Analysis of Variola and Vaccinia Virus Neutralization Assays for Smallpox Vaccines

Abstract

P revious studies (6) suggested that the quantitative titers of human serum plaque reduction neutralization tests (PRNTs) differed when variola or vaccinia virus was used as the neutralization target. Similar observations were made during Acambis2000 vaccine studies in postimmunization nonhuman primate sera (10). This study was designed to evaluate the correlation between PRNTs when different virus species or strains were used as the neutralization target. This study is the first to look extensively at these differences using the same set of well-defined human postvaccination sera. If the ability to neutralize variola is the desired outcome of smallpox vaccination, then understanding the relative significances of variola and vaccinia neutralization titers is a critical surrogate measure, especially if previous measures of vaccine efficacy (i.e., the Jennerian pustule or "take") are not available for third-generation vaccines and if variola stocks are destroyed. The comparable efficacy of a vaccination regimen using vaccinia (Dryvax or modified vaccinia Ankara [MVA]) or variola virus as the neutralizing target is presented elsewhere (3, 4). Sera (from 46 participants) from a National Institutes of Health-funded smallpox vaccine trial (DMID 02-017) were evaluated at Saint Louis University (SLU) and at the Centers for Disease Control and Prevention (CDC). Twenty participants received MVA (IMVAMUNE) subcutaneously (SC) (1 ϫ 10 8 50% tissue culture infective dose units [TCID 50 ]; 2 doses, 1 month apart), 15 received MVA intramuscularly (IM) (1 ϫ 10 8 TCID 50 ; 2 doses, 1 month apart), and 11 received Dryvax vaccination by scarification (1 dose) (4). MVA is a replication-deficient, less-reactogenic third-generation smallpox vaccine (1, 7, 9). Sera at peak response times postvaccination were evaluated using variola, Dryvax, and MVA PRNTs. Individuals were evaluated 28 to 30 days postDryvax vaccination or 14 days after the second MVA dose.At SLU, serum samples were tested in a qualified PRNT assay using an American Type Culture Collection (ATCC) strain of MVA (catalog number VR-1508) or Dryvax (2, 8) as the neutralization reference virus. The Dryvax PRNT was modified by substituting MVA for Dryvax as the neutralizing target and by identifying plaques through immunostaining in place of crystal violet staining. Sonicated MVA virus was diluted to ϳ30 to 50 PFU/well. An equal volume of diluted MVA was mixed with each serial 2-fold dilution of heat-inactivated serum or medium and incubated overnight at 37°C. Each serum-virus mixture and virus-medium mixture (virus-only control) was inoculated onto

Comparison of lyophilized versus liquid modified vaccinia Ankara (MVA) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naïve subjects

“…However, a two-dose regimen of IMVAMUNE ® has been shown to provide a similar antibody response as Dryvax ® [9], reduced the size of replicating vaccinia takes [9,28] and was as effective if not better than Dryvax ® in producing peak 90% variola virus neutralization GMTs [39,40]. The two-dose SC MVA regimen remains the standard against which other regimens/doses are compared until more data are available.…”

Safety and immunogenicity of IMVAMUNE® smallpox vaccine using different strategies for a post event scenario

“…Serum from the same clinical trial was used to correlate between PRNTs when different viruses and different strains of virus were used as the neutralization target. Use of Dryvax as the neutralization target resulted in significantly lower 90% PRNTs than use of variola as the neutralizing target and use of MVA resulted in significantly higher PRNTs than using variola [19]. …”

Humoral Immunity to Smallpox Vaccines and Monkeypox Virus Challenge: Proteomic Assessment and Clinical Correlations

“…Keasey et al observed greater increases in antibody responses to several MPXV proteins than to VACV proteins by aerosol MPXV-challenged macaques (59), indicating that although broad cross-reactivity exists, a stronger response may be directed toward MPXV-specific proteins. Differences in neutralization titers have also been observed, depending on the strain of OPV utilized in the PRNT assays (68,69). While a direct comparison between titration methods is not possible using the vaccinia virus-based HCS-GFP assay, future experiments that shed light on the virus specificity of this broader response and the virus-specific neutralization capacity after vaccination and challenge would be of interest.…”