Monkeypox infection elicits strong antibody and B cell response against A35R and H3L antigens

Abstract

Monkeypox virus (MPXV) resides in two forms; mature and enveloped, and depending on it, distinct proteins are displayed on the viral surface. Here, we expressed two MPXV antigens from the mature, and one from the enveloped form, and tested their reactivity to sera of 11 MPXV recoverees while comparing to sera from recently and past vaccinated individuals. 8 out of 11 recoverees exhibited detectable neutralization levels against Vaccinia Lister. Sera from all recoverees bound strongly to A35R and H3L antigens. Moreover, the responses to A35R were significantly higher within the recoverees compared to both recently and past vaccinated donors. Lastly, A35R- and H3L-specific IgG⁺ B cells ranging from 0.03-0.46% and 0.11-0.36%, respectively, were detected in all recoverees (A35R), and in 9 out of 11 recoverees (H3L). Therefore, A35R and H3L represent MPXV immune targets and could be used in a heat-inactivated serological ELISA for the identification of recent MPXV infection.

Keywords: Immunological methods; Immunology; Virology.

Graphical abstract

Figure 1

Sera from MPXV recoverees bind VACV and exhibit some VACV neutralization (A) Left: binding curves as measured by ELISA at O.D._650nm demonstrating serum response against VACV IHDJ strain. MPXV recoverees are in red (n = 11), VPXV are in purple (n = 6), uninfected >45yo are in blue (n = 11) and uninfected <45yo are in gray (n = 11). The mean serum response and standard error of the mean (SEM) are depicted in bold line and shadow, respectively, for each group. Four consecutive serum dilutions, starting from 1:10, for every group of donors were tested. Right panel: Area under the curve (AUC) values depicting each donor separately. Statistical analysis was performed using One-way ANOVA. ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001. (B) Left panel: NT₅₀ values of purified IgG from 11 MPXV recoverees as measured in a PRNT using VACV Lister, with and without the addition of complement. Right panel: graphical representation of the NT₅₀ values. Dashed line represents the PRNT’s measurement cutoff values of 300 μg/mL. NA – non applicable. Statistical analysis was performed using Ratio paired t-test. ∗∗p< 0.01.

Figure 2

Sera and purified IgG from MPXV recoverees bind recombinantly expressed MPXV antigens (A) Table listing the three MPXV antigens A35R, M1R and H3L produced in this study. Published PDB IDs of VACV homologs are given.^,, Amino-acid sequence alignment scores between MPXV and VACV antigens were calculated using the Clustal Omega web tool. (B) Construct design of the three recombinantly expressed antigens, A35R, M1R and H3L. Signal peptide, His-tag and Avi-tag are in black, light gray and dark gray, respectively. The amino acid section produced for each antigen is stated. (C) SDS-PAGE (left) and western blot using mouse anti-Avi-tag antibody (right) of the recombinantly expressed MPXV antigens A35R, M1R and H3L after purification. The antigens are indicated on top, the protein marker is on the right of each gel/blot, and protein sizes are indicated on the right in kDa. (D) Serum binding to MPXV antigens A35R, M1R and H3L as detected by ELISA. MPXV recoverees are in red (n = 11), VPXV are in purple (n = 6), uninfected >45yo are in blue (n = 11) and uninfected <45yo are in gray (n = 11). Upper panel: The bold lines and the shadowed areas on the graph represent the mean serum response and SEM, respectively, of 4 consecutive serum dilutions for every group of donors, starting from 1:75 dilution. Lower panel: AUC values for every individual donor. Statistical analysis was performed using One-way ANOVA. ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001. (E) Purified IgG binding to MPXV antigens A35R, M1R and H3L as detected by ELISA. MPXV recoverees are in red (n = 11), VPXV are in purple (n = 6), uninfected <45yo are in gray (n = 2), and VIG is represented by the dashed black line. Eight consecutive IgG dilutions starting from 100 μg/mL were tested (x axis). Upper panel: The bold lines and the shadowed areas on the graph represent the mean IgG response and SEM, respectively, for every group of donors. Lower panel: AUC values for every individual donor. Statistical analysis was performed between MPXV and VPXV groups using Welch’s t test. ∗∗p< 0.01.

Figure 3

IgG⁺ B cells from MPXV recoverees bind A35R and H3L antigens (A) Flow cytometry gating strategy for staining of A35R- and H3L-specific IgG⁺ B cells. Representative plots for MPXV recoveree (Mpx01), VPXV (Vpx05) and uninfected <45yo (<45yo-2) are shown. (B) Frequency of A35R-specific IgG⁺/CD19⁺ cells of MPXV recoverees are in red (n = 11), VPXV are in purple (n = 6) and uninfected <45yo are in gray (n = 3). Statistical analysis was performed between MPXV and VPXV groups using Unpaired t test. ∗p< 0.05, ∗∗p< 0.01. (C) The same as (B), but for H3L-specific IgG⁺/CD19⁺ cells.