Humoral immunity to smallpox vaccines and monkeypox virus challenge: proteomic assessment and clinical correlations

Abstract

Despite the eradication of smallpox, orthopoxviruses (OPV) remain public health concerns. Efforts to develop new therapeutics and vaccines for smallpox continue through their evaluation in animal models despite limited understanding of the specific correlates of protective immunity. Recent monkeypox virus challenge studies have established the black-tailed prairie dog (Cynomys ludovicianus) as a model of human systemic OPV infections. In this study, we assess the induction of humoral immunity in humans and prairie dogs receiving Dryvax, Acam2000, or Imvamune vaccine and characterize the proteomic profile of immune recognition using enzyme-linked immunosorbent assays (ELISA), neutralization assays, and protein microarrays. We confirm anticipated similarities of antigenic protein targets of smallpox vaccine-induced responses in humans and prairie dogs and identify several differences. Subsequent monkeypox virus intranasal infection of vaccinated prairie dogs resulted in a significant boost in humoral immunity characterized by a shift in reactivity of increased intensity to a broader range of OPV proteins. This work provides evidence of similarities between the vaccine responses in prairie dogs and humans that enhance the value of the prairie dog model system as an OPV vaccination model and offers novel findings that form a framework for examining the humoral immune response induced by systemic orthopoxvirus infection.

Fig 1

Normalized proteome view associated with vaccination using Dryvax, Acam2000, or two-dose Imvamune in humans and prairie dogs. The proteins are sorted by function and location (if known), with signal intensities represented by pale green for no response and red for increasingly intense reactions. Average raw values for naïve human and prairie dog serum profiles were included to provide visual controls for background proteome-wide antibody reactivity, whereas VIG and the remaining sample profiles are shown after subtraction of naive reactivity. Each column is an average of measurements for individual vaccinees, with the time point postvaccination indicated above each column. The normalization procedure for comparing human and prairie dog responses is described in Materials and Methods.

Fig 2

Antibody response to known neutralizing or protective antigenic targets and induction of neutralizing antibodies after Dryvax, Acam2000, or two-dose Imvamune vaccination. (a) Fluorescence intensities above the microarray cutoff for human and prairie dog sera in response to eight known neutralizing targets, H3, D8, A17, A27, and B5 and protective targets A33, A4, and A10, were scored positive or negative, counted for each vaccinee, and averaged (Wilcoxon rank sum; *, P < 0.05). (b) Neutralizing antibody titers for vaccinees were evaluated by an HCS-GFP neutralization assay and are displayed as the 50% RPR titer (50% neutralization). Human sera were not heat inactivated prior to analysis. Insufficient quantities of prevaccination sera were available for prairie dog NAb testing with the HCS-GFP assay. Time points for both assays are as shown in Fig. 1 and range from 21 to 49 (median = 30) days postvaccination. The error bars indicate standard deviation (SD).

Fig 3

Kinetic view of responses to MV proteins at days 0, 7, 14, 21, and 28 or 30 and correlation with ELISA results; shown are data for an Acam200-vaccinated human and a Dryvax-vaccinated prairie dog. Raw microarray signal intensities for select MV surface proteins are plotted, along with the array cutoff (dashed blue lines) and endpoint titer ELISA values (solid purple lines). The data are typical for each species regardless of the vaccination strain.

Fig 4

Microarray fluorescence in response to MV proteins correlates with the ELISA endpoint antibody titer. The intensities for all MV proteins present on the array were analyzed at days 0, 7, 14, and 21 to 49 after Dryvax or Acam2000 vaccination of humans (n = 6) and prairie dogs (n = 6) and compared with the corresponding ELISA titers. The data points are shown in color and represent the date of sampling, i.e., days 0, 7, 14, 21, and 29 to 49. The inset in the prairie dog graph shows a close-up view of the data with a 10⁰ ELISA endpoint titer. Data for all time points were not available for all individuals.

Fig 5

Heat map of reactivities to microarray proteins of prairie dog sera postvaccination and post-MPXV challenge. The animals were vaccinated with Dryvax, Acam2000, or Imvamune; allowed to seroconvert; and then challenged with MPXV. The animals providing MPXV-only samples were not vaccinated. The samples were collected between 27 and 30 days postchallenge and had prevaccination reactivity subtracted. VIG is shown for visual comparison with the human response to vaccination.