Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis.

Keywords: B cell; T cell; antibodies; memory; neutrophil.

Figure 1

Interferon‐γ (IFN‐γ) production upon stimulation of human peripheral blood mononuclear cells (PBMCs) from seronegative and seropositive individuals. Isolated human PBMCs (5 × 10⁵) from seronegative (n = 3) and seropositive (n = 5) individuals were stimulated at 37° for 48 hr in duplicate with dead B. pseudomallei whole bacteria (Bp), 3 μg/ml phytohaemagglutinin (PHA) or 10 μg/ml B. pseudomallei proteins; FliC, OmpA or N‐PilO2. IFN‐γ production upon stimulation was determined by ELISA. Statistical significance was analysed using one‐way analysis of variance, and post‐test using Bonferroni's Multiple Comparison test. *P < 0·05, **P < 0·01, ns, not significant.

Figure 2

Expansion of human B‐cell population induced by Burkholderia pseudomallei FliC, OmpA and N‐PilO2 protein antigens in vivo in hu‐PBL‐SCID mice. NOD/SCID/Jak3^null (SCID) mice were reconstituted with peripheral blood mononuclear cells (PBMCs) from seropositive donors and boosted with PBS (non‐boosted controls) or B. pseudomallei FliC, OmpA or N‐PilO2 proteins. After 14 days, spleen cells from hu‐PBL‐SCID mice were collected, processed and stained with the viability marker (AmCyan), and markers of human CD45, CD19, CD3 and CD4, before analysis by flow cytometry. Statistically significant differences between the results obtained from non‐boosted mice versus antigen‐boosted mice were analysed, using one‐way analysis of variance, and post‐test, using Bonferroni's Multiple Comparison test. *P < 0·05, **P < 0·01, ns, not significant.

Figure 3

In vivo human IgM and IgG antibodies are induced by boosting hu‐PBL‐SCID mice with FliC, OmpA and N‐PilO2. Mouse spleen and sera were collected at day 14 after boosting with PBS, FliC, OmpA or N‐PilO2. Levels of specific human antibodies, produced against boosting antigens in hu‐PBL‐SCID mice sera, were measured by indirect ELISA (a). The numbers of specific antibody secreting cells (ASCs) in antigen‐boosted or control mice were enumerated by ELISpot assay (b). Statistically significant differences between results from control and antigen‐boosted hu‐PBL‐SCID mice were analysed, using one‐way analysis of variance, and post‐test, using Bonferroni's Multiple Comparison test. *P < 0·05, **P < 0·01, ***P < 0·001, ns, not significant.

Figure 4

Burkholderia pseudomallei antigen‐specific interferon‐γ (IFN‐γ) ‐secreting cells are present in hu‐PBL‐SCID mice and are increased by boosting in vivo with FliC, OmpA and N‐PilO2. After boosting for 14 days, spleen cells were removed from hu‐PBL‐SCID mice, and were restimulated in vitro with cell culture medium, containing FliC, OmpA or N‐PilO2 for 48 hr, before IFN‐γ detection, counting the number of IFN‐γ spot forming units (SFU) by ELISpot assay. Statistically significant differences were analysed by using one‐way analysis of variance, and post‐test by using Bonferroni's Multiple Comparison test. *P < 0·05, **P < 0·01, ***P < 0·001, ns, not significant.

Figure 5

Affinity maturation of anti‐Burkholderia pseudomallei IgG antibody after boosting hu‐PBL‐SCID mice with FliC, OmpA and N‐PilO2. Antibody avidities of IgM and IgG were evaluated by using indirect ELISA following treatment with 7 m urea (n = 5). % Urea resistance of IgM and IgG antibodies from human sera (Human) and antigen boosted hu‐PBL‐SCID mice sera (Hu‐mice) were compared, statistically significant differences were analysed, using one‐way analysis of variance, and post‐test, using Bonferroni's Multiple Comparison test. **P < 0·01, ****P < 0·0001, ns, not significant.

Figure 6

Burkholderia pseudomallei specific antibody in hu‐PBL‐SCID mice promotes phagocytosis and oxidative burst activities of human polymorphonuclear cells (PMNs), in a concentration‐dependent and affinity‐dependent manner. Antigen‐specific purified pooled human plasma antibodies by affinity chromatography (a) and sera from hu‐PBL‐SCID mice 14 days after boosting with either PBS (non‐boosted), FliC, OmpA and N‐PilO2 (b) were used for FITC‐labelled opsonization of dead, intact B. pseudomallei. Elution buffer passed through a column with uncoated beads (no antibody; No Ab) was used as a negative control for purified human antibody (a), whereas sera from non‐boosted hu‐PBL‐SCID mice (Non‐boosted) were base line control for hu‐PBL‐SCID sera (b). Whole blood from seropositive donors was cultured with pre‐opsonized FITC B. pseudomallei, and oxidative burst activities from human PMNs were detected by flow cytometry. Statistical significance was analysed using one‐way analysis of variance, and post‐test using Bonferroni's Multiple Comparison test. *P < 0·05, **P < 0·01, ***P < 0·001, ns, not significant compared between results from conditions with and without antibody. The correlation between level (c) or avidity (d) of IgG antibody in each antigen boosted hu‐PBL‐SCID mice and % total phagocytosis (closed circles) and % Oxidative burst in phagocytosed cells (open circle) was analysed by linear regression.

Figure 7

Summary of the humanized mouse model (hu‐PBL‐SCID) reconstituted with human peripheral blood mononuclear cells (PBMCs) from Burkholderia pseudomallei seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N‐PilO2) for boosting both T‐cell and B‐cell immune responses.