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. Author manuscript; available in PMC: 2021 Jun 11.
Published in final edited form as: Cell Host Microbe. 2019 Jun 18;26(1):100–113.e8. doi: 10.1016/j.chom.2019.05.003

Figure 5. Immunization of BALB/c Mice with R. int Induces Human β2GPI Cross-Reactivity.

Figure 5.

(A) Clustal Omega alignment of RGGMR DI epitope in β2GPI, Roseburia intestinalis L1-82 (R. int) and Bacteroides thetaiotaomicron VPI-5482 (B. theta). B. theta lacks T cell mimotope homology but contains a partial B cell mimotope (GGMR) homology.

(B and C) Proliferative response to immunization was determined using ATP luminescence in triplicates. Fold increase was determined by averaging triplicates from stimulated populations and dividing by the average of the unstimulated (“no antigen”) cells in RLU. Splenocytes (B) and peripheral lymphocytes isolated from inguinal and axillary lymph nodes (C) were restimulated ex vivo with rhβ2GPI.

(B) Splenocytes from R. int-immunized mice (n = 7) respond significantly more to rhβ2GPI than sham-immunized mice (n = 7, p = 0.0017) and B. Theta-immunized mice (n = 6, p = 0.0047).

(C) Peripheral lymphocytes from R. int-immunized mice (n = 7) respond significantly more to rhβ2GPI than sham-immunized mice (n = 7, p = 0.0006) and B. theta-immunized mice (n = 6, p = 0.0012).

(D and E) Mice were immunized s.c. with lysates from R. int, B. theta, or no lysates (sham) in IFA followed by three s.c. IFA boosts. Representative ELISA using 1:100 diluted serum. Values represent the average OD450 of individual mice in triplicates.

(D) R. int-immunized mice have significantly more serum anti-β2GPI IgG (n = 8) compared to B. theta-immunized mice (n = 7, p = 0.0003) and sham-immunized mice (n = 7, p = 0.0003). R. int-immunized and B. theta-immunized sera lose significantly binding to rhβ2GPI39-43 compared to rhβ2GPI.

(E) R. int-immunized mice have significantly more serum anti-R. int DNMT IgG (n = 8) compared to B. theta-immunized mice (n = 7, p = 0.0205) and sham-immunized mice (n = 7, p = 0.0003). R. int-immunized and B. theta-immunized sera lose significantly binding to R. int DNMTΔ122-126 compared to R. int DNMT. Two-tailed Mann-Whitney U test performed for inter-group comparison and two-tailed Wilcoxon signed-rank test performed for intra-group comparison. Error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.