Francisella And Antibodies Part 2

4.2. Initiation of Immune Responses

If the key to the binding of complement components to the surface of F. tularensis is indeed a consequence of previous interaction between the bacterium and antibody, then the antibodies already influence or control the bacterium’s primary interaction with the host cell. If only the actual primary host–pathogen interactions are considered, then isotypes of natural antibodies will control the nature of the infection. 

The relationship between bacteria and immunity is very close. While we often think of bacteria as scary pathogens, not all bacteria are bad. Conversely, some bacteria are good and help us maintain good immune health.

First, bacteria play a key role in our gut. There are a large number of bacteria in our intestines. On the one hand, they can help us digest food, and on the other hand, they can also enhance our immunity. These bacteria interact with our immune system to help us find and destroy pathogens to keep us healthy.

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Therefore, we cannot simply regard all bacteria as unfavorable. We should be thankful for those good-for-us bacteria. We need to promote the growth of these beneficial bacteria through a healthy diet and lifestyle to boost our immunity.

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This opinion relates to the distribution of complement receptors (CRs) and Fc receptors of mammalian cell types. While macrophages and monocytes express complementary CR1, CR3, and CR4 receptors on their surfaces, follicular dendritic cells express CR1, CR2, and CR3 receptors, and B cells express only CR1 and CR2 receptors [108]. Similarly, Fc-receptor (FcR) types for IgG and for other switched Ig isotypes are dominantly expressed on phagocytic cells, including neutrophils and eosinophil granulocytes, but the FcµR binding IgM is selectively expressed only on T and B lymphocytes and natural killer (NK) cells in humans and exclusively on B cells in mice [109,110]. FcµR signaling is critical not only for B-cell survival and activation, IgM homeostasis, and regulation of humoral immune response but also for the resolution of infections [109,111–113].

Natural antibodies of the IgM isotype are critical components during the primary interaction of Francisella with human neutrophils and macrophages. Natural IgM binds to surface capsular and O antigen polysaccharides of F. tularensis and activates the classical complement cascade via C1q. That is followed by C3-opsonization of the bacterium. Francisella opsonized by C3 complement fragments is finally phagocytosed by human neutrophils via CR1 and CR3 acting in concert and by human monocyte-derived macrophages via CR3 and CR4 [114,115]. 

The IgM surface antigen receptor and CR1/2 of B cells are needed for Francisella’s internalization within the B cells [20]. Thus, natural IgM antibodies that opsonize the Francisella surface components initiate the classical complement cascade via C1q and promote the internalization of Francisella through different complement receptors in a phagocyte-specific manner. Internalization of Francisella into the phagocytic cell is a prerequisite step for both dissemination and progression of infection on the one hand and antigen presentation to T lymphocytes on the other hand. 

The importance of fully functional B1a cells producing natural IgM antibodies has been demonstrated using µMT−/− mice, which lack the µ chain in B cell development [116]. The µMT−/− mice, having no mature B cells, exhibit increased susceptibility to primary infection with LVS, as well as reduced resistance to secondary infection and greater susceptibility to infection with virulent F. tularensis strain SchuS4, than do wild-type mice [117,118].

Thus, the IgM antibodies, as the first antibody isotype to appear during evolution, as well as during ontogeny, and the first isotype responding to antigenic stimulation, might be a leader in immune responsiveness. It has been demonstrated, using a murine model and classical model immunogens, that binding of complement onto the IgM antibody specific to immunogen enhances the humoral but not T-cell-dependent cellular responses [119]. IgM, similar to IgG3, as both are dominant natural antibody isotypes, regulates antibody response via complement and the complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells [120].

As previously presented, Francisella interacts directly with peritoneal murine B cells [18–20,121] and, as early as 12 h post-infection, both germ-free and specific pathogen-free mice infected with F. tularensis produce infection-induced antibody clones reacting with F. tularensis proteins and having the character of natural antibodies. According to data in the literature, we characterized their functional profile as inducers of homeostasis restoration (Table 1). We also suggest that a phylogenetically stabilized defense mechanism utilizing early infection-induced antibody specificities can be activated not only to eliminate a pathogen but also to remove debris and molecular residues of infection-damaged self-cells [98].

4.3. Adaptive Immune Response: Antibody Functions

Antibody functions within the framework of immune mechanisms are specified by antibody molecule structure and by cellular antibody receptors, which ensure signal transduction and cellular response. Antibody molecules possess two functional domains. The antigen-binding fragment (Fab) confers antigen specificity. The Fc fragment (crystallizable fragment), generally known also as the constant fragment, drives other antibody functions. The Fc fragment variants, IgM, IgD, IgG, IgA, and IgE isotypes, have unique structural features that impact antibody function. 

Moreover, the IgG isotype has four subclasses (IgG1, IgG2, IgG3, and IgG4) and the IgA isotype has two subclasses (IgA1 and IgA2) [122]. The specific effector functions are triggered by the binding of an antibody molecule to the appropriate receptor on the specific cell type, to which the antibody Fc domain binds [123,124]. These sensors include both classical FcRs and nonclassical C-type lectin receptors (CLRs), which are differentially expressed on immune cell subsets. The targeting of antibody function is further specified by posttranslational modification of FcRs [125]. 

Such a complex molecular basis of antibody function expression enables response to countless molecular and cellular targets that could compromise the integrity of the vertebrate organism. If we accept a simple classification of antibody functions, then antibodies are capable of neutralizing pathogen entry into the cell and its replication, neutralizing microbial virulence factors, ensuring antigen uptake, inducing antibody-mediated complement activation or antibody-dependent cellular cytotoxicity and realizing antibody-dependent cellular phagocytosis, as well as antibody-mediated granulocyte degranulation and release of vasoactive mediators, chemoattractants, and cytokines. The dark side of their effects can involve antibody-dependent disease enhancement by altering the targeting of immune defense mechanisms [126].

Collectively, the data obtained from different F. tularensis infection models suggest that the antibodies against structural components of F. tularensis participate during both the innate and the adaptive phases of the immune response. The functions of IgM isotype antibodies produced by B1a cells dominate during innate immune response [96,127], while functions of other isotypes seem to be needed during the adaptive phase of the immune response against F. tularensis.

Results from a study by Furuya et al. showed that IgA−/− mice were more susceptible than IgA+/+ mice to intranasal F. tularensis LVS infection that started in the second-week post-infection, despite developing higher levels of anti-LVS total, IgG, and IgM isotype antibodies in bronchoalveolar lavage [128]. 

Furthermore, a comparison of vaccination via different routes (intradermal versus intranasal) suggests the involvement of IgA isotype antibodies in protective efficacy against the lethal effect of F. tularensis infection [129,130]. 

The protective efficacy of the IgA antibody isotype seems to involve a complex event encompassing, inter alia, cellular IFN-γ responses [128]. Experiments with F. tularensis lipopolysaccharide further demonstrated the role of IgA in IgG class switching after lipopolysaccharide vaccination. This modulatory effect, demonstrated on IgA−/− mice, can be overcome by immunization with whole bacteria [131].

The IgG antibody isotype dominates during the adaptive response to F. tularensis natural infection (i.e., from the second week after infection) in humans. Such a conclusion is supported by numerous studies, collectively discussed in a review by Maurin et al. [63]. Among the IgG subclasses, the IgG2 subclass prevailed, having been diagnosed in 92.9% of patients with serologically confirmed tularemia. 

The serum levels of IgG2 prevailed over those of IgG1 and IgG3, and the level of IgG4 was below the detection level [66]. The effects of IgG antibodies during the adaptive phase of immunity are produced mostly through receptors for their Fc fragments. The recognition of an FcR-targeted immunogen has a significant impact on the expression of immune defense mechanisms. Targeting of an inactivated F. tularensis live vaccine strain using mouse IgG2a anti-F. tularensis LPS mAb upon FcRs at mucosal sites (via intranasal immunization) enhances immunogen-specific IgA production and confers protection against subsequent infection in an IgA-dependent manner. Moreover, it enhances protection against the highly virulent SchuS4 strain of F. tularensis. 

Two types of FcRs, specifically FcgammaR and neonatal FcR, are crucial to this protection [132]. An effect similar to the induction of a protective response as utilization of opsonized inactivated F. tularensis has been shown to occur following the independent intranasal application of IgG2a anti-F. tularensis mAb and inactivated F. tularensis live vaccine strain [133]. This study design also showed an increase in protection against subsequent F. tularensis challenges that is FcR-dependent and requires a physical linkage between the monoclonal antibody and the inactivated F. tularensis immunogen [133].

If the role of anti-F. tularensis antibodies during the murine innate phase of the immune response are mostly dependent on complement activation, then such a dependency has not been demonstrated using sera obtained during the adaptive phase of the immune response or using convalescent sera. In this case, the protective effect of anti-Francisella antibodies is independent of complement activation but the dependency on Fc receptors and phagocytosis was demonstrated [134]. 

Studies on a combination of inactivated F. tularensis immunogen and immune sera or anti-F. tularensis mAb has revealed modulatory effects on adaptive immune mechanisms, starting from better protection against subsequent F. tularensis challenge, also in addition to including enhanced binding and internalization of inactivated F. tularensis by antigen-presenting cells through engagement of different FcR types, enhanced dendritic cell maturation, a prolonged period through which Ag-presenting cells stimulate T cells, and modulated kinetics of inactivated F. tularensis immunogen transport from the periphery to lymphoid tissues [132–134].

Antibacterial antibody-dependent cell-mediated cytotoxicity (ADCC) is a complex immune mechanism that can limit the proliferation of microbial pathogens inside the infected organism. Downregulation of microbial proliferation is effected through the limitation of microbial proliferation inside the cells [135] or directly by killing free bacteria [136]. In contrast to complement, which also lyses targets but does not require any other cell, ADCC requires an effector cell that dominantly interacts with IgG antibodies bound to the surface of target cells. In this sense, the ADCC, utilizing the antibody as a critical component, is independent of the complement system. The typical effector cell in ADCC is an NK cell expressing an Fcγ receptor. Nevertheless, other cell types such as macrophages, neutrophils, or eosinophils [137], as well as other antibody isotypes, can also mediate antibacterial ADCC [138]. 

The NK cells having appropriate Fc receptors will bind to the corresponding antibody and will release proteins, such as perforin and granzymes, which cause lysis of the infected cell. One of the Francisella models, F. novicida ∆fopC, allowed documenting perforin-mediated inhibition of F. tularensis LVS replication in macrophages while identifying the NK cells as the critical cell type producing perforins [139]. This is one of the examples where anti-F. tularensis antibodies limit the proliferation of bacteria inside the body via the ADCC mechanism. Our unpublished data from the 1980s suggested the existence of ADCC-mediated limitation of F. tularensis proliferation in macrophages. The nonadherent spleen cells isolated from vaccinated mice 21 days post-vaccination limited the number of F. tularensis in the cultures of in vivo infected peritoneal macrophages from naïve mice, but only when the sera from vaccinated mice were added to the cultures.

4.4. Protective Value of Anti-F. tularensis Antibodies

In early studies, the contribution of antibodies to host protection against F. tularensis appeared somewhat ambiguous. Passively transferred antibodies have been shown to confer protection against F. tularensis subsp. holarctic, including the LVS strains, but not against F. tularensis subsp. tularensis strains. This was documented mostly in the SCHU S4 infection model. At present, however, there remains controversy as to the contribution of B cells at a molecular level to protective immunity against F. tularensis infections. 

Both the production of cytokines affecting the functional profile of immunocompetent cells and the production of antibody molecules capable of modulating effector mechanisms of immunity comes into consideration. Recently, it has become clear that specific antibodies against F. tularensis used for passive immunization are to some extent able to protect against lethal F. tularensis infection. Early studies using immune sera have already shown the indisputable protective effectiveness of the humoral components of the serum [140–142]. Later studies demonstrated that natural infection and vaccination-induced long-lasting humoral and cell-mediated protective immunity; however, the humoral immunity gave protection only against strains having reduced virulence [143,144]. 

For this reason, interest has turned to the study of cell-mediated immunity mechanisms. At the turn of the millennium, several publications again stimulated interest to further study the humoral immunity in tularemia [145–148]. Different models of experimental tularemia gave rise to basic knowledge of the protective value of anti-F. tularensis antibodies. Passive transfer of immunity by specific antibodies against F. tularensis provided direct evidence that, due to the presence of pathogen-specific antibodies, this will benefit the host during infection caused by intracellular pathogens [149]. 

However, no protection was obtained in BALB/c mice against F. tularensis pulmonary infection by serum transfer from F. tularensis subsp. holarctic LVS-immune animals [150]. Passive transfer of immune sera also protected immunocompromised mice to some extent. Mice irradiated by sublethal gamma irradiation (3Gy) were protected against low lethal doses of the F. tularensis LVS, as well as against the original Soviet vaccine strain 15, which is more virulent for mice than the LVS strain [151]. The protective effect of anti-Francisella antibodies is dependent upon IFN-gamma production and on FcγR-mediated opsonophagocytosis and is independent of complement activation. This might suggest a dominant role of ADCC in the protective efficacy against F. tularensis infection [134].

4.5. Secondary Immune Response: B Cells and Antibodies

Passive protection of sub-lethally irradiated mice against primary F. tularensis LVS infection also protected those mice that survived primary LVS infection against further secondary challenges with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Meanwhile, significantly fewer mice survived that were only LVS-vaccinated without an initial passive transfer of immunity [151,152]. Thus, the initial participation of the humoral immune response to F. tularensis infection seems to play a substantial role in an effective protective secondary response. Parenteral intradermal and intraperitoneal F. novicida infections of wild-type mice or B-cell knockout mice did not appreciably impact survival after subsequent lethal F. novicida challenge, thus demonstrating that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice [153]. Studies have shown that, in constructing vaccines against tularemia, emphasis must also be given to inducing humoral immunity, which participates in a protective immune response. This is evidenced by a study with a combination vaccine containing ingredients that induce both humoral and cellular branches of immunity and protect against otherwise lethal intranasal and intradermal challenges with wild-type F. tularensis strains Schu S4 /type A/ and FSC 108 /type B/ [154].

5. Conclusions

The experimental data presented in the studies cited above clearly document that antibodies must be an integral part of induced immunity if it is to be truly protective. Protection against F. tularensis infection requires several discrete events. Early production of anti-F. tularensis antibody [98], which might be characterized as a booster of opsonophagocytosis of pathogens, is one of the key events in the induction of acquired immunity against bacterial pathogens. The second one is innate immune recognition and activation of antigen presentation. As antigen-presenting cells, dendritic cells, macrophages, and B cells are equipped with receptors recognizing pathogen-associated molecular patterns that mediate innate immune recognition [4]. Another link in the functional chain of events providing protective immunity is the activation of effector cells that eliminate bacteria from the cells and tissues of the infected host. The last desirable protective event is the establishment of immune memory. Available data indicate that antibodies can initiate, regulate, or directly mediate these events.

There exist several approaches to harnessing the ability of antibodies to create effective and safe means of protection against highly virulent strains of F. tularensis. The simplest of these is a combination of passive and active immunization, which not only eliminates the side effects of vaccination with live strains but also provides greater protection against subsequent virulent challenges. The second one consists of a generation of pathogen-specific IgM antibody clones produced by B1a B cells, which have been shown to induce protection while assuming the mutual interaction of cellular and humoral immune mechanisms [155], or in preparing antibody clones based on the knowledge of B-cell-activating epitopes on F. tularensis proteins [156]. 

Another more sophisticated, approach is to construct a combined bacterial protein or whole bacterium with targeting (homing) components of eukaryotic molecules, such as Fc fragments of antibodies or the C3 component of complement, to provide directed opsonophagocytosis of tularemic antigens or whole microbes and initiate the effective protective response (see, for example, Holland-Tummillo et al. [157]. 

However, we should once more repeat that, about effective immunoprophylaxis of tularemia, we still have substantial gaps in our knowledge regarding the effective immune mechanisms, their collaboration and precise timing during innate and adaptive phases of the immune response, and the bacterial molecular mechanisms interfering with induced immune responses. Thus, despite several very sophisticated studies on mutual host–pathogen interactions on cellular and molecular levels, it can be valuable to conduct further targeted studies regarding individual mechanisms of immunity enabling the elimination of the microbe, including the role of B cells and their products.

Author Contributions:

Writing—original draft preparation, K.K. and A.M. Both authors contributed equally to the work. Both authors have read and agreed to the published version of the manuscript.

Funding:

This work was funded by the Ministry of Interior of the Czech Republic, grant number VJ01030003.

Institutional Review Board Statement:

Ethical review and approval have been waived for this publication because it is a summarizing review article.

Informed Consent Statement:

Not applicable.

Data Availability Statement:

Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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