProteinSimple manual and included purified proteins or in-house generated un-purified proteins. Sensitivity and specificity analyses were conducted and compared to corresponding ELISA results.

Our study found that vaccinia proteins A27, H3, D13, and A25 correlated to the band pattern observed previously at 13, 42, 62, and 91 kilodaltons, respectively, in both VIG and post-vaccination serum samples. Samples were tested using a mixture containing purified A27 and H3 resulting in a sensitivity and specificity of 63% and 100%, respectively, using one-of-two peak criteria. Use of un-purified D13 and A25 proteins failed to increase assay sensitivity, but efforts to purify and incorporate these proteins is ongoing.