Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection

ABSTRACT

Brucellosis, caused by the bacterium Brucella, poses a significant global threat to both animal and human health. Although commercial live Brucella vaccines including S19, RB51, and Rev1 are available for animals, their unsuitability for human use and incomplete efficacy in animals necessitate the further study of vaccine-mediated immunity to Brucella. In this study, we employed in vivo B-cell depletion, as well as immunodeficient and transgenic mouse models, to comprehensively investigate the roles of B cells, antigen uptake and presentation, antibody production, and class switching in the context of S19-mediated immunity against brucellosis. We found that antibody production, and in particular secretory IgM plays a protective role in S19-mediated immunity against virulent Brucella melitensis early after the challenge in a manner associated with complement activation. While T follicular helper cell deficiency dampened IgG production and vaccine efficacy at later stages of the challenge, this effect appeared to be independent of antibody production and rather was associated with altered T-cell function. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy at later timepoints after the challenge. In addition, B-cell depletion after vaccination, but before the challenge, enhanced S19-mediated protection against brucellosis, suggesting a deleterious role of B cells during the challenge phase. Collectively, our findings indicate antibody production is protective, while B-cell MHCII expression is deleterious, to live vaccine-mediated immunity against brucellosis.

IMPORTANCE

Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

INTRODUCTION

Brucellosis, caused by the Gram-negative bacterium Brucella, stands as a significant zoonotic disease that carries extensive implications for global human and animal health. Despite its profound impact, it remains a disease that has not garnered sufficient attention (1). This facultative intracellular pathogen has adeptly broadened its reservoirs to encompass a variety of domestic and wildlife animals (2), rendering its eradication a formidable challenge. Brucellosis affects an estimated 2.1 million humans annually worldwide, with the pathogen also infecting over 300 million cattle out of the 1.4 billion total cattle population (3, 4). In animals, brucellosis primarily results in abortion and other manifestations such as retained placentas, decreased milk yield, and decreased fertility (5). In humans, acquiring the disease can occur through various routes, including contact with infected animals, consumption of contaminated animal products, and inhalation of airborne agents (6). The impact of brucellosis on human health is wide ranging, spanning from the characteristic undulant fever to more debilitating effects such as arthritis, orchitis, hepatitis, and endocarditis (7–11).

The lack of effective treatments for animal brucellosis and the high cost of treating infected humans (5, 12) emphasize the necessity for robust disease prevention. Current vaccines such as S19, RB51, and Rev1 exhibit around 70% efficacy against animal brucellosis (13–15), underscoring the need for further research on vaccine-mediated immunity.

Various investigations, including our own, have found that B-cell deficiency renders mice more resistant to brucellosis (16–18), indicating a detrimental role of B cells in primary infection. This negative role imposed by B cells on resistance to Brucella is antibody independent (16, 18). By contrast, in a model of secondary brucellosis, a prior study has indicated an indispensable role for B cells in controlling infection (19, 20). Other studies have provided evidence that passive antibody transfer from infected or immunized animals protects naïve mice from brucellosis (21–26). However, in bovine, high levels of IgG1 and IgG2 antibodies following infection were found to hinder complement-mediated killing of B. abortus in vitro presumably due to the “prozone effect” and there is generally thought to be a lack of positive correlation between antibody titers and resistance to brucellosis (27).

The S19 vaccine is cross-protective against B. melitensis in laboratory animals and dairy cattle (28–30). As we previously investigated B-cell effector functions during primary infection with B. melitensis (18, 31) here we employed a S19 vaccination/B. melitensis challenge model to determine the role of B-cell effector functions in vaccine-mediated immunity against Brucella. Our comprehensive investigation employed a range of immunodeficient and transgenic mouse models, along with depletion assays, to explore the contributions of B-cell and T follicular helper cell responses to vaccine-mediated immunity against brucellosis.

RESULTS

B cells exert a phase-dependent detrimental impact on S19 vaccine-mediated efficacy against brucellosis

Previously, we found that B-cell deficiency or depletion enhanced the resistance of mice to primary infection with B. melitensis (18, 31). However, in secondary brucellosis, prior research utilizing B-cell-deficient mice, where infected animals were treated with antibiotics and then rechallenged with Brucella, has suggested somewhat variable roles on the contributions of B cells which might be related to the route of infection or time post-challenge when control of infection was evaluated (19, 32). Thus, we employed a vaccination/challenge approach (33), combined with B-cell depletion, to evaluate the impact of temporal B-cell deficiency on vaccine-mediated immunity against brucellosis. B cells were depleted either 7 days prior to vaccination or 21 days post-vaccination (one week before challenge, Fig. 1A) with anti-CD20 antibody. B-cell depletion in blood was found to be ≥90% effective at the time of challenge (Fig. S1A) and significantly reduced anti-Brucella IgM and IgG levels (Fig. S1B and C). B-cell depletion prior to vaccination raised splenic B. melitensis burdens 2 weeks post-challenge, though this difference was not significant (Fig. 1B). Conversely, depleting B cells from mice at day 21 after vaccination (1 week before challenge) significantly reduced splenic burdens compared to isotype treated mice at this timepoint (Fig. 1B). This indicates that the presence of B cells during vaccination, followed by their removal before challenge is advantageous in protecting against virulent Brucella infection. Similarly, after 4 weeks of vaccination and 4 weeks post-challenge, we again observed that B-cell depletion prior to the challenge improved resistance to brucellosis (Fig. 1C), further validating the deleterious role of B cells during the challenge phase. Taken together, these findings demonstrate that B cells have time-dependent roles in the setting of S19 vaccination and Brucella infection.

Fig 1.

Impact of temporal B-cell deficiency on S19-mediated efficacy against brucellosis. (A) Schematic of the B-cell depletion, vaccination, and challenge strategy employed. Mice were s.c. vaccinated with 2 × 10⁵ CFUs of S19 vaccine and were challenged i.p. 4 weeks later with 1 × 10⁵ CFUs of B. melitensis 16M. For B-cell depletion, an anti-CD20 antibody was injected i.p to WT mice either at 7 days before vaccination (referred to the figures as Anti-CD20 _-7preS19) or 21 days after vaccination (referred in the figures as Anti-CD20 _+21postS19). (B) Splenic B. melitensis burdens in B-cell depleted and IgG-treated vaccinated WT mice (n = 9–10 mice/per group) measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (C) Splenic B. melitensis burdens in B-cell depleted and IgG-treated vaccinated WT mice (n = 10 mice/per group) measured after 4 weeks of vaccination and 4 weeks of challenge (4 + 4). Data in (C) are combined from two experiments. Dashed lines indicate the limit of detection.

B cells are deleterious during the Brucella challenge phase in an MHCII-dependent manner

We previously demonstrated B-cell MHCII expression enhances susceptibility to primary brucellosis (18). Therefore, we utilized CD19^CreiAB^fl/fl mice that specifically lack B-cell MHCII expression (31) to determine whether the detrimental effect exerted by B cells during the challenge following S19 vaccination is contingent on their antigen presentation capability. Similar to what we observed during primary infection (31), anti-Brucella IgG levels were reduced in CD19^CreiAB^fl/fl mice following vaccination with S19 (Fig. S1D). After 4 weeks of vaccination and 2 weeks of challenge, we observed similar levels of B. melitensis in iAB^fl/fl and CD19^CreiAB^fl/fl mice, which were reduced ~90- to 180-fold relative to PBS-treated iAB^fl/fl control mice (Fig. 2A). However, after 4 weeks of vaccination and 4 weeks of challenge, we observed a significant reduction in the splenic burden of S19-vaccinated B-cell MHCII-deficient mice in relation to vaccinated control animals (Fig. 2B), indicating B-cell MHCII expression is detrimental to S19-mediated efficacy at later stages after challenge. Taken together, these observations suggest MHCII expression on B cells undermines the effectiveness of the S19 vaccine against brucellosis.

Fig 2.

B-cell MHCII expression is deleterious to S19-mediated efficacy. Mice were s.c vaccinated with 2 × 10⁵ CFUs of S19 and were challenged 4 weeks later with 1 × 10⁵ CFUs i.p. of B. melitensis 16M. (A) Splenic B. melitensis burdens in CD19^CreiAB^fl/fl and iAB^fl/fl mice (n = 8–12 mice/per group) measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (B) Splenic B. melitensis burdens in CD19^CreiAB^fl/fl and iAB^fl/fl mice (n = 7–14 mice/per group) measured after 4 weeks of vaccination and 4 weeks of challenge (4 + 4). Data are combined from two experiments. Dashed lines indicate the limit of detection.

BCR specificity has a dispensable role in S19-mediated efficacy against brucellosis

BCR-mediated mechanisms of antigen uptake are 100–1,000 times more efficient in inducing cognate T-cell activation through MHCII compared to BCR-independent mechanisms of antigen uptake (34). In light of our finding that B-cell MHCII expression diminishes S19 vaccine-mediated protection (Fig. 2B), we investigated the influence of BCR specificity on S19-mediated immunity against Brucella by employing MD4 mice, in which ~90% of B cells express a BCR specific for the irrelevant antigen hen egg lysozyme (HEL) (35). First, we compared the ability of B cells from WT and MD4 mice to uptake Brucella by exposing splenocytes obtained from WT or MD4 mice to GFP-expressing S19 in vitro and employing confocal microscopy to visualize the presence of bacteria within B cells. Our results revealed that B cells from MD4 mice can uptake GFP-S19, albeit at a level ~3 times lower than B cells from WT mice (Fig. 3A through C), suggesting BCR specificity plays a significant role in the uptake of S19.

Fig 3.

BCR specificity impacts S19 uptake by B cells but does not influence S19-mediated efficacy against brucellosis. (A–C) Confocal images (A and B) and quantification (C) of GFP-S19 in B cells from WT (A) or MD4 (B) mice in vitro following 4.5 hours of infection (MOI = 500). (D & E) Mice were s.c vaccinated with 2 × 10⁵ CFUs of S19 and were challenged 4 weeks later with 1 × 10⁵ CFUs i.p. of B. melitensis 16M. (D) Splenic B. melitensis burdens in MD4 and WT mice (n = 4–10 mice/per group) were measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (E) Splenic B. melitensis burdens in MD4 and WT mice (n = 8–15 mice/per group) were measured after 4 weeks of vaccination and 4 weeks of challenge (4 + 4). Data in (E) are combined from two experiments. Dashed lines indicate the limit of detection.

Consequently, we investigated whether BCR specificity affects S19 efficacy by vaccinating MD4 and WT littermate control mice and comparing splenic bacterial loads at 2 and 4 weeks post-challenge. Surprisingly, at both times post-challenge, the lack of BCR specificity to Brucella antigen did not significantly alter B. melitensis levels in S19-vaccinated mice (Fig. 3D and E). These findings could suggest that BCR specificity does not mediate the detrimental effects of B-cell MHCII expression on vaccine- mediated Brucella. However, BCR specificity also mediates antibody specificity, and we previously found impaired anti-Brucella antibody production in MD4 mice (31). Thus, we sought to determine whether impaired Brucella-specific antibody production in MD4 mice could mask a potential deleterious role of BCR specificity on vaccine-mediated immunity against Brucella.

Vaccine-elicited IgM and complement are crucial to control the early dissemination of Brucella

Passive transfer of immune sera from infected or vaccinated animals has indicated antibodies can protect recipients against Brucella (21–26). In addition, by comparing results in B-cell-deficient mice and mice unable to undergo class switching others have suggested a protective role of IgM in secondary Brucella infection (19). By contrast, in primary infection, we found an inability to secrete antibodies does not affect the control of B. melitensis within the first month after infection (Fig. S2A) ( 31). To clarify the role of humoral immunity in vaccine-mediated immunity against Brucella, we used sIgM^−/−/AID^−/− mice which express a polyclonal B-cell receptor but are unable to secrete IgM or class-switched antibodies (Fig. S2B and C) (36). Our findings revealed that 2 weeks post-challenge, S19-vaccinated sIgM^−/−/AID^−/− mice, vaccinated 4 weeks or 8 weeks before the challenge, had significantly higher splenic loads relative to WT mice (Fig. 4A and C). By contrast, we observed that the absence of antibodies had no impact on the efficacy of the S19 vaccine at 4 weeks post-challenge (Fig. 4B). These findings indicate that anti-S19 secretory antibodies play a crucial role in limiting the early dissemination of virulent Brucella; however, the role of antibodies diminishes as infection advances.

Fig 4.

Vaccine elicited antibodies are necessary for protecting against brucellosis at the early stage of infection. Mice were s.c vaccinated with 2 × 10⁵ CFUs of S19 and were challenged 4 or 8 weeks later with 1 × 10⁵ CFUs i.p. of B. melitensis 16M. (A) Splenic B. melitensis burdens in sIgM^−/−/AID^−/− and WT mice (n = 4–10 mice/per group) measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (B) Splenic B. melitensis burdens in sIgM^−/−/AID^−/− and WT mice (n = 7–10 mice/per group) measured after 4 weeks of vaccination and 4 weeks of challenge (4 + 4). (C) Splenic B. melitensis burdens in sIgM^−/−/AID^−/− and WT mice (n = 4–10 mice/per group) measured after 8 weeks of vaccination and 2 weeks of challenge (8 + 2). Data in (B) are combined from two experiments.

We proceeded to investigate the specific contribution of IgM and class-switched antibodies by generating mice unable to secrete class-switched antibodies (sIgM^−/+/AID^−/−) or IgM (sIgM^−/−/AID^−/+). We then evaluated the ability of S19 to protect these strains against Brucella. Serological analysis (Fig. S2C and D) confirmed IgM and IgG production was ablated in sIgM^−/−/AID^−/+ and sIgM^−/+/AID^−/− mice, respectively. Bacterial levels trended higher in S19-vaccinated mice unable to produce class-switched antibodies relative to controls, though this difference was not statistically significant (Fig. 5A). However, vaccine efficacy was significantly impaired in mice unable to secrete IgM (Fig. 5A) indicating a pivotal role of IgM in vaccine-mediated immunity against Brucella. A key function of IgM is the activation of complement (37). Therefore, we depleted S19 vaccinated mice of complement by treating them with cobra venom factor (CVF). As revealed in Fig. 5B, complement depletion in vaccinated mice significantly increased splenic B. melitensis burdens 2 weeks post-challenge relative to controls, indicating a crucial role of complement in vaccine-mediated immunity against brucellosis.

Fig 5.

IgM and complement contribute to S19-mediated efficacy against brucellosis. Mice were s.c vaccinated with 2 × 10⁵ CFUs of S19 and were challenged 4 weeks later with 1 × 10⁵ CFUs i.p. of B. melitensis 16M. (A) Splenic B. melitensis burdens in mice lacking the ability to secrete IgM (sIgM^−/−/AID^−/+), class-switched antibodies (sIgM^−/+/AID^−/−), and heterozygous control sIgM^−/+/AID^−/+ mice (n = 11–12 mice/per group) measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (B) WT mice (n = 9–10 mice/per group) were s.c vaccinated with 2 × 10⁵ CFUs of S19 and challenged i.p. 4 weeks later with 1 × 10⁵ CFUs of B. melitensis 16M. Some mice were also depleted of complement with cobra venom factor (CVF). B. melitensis was measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). Data in (A) are combined from two experiments. Dashed lines indicate the limit of detection.

Bcl6 deficiency is associated with impaired vaccine efficacy and altered CD4⁺ T-cell transcriptomic profile

Our investigation on the role of antibodies was furthered by employing CD4^CreBcl6^fl/fl mice, in which Bcl6 deletion in CD4⁺ T cells results in T follicular helper cell (T_FH) deficiency. Isotype switching and germinal center responses are also impaired in mice lacking Bcl6 expression in CD4⁺ T cells (38). Analysis of antibody levels 4 weeks post-vaccination indicated T_FH deficient mice exhibited comparable anti-Brucella IgM levels but significantly dampened anti-Brucella IgG levels relative to their vaccinated Bcl6^fl/fl counterparts (Fig. S3A and B). Interestingly, at 2 weeks post-challenge, the absence of the T_FH did not significantly influence the efficacy of S19 vaccination (Fig. 6A). This reinforced the idea that the protective role conferred by humoral immunity is predominantly linked to IgM rather than IgG. However, as infection progressed to the 4-week stage, the efficacy of S19 was significantly diminished in T_FH-deficient mice compared to Bcl6^fl/fl mice (Fig. 6B). While Bcl6 deficiency in CD4⁺ T cells results in T_FH deficiency, impaired germinal center formation, and defects in class-switched antibody production (39), at 4 weeks post-challenge an inability to secrete antibodies did not affect S19-mediated immunity against B. melitensis (Fig. 4B). Therefore, we investigated the effects of Bcl6 deficiency on the function of CD4⁺ T cells in the context of vaccination and Brucella challenge.

Fig 6.

CD4⁺ T-cell Bcl6 deficiency is associated with impaired control of Brucella in S19-vaccinated mice. (A) Splenic B. melitensis burdens in WT, CD4^creBcl6^fl/fl, and Bcl6^fl/fl mice (n = 9–16 mice/per group) measured after 4 weeks of vaccination and 2 weeks of challenge (4 + 2). (B) Splenic B. melitensis burdens in CD4^creBcl6^fl/fl and Bcl6^fl/fl mice (n = 10–15 mice/per group) measured after 4 weeks of vaccination and 4 weeks of challenge (4 + 4). Data are combined from two experiments. Dashed lines indicate the limit of detection.

Bcl6 deficiency is associated with altered CD4⁺ T-cell transcriptomic profile

We first measured CD44 expression as a marker of activation on CD4⁺ T cells after 4 weeks of vaccination in both CD4^CreBcl6^fl/fl and Bcl6^fl/fl mice. We observed a significant decrease in the percentage of activated CD4⁺ T cells (CD44⁺CD4⁺) in vaccinated CD4^CreBcl6^fl/fl mice when compared to vaccinated Bcl6^fl/fl counterparts (Fig. S3C). To further examine the role of Bcl6 deficiency on CD4⁺ T-cell function, we performed RNA-seq on CD4⁺ T cells isolated from Bcl6^fl/fl and CD4^CreBcl6^fl/fl mice after 4 weeks of vaccination and either 2 or 4 weeks of challenge. We identified a total of 1,565 differentially expressed genes (Log2FC > 0.5 and FDR < 0.05) in Bcl6-deficient CD4⁺ T cells, with 298 genes upregulated and 1,267 genes downregulated compared to their Bcl6^fl/fl CD4⁺ T-cell counterparts at 2 weeks post-challenge (Fig. 7A; Table S1). At 4 weeks post-challenge, 1,390 genes showed differential expression in Bcl6-deficient CD4⁺ T cells, with 994 genes upregulated and 396 genes downregulated (Fig. 7C; Table S2).

Fig 7.

Bcl6 deficiency is associated with an altered CD4⁺ T-cell transcriptomic profile. Mice were s.c vaccinated with 2 × 10⁵ CFUs of S19 vaccine and were challenged 4 weeks later with 1 × 10⁵ CFUs i.p. of B. melitensis 16M. On 2 and 4 weeks post-infection, CD4⁺ T cells were purified from spleens and RNA was extracted for RNA-Seq analysis. (A, C) Volcano plot showing genes with altered gene expression in CD4^CreBcl6^fl/fl and Bcl6^fl/fl mice CD4⁺ T cells at 2 weeks (A) or 4 weeks (C) post-challenge in previously vaccinated mice. The horizontal dashed line indicates a false discovery rate (FDR) of 0.05, and genes outside the vertical dashed lines have an absolute log2 fold change of >0.5. (B & D) Heat map of 23 selected genes whose expression was altered by CD4⁺ T-cell Bcl6 deficiency after vaccination and either 2 (B) or 4 (D) weeks post-B. melitensis challenge. (E) Table depicting a comparison of 23 selected genes altered in the context of CD4⁺ T-cell Bcl6 deficiency at 2 and 4 weeks post-infection in S19 vaccinated mice. Data are from one experiment.

In our investigation, we identified a subset of genes (Fig. 7B, D and E) to highlight their possible impact within our vaccination/infection model. Of particular interest, the expression of Ifng remained unaltered between Bcl6-deficient and control CD4⁺ T cells at both time points. However, the expression of Il10, which is deleterious to host control of Brucella (40–43), and Gata3, which promotes IL-4 production (44), was significantly upregulated at both timepoints in CD4⁺ T cells lacking Bcl6. By contrast, Bcl6 deficiency led to decreased expression of Cxcr5, which is associated with T_FH, and Tox2 (39), which signals downstream of BCL6, at both timepoints. Collectively, these findings indicate Bcl6 deficiency in CD4⁺ T cells is associated with a disrupted transcriptomic profile that could negatively impact S19 vaccine-mediated efficacy. The differential expression of some T-cell-related genes such as Foxp3, Ccr7, Sell, Cd127, Cd44, Tnf, Tbx21, P2r × 7, Il22, and Il17re varied between 2 and 4 weeks post-challenge. While CFU levels are similar in Bcl6^fl/fl and CD4^CreBcl6^fl/fl mice after 4 weeks of vaccination and 2 weeks of challenge, after 4 weeks of challenge CFU levels are significantly higher in CD4^CreBcl6^fl/fl mice (Fig. 6B and C). Therefore, differences in CFU levels between the two timepoints could impact the inflammatory environment and consequently alter gene expression by CD4⁺ T cells.

DISCUSSION

Here, we reveal the multifaceted contributions of B cells in the context of S19 vaccination and Brucella infection. Our investigation demonstrates a phase-specific influence of B cells and antibodies on the effectiveness of S19 against brucellosis. Interestingly, the presence of B cells during the challenge phase is associated with a detrimental effect on vaccine efficacy (Fig. 1). By contrast, at the early stage of the challenge, we show IgM plays a pivotal role in protecting against Brucella; however, the role of antibodies diminishes as the infection progresses (Fig. 4). These observations indicate that the presence of B cells during vaccination is necessary for mounting a humoral response that impedes early dissemination of B. melitensis; however, the presence of B cell during the challenge phase may alter the immune response to create a favorable environment for the pathogen (16, 45, 46).

When investigating mechanisms by which B cells inhibit vaccine-mediated control of Brucella, we found S19-vaccinated mice lacking B-cell MHCII expression demonstrated enhanced resistance to B. melitensis during the later stage of challenge (Fig. 2B). This detrimental role aligns with a prior discovery from our laboratory, wherein a deleterious influence of B-cell MHCII expression during primary brucellosis was documented (18). The cumulative findings from our studies implicate B-cell antigen presentation as a limiting factor for both primary and vaccine-mediated immunity to Brucella (31).

Subsequently, our focus shifted toward the role of antibodies in S19-mediated efficacy. Notably, we observed S19-vaccinated sIgM^−/−/AID^−/− mice, which lack the capacity to produce both IgM and class-switched antibodies, demonstrated enhanced susceptibility to brucellosis 2 weeks after challenge (Fig. 4A and C). The concept of IgM offering a protective role against secondary brucellosis has been proposed by other researchers (19). In alignment with this perspective, our investigation using sIgM^−/−/AID^−/+ animals confirmed IgM originating from S19 vaccination provides protection against brucellosis (Fig. 5A). IgM is a potent activator of complement (37), and unlike the reported deleterious role of complement in control of primary B. abortus infection (47), here we found complement depletion impaired vaccine-mediated immunity against B. melitensis (Fig. 5B). Therefore, in the future, we will investigate mechanisms by which complement activation alters control of primary and secondary Brucella infection.

As infection progressed, the role of antibody production in vaccine-mediated immunity was diminished. This change in antibody function across different infection stages could be attributed to the fact that following intraperitoneal inoculation, brucellae undergo a brief extracellular phase in the blood where they can be susceptible to specific circulating antibodies in immunized mice, thus reducing bacterial dissemination (48). Other intracellular pathogens that exhibit an in vivo extracellular stage are also susceptible to the impact of antibodies (49, 50). Nevertheless, the role of humoral immunity weakens as bacteria shift to an intracellular life where antibodies cannot access them, and host defenses predominantly rely on cellular immunity to control infection (51).

Our observation that IgM plays a greater role than IgG in S19-mediated efficacy against brucellosis was corroborated by studies in T_FH-deficient mice which lack the ability to form germinal centers and undergo class switching (52) but can produce IgM (53). Our results indicate S19 vaccination continues to offer protection for T_FH-deficient mice against brucellosis similar to Bcl6^fl/fl animals at the early stage of infection (Fig. 6A) confirming class switching is not essential for the efficacy of S19. Furthermore, it implies protective IgM elicited by S19 vaccination does not require germinal center formation. While class- switched antibodies do not appear essential for vaccine-mediated immunity, S19 vaccine efficacy does wane in CD4^CreBcl6^fl/fl mice at later time points after challenge (Fig. 6B). In line with a prior report (38), our RNA-seq analysis (Fig. 6A through E) demonstrated an increased expression of Il10 in Bcl6-deficient CD4 cells. This was of significance, as CD4⁺ T cell-derived IL-10 is deleterious to host control of Brucella (43). Il10 is expressed by various immune cells including Th1, Th2, and Th17 cells, Treg cells, CD8⁺ T cells, and B cells (54). Although the specific Il10-producing cells in our model require further identification, the upregulation of Gata3 and Foxp3 in CD4⁺ T cells lacking Bcl6 suggests that Th2 and Treg cells could be sources.

Collectively, this study sheds light on the protective role of IgM and the deleterious impact of B-cell MHCII expression in the context of live vaccine-mediated immunity against brucellosis. In addition, it underscores the detrimental role of B cells during the challenge phase and highlights the significance of Bcl6 presence for CD4⁺ T-cell activation and the maintenance of a well-balanced inflammatory response throughout vaccination and challenge. Overall, our study improves our understanding of vaccine-mediated effector mechanisms against Brucella infection.

MATERIALS AND METHODS

Mice and animal use

Animal studies were conducted using sex-matched mice aged between 6 and 12 weeks of age. Mice were maintained in individually ventilated caging under high-efficiency particulate air-filtered barrier conditions with 12-h light and dark cycles within ABSL-3 facilities at the University of Missouri. Food and water were provided to animals ad libitum. All experiments were conducted in accordance with the University of Missouri Animal Care and Use Committee. Various immunodeficient and transgenic mouse strains on a C57BL/6J background (Table 1) were employed in this study. C57BL/6-Tg(IghelMD4)4Ccg/J (MD4), B6.129 × 1-H2-Ab1^tm1Koni/J (iAB^fl/flB6.129S(FVB)-Bcl6tm1.1Dent/J (Bcl6^fl/fl), and C57BL/6J (WT) mice were originally obtained from the Jackson Laboratory. sIgM^−/−/AID^−/− mice were a gift from Dr. Nicole Baumgarth at the University of California, Davis. AID^−/− mice were originally generated at Kyoto University (55) and were bred to sIgM^−/− mice at the Trudeau Institute (56). Mice with a singular deficiency in IgM secretion or class switching were generated by breeding sIgM^−/−/AID^−/− mice with WT C57BL/6J mice (Table 1). B6.129P2(C)-Cd19tm1(cre)Cgn/J (CD19^Cre), and B6.Cg-Tg(Cd4-cre)1Cwi/BfluJmice (CD4^Cre) were a gift from Dr. Mark Daniels (University of Missouri). CD19^Cre animals were intercrossed with iAB^fl/fl animals to generate CD19^CreiAB^fl/fl mice. CD4^Cre mice were intercrossed with Bcl6^fl/fl mice to generate Cd4^CreBcl6^fl/fl mice. Experiments involving the challenge of MD4, or CD19^CreiAB^fl/fl mice utilized HEL-negative or iAB^fl/fl litter mates as control animals, respectively. WT and Bcl6^fl/fl mice displayed similar phenotypes and were both used as controls for CD4^CreBcl6^fl/fl animals.

TABLE 1.

Mouse strains used in this study

Mouse strain	Phenotype
C57BL/6	Wild-type (WT) mice
MD4	Express BCR specific for hen egg lysozyme
sIgM−/−/AID−/−	Cannot secrete IgM or class switched antibodies
sIgM−/−/AID−/+	Cannot secrete IgM
sIgM−/+/AID−/−	Cannot secrete class-switched antibodies
sIgM−/+/AID−/+	Littermate control for sIgM−/+/AID−/− and sIgM−/−/AID−/+ mice
CD19CreiABfl/fl	Lack B-cell MHCII expression
iAB fl/fl	Control for CD19CreiABfl/fl mice
CD4creBcl6fl/fl	Lack T follicular helper cells
Bcl6 fl/fl	Control for CD4creBcl6fl/fl mice

Bacteria and culture conditions

Brucella melitensis 16M was obtained from Montana State University (Bozeman, MT). All experiments with B. melitensis were performed in biosafety level 3 (BSL-3) facilities. The Brucella abortus S19 vaccine strain was obtained from the University of Wyoming (Laramie, WY). GFP-expressing S19 (GFP-S19) was generated by transforming pBBR1MCS6-Y (57) into S19 and followed by selection on agar with chloramphenicol (5 µg/mL). Bacteria were grown on Brucella agar (Becton Dickinson) at 37°C/5% CO₂ before colonies were picked and cultured in Brucella broth overnight at 37°C in an orbital shaker. Challenge/vaccination doses were approximated by measurement of optical density at 600 nm and diluted using sterile Dulbecco’s phosphate-buffered saline (DPBS) (Thermofisher).

Vaccination and challenge

Each animal received a single subcutaneous (s.c.) vaccination of 200 µL DPBS containing 2 × 10⁵ CFU Brucella abortus S19. The control group received DPBS only. Four or eight weeks post-vaccination, animals were challenged intraperitoneally (i.p.) with 1 × 10⁵ CFU Brucella melitensis 16M in a volume of 200 µL PBS. The vaccination and challenge doses were confirmed through 10-fold serial dilution and plating of inoculum onto Brucella agar plates.

Immunoassay

Four weeks post-vaccination, peripheral blood samples were collected and centrifuged at 10,000 × g for 10 minutes at room temperature to obtain sera, which were stored at −80°C until further processing. An optimized indirect ELISA (31) was employed to measure antibody titers. To measure Brucella-specific antibody levels, ELISA plates were coated with 0.05 M carbonate/bicarbonate buffer containing heat-killed S19 at a concentration of 1 × 10⁸ CFU per well overnight at 4°C. For the standard curve, either unlabeled rat anti-mouse IgM (5 µg/mL) or goat anti-mouse IgG (0.5 µg/mL) (Southern Biotech) were used to coat IgM or IgG ELISA plates, respectively. The plates were then washed with 0.05% Tween and blocked with 1% BSA at 37°C. Diluted sera (1:50 or 1:2,000) or standards (mouse IgM or IgG) were added to the plates, which were maintained at room temperature for 2 h. After washing, HRP-conjugated secondary antibodies (1:1,000 for IgM or 1:4,000 for IgG) were added and incubated for an additional hour. TMB substrate (Invitrogen) was used to initiate the colorimetric reaction, and the reaction was stopped using 2N sulfuric acid. The plates were read at 450 nm using a SpectraMax Plus reader (Molecular Devices, San Jose, CA). All samples were tested in duplicate reactions. The limit of detection for anti-Brucella antibody was 30.9 pg/mL for IgM and 3.43 pg/mL for IgG.

In vivo B-cell and complement depletion

We employed i.p. administration of 250 µg of anti-CD20 antibody (clone MB20-11, Southern Biotech) to deplete B cells from mice (58, 59). Animals were treated either on day 7 prior to vaccination or on day 21 post-vaccination (7 days before the challenge). Control animals received unlabeled mouse IgG as an isotype (Southern Biotech). For complement depletion, mice were treated i.p. with 10 µg of CVF (Complement Technology) twice at 4-h intervals both 1 day prior to challenge and 1 week post-challenge (60).

Bacterial enumeration

At 2- or 4-week intervals after the challenge, the whole spleen was extracted from each mouse, weighed, and homogenized. Homogenates were 10-fold serially diluted and plated on erythritol-supplemented Brucella agar plates (1 mg/mL) to exclude S19 growth (61). Following 3–5 days of incubation at 37°C/5% CO₂, colonies were counted.

Flow cytometry

Spleens were homogenized and cell suspensions were filtered through a sterile 40-µm mesh following red blood cell lysis. Splenocytes were Fc blocked (2.4G2 Leinco) in fluorescence-activated cell-sorting (FACS) buffer (2% heat-inactivated fetal bovine serum [FBS] in DPBS) before extracellular staining. Blood samples were collected in 6.32 USP units per mL of sodium heparin (62), prior to RBC lysis and resuspension in FACS buffer and Fc block. After the Fc block, cells were stained with fluorochrome-conjugated mAbs: anti-CD4 (GK1.5 Biolegend), anti-CD44 (IM7 Biolegend), and anti-CD19 (1D3 Biolegend). Subsequently, the samples were washed and fixed with 4% paraformaldehyde (PFA) before being processed using a BD LSRFortessa X-20 flow cytometer. FlowJo software (Tree Star) was utilized to analyze the flow cytometry data.

Confocal microscopy

Splenocytes were isolated from mice as described above and cultured in complete media (RPMI 1640 supplemented with HEPES, MEM, sodium pyruvate, and 10% FBS). A total of 3 × 10⁶ splenocytes per well were then added to an µ-Slide 8 Well chambered coverslip (ibidi) that had been pre-coated with 25 µg/mL Poly-L-lysine (Sigma). The cells were incubated for 1 h at 37°C/5% CO₂ to ensure cell attachment, which was confirmed by inverted microscopy. After adherence, the cell culture media was removed and replaced with fresh media containing S19-GFP at a multiplicity of infection (MOI) of 500 and then incubated for 4 h at 37°C/5% CO₂ before applying gentamicin (50 µg/mL) for an additional 30 minutes. Uninfected cells were used as a negative control.

For processing samples for confocal microscopy, the cells were washed with PBS, fixed with 2% PFA for 10 minutes, and blocked with 1% BSA for 30 minutes. The cells were then stained with APC-CD19 antibody (Biolegend) at a dilution of 1:100 in 1% BSA for 1 h at room temperature. Finally, the slide was washed three times to remove unbound antibodies and mounted using Calbiochem MOWIOL 4–88 anti-fade reagent (MilliporeSigma). The images were obtained using a TCS LeicaSP8 Confocal Microscope (Leica technologies) and analyzed using LAS X software (Leica technologies).

Cell sorting

Splenocytes were isolated from mice as described above and CD4⁺ T cells were sorted using the EasySep Mouse CD4-Positive Selection Kit II (Stem Cell Technologies) following the manufacturer’s instructions. Aliquots of sorted cells were stained with anti-CD4 (GK1.5 Biolegend) to assess the cell purity by flow cytometry and the remaining samples were then preserved in RNAlater (Invitrogen) and kept at 4°C for subsequent RNA isolation. The purity of sorted splenic CD4⁺ T cells was confirmed to be ~90% by flow cytometry.

RNA sequencing

RNA was extracted from sorted CD4⁺ T cells in RNAlater via the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. Poly A enriched stranded mRNA libraries were generated from extracted RNA and sequenced on a NovaSeq 6000 (Illumina) at the University of Missouri Genomics Technology Core. The RNA-seq data were analyzed using the OneStopRNAseq v1.0.0 server (https://mccb.umassmed.edu/OneStopRNAseq/)(63). Detailed parameter settings were followed as we recently described (64). Gencode.vM25.primary assembly was used as a mouse reference genome. Differentially expressed genes were filtered and selected using a false discovery rate (FDR) threshold of less than 0.05 and an absolute log2 fold change (log2FC) greater than 0.5. Heatmaps for selected genes were generated from the TPMs using the ClustVis server (https://biit.cs.ut.ee/clustvis/)(65).

Statistical analyses

Statistical analyses were performed using GraphPad Prism software (version 9.2, GraphPad). Student t-tests were used to compare means between the two groups, with significance set at P ≤ 0.05. For comparisons involving three or more groups, one-way ANOVA was applied, followed by Turkey’s test for correction of multiple comparisons. Data are presented as average ±SD and statistical differences are indicated as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, not significant.

DATA AVAILABILITY

Data supporting the findings of this study are available in this paper, Supplementary information, or are available from the corresponding author upon request. The raw RNA-seq files used to generate the data in this manuscript have been deposited in the NCBI Gene Expression Omnibus (GSE243863).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/msphere.00750-23.

Figure S1. msphere.00750-23-s0001.tif.

Efficacy of B cell depletion.

Figure S2. msphere.00750-23-s0002.tif.

Antibody levels following S19 vaccination.

Figure S3. msphere.00750-23-s0003.tif.

Antibody levels and CD4+ T cell responses in TFH deficient mice.

Supplemental Material Legends. msphere.00750-23-s0004.docx.

Legends for supplemental figures and tables.

Table S1. msphere.00750-23-s0005.xlsx.

Gene expression in CD4+ T cells from vaccinated mice at two weeks post-challenge.

Table S2. msphere.00750-23-s0006.xlsx.

Gene expression in CD4+ T cells in vaccinated mice at at four weeks post-challenge.

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