**4.2. Experimental studies with antibodies**

A substantial number of studies utilizing anti mycobacterial antibodies have been conducted as far back as the end of the 19th century. These experiments can be grouped into several categories: serum therapies, mouse polyclonal antibodies, human polyclonal antibodies (including commercial human gamma globulins), secretory human IgA (hsIgA) and studies with monoclonal antibodies.

#### *4.2.1. Serum therapies*

Serum therapy experiments were conducted from the second half of the 19th century (reviewed in [39,40]). Immune sera was generated by immunizing animals with different mycobacterial fractions and administered either to animals or humans [39,40] The results obtained were either beneficiary, variable, inconclusive or contradictory, [39,40]. These variable results led to the perceived minor role of antibodies in the defense against *M. tuberculosis*.

What factors could have led to the heterogeneity in study results? Recognizable differences in the methods used for serum preparations and their administration, as well as the lack of appropriate experimental controls probably accounted in part for the studies outcomes. Furthermore, it is important to recognize that immune serum is a polyclonal preparation that includes antibodies with multiple specificities and isotypes. Consequently, polyclonal sera may contain antibodies of different subclasses and functional categories that can affect theoutcome of infection. For example, IgG3 murine monoclonal antibodies protected against *M.tuberculosis* [27] but failed to protect against *Cryptococcus neoformans* [41]. An IgG3 nonprotectivemonoclonal antibody to *C. neoformans*, became protective upon subclass switching to IgG1 [41]. In addition to intrinsic factors associated with the antibody structure, other parameters such as the genetic background of the microbe and the immunocompetence of the host could alter the outcome of antibody protection experiments.

In this study, serum samples containing specific antimycobacterial antibodies were obtained from volunteers vaccinated twice with BCG by the intradermal route. Significant titres of IgG antibodies against lipoarabinomannan (LAM) were detected in the sera. BCG internalization into phagocytic cells was significantly increased in the presence of these BCG induced antibodies as were the growth inhibitory effects of neutrophils and macrophages on myco‐ bacteria. Furthermore, these antibodies induced significant production of IFN-γ by CD4+ and

The Role of Antibodies in the Defense Against Tuberculosis

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Human Intravenous Immunoglobulin (IVIG) has been used to treat individuals with immune deficiencies and patients with inflammatory, autoimmune and infectious conditions [49, 50, 51]. Several groups tested the effect of human immunoglobulin preparation on mycobacterial infection. Roy and colleagues showed that treatment of *M. tuberculosis* infected mice with one cycle of IVIG led to the substantially lower bacterial loads in the spleen and lungs following its administration either at early or at late stage of infection 52]. The effect of the administration of a commercial preparation of human immunoglobulin (hIg) in a mouse model of intranasal infection with BCG was evaluated by Olivares and colleagues [33]. This group demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration of human hIg to mice. This treatment inhibited BCG coloniza‐ tion of the lungs of treated mice. A similar inhibitory effect was observed after infection of

The same formulation was evaluated also in a mouse model of intratracheal infection with *M. tuberculosis*. Animals receiving human hIg intranasally 2h prior to intratracheal challenge demonstrated a significant decrease in lung bacillary load as compared with non-treated animals [29]. When *M. tuberculosis* was pre-incubated with hIg prior to challenge the same

The protective effect of the hIg formulation was abolished following pre-incubation with *M. tuberculosis* [29]. These results are suggestive of a potential role for specific human antibodies

Taken together, these studies provide support for the potential use of immunoglobulins

Human secretory IgA (hsIgA) is the major class of antibody associated with immune protection of the mucosal surfaces [53]. Colostrum volume is above 102 mL in humans during the first three days after delivery [54]. The high percentage of (hsIgA) in human colostrum [55] strongly suggests its important role in passive immune protection against gastrointestinal and respi‐ ratory infections [56]. In one study performed by Alvarez and colleagues, hsIgA from human colostrum was obtained by anion exchange and gel filtration chromatographic methods, using

DEAE Sepharose FF and Superose 6 preparative grade, respectively [57].

CD8+ T cells [15].

*4.2.3.2. Commercial immunoglobulin formulations*

mice with hIg -opsonized BCG [33].

in the defense against mycobacterial infections.

effect was observed [29].

against *M. tuberculosis*.

*4.2.3.3. Human secretory IgA*

For some microorganisms, such as *Samonella typhimurium* and *C. neoformans,* passive antibody therapy efficacy depends on the mouse strain used [42, 43]. In the same way, some microbial strains are more susceptible to the effects of antibodies [44].

The animal model used is another important parameter that varies between different experi‐ ments cited in the literature [45]. Timing, the route of infection, the magnitude of the infecting inoculum are some additional variables that could affect antibody protection studies [46].

Despite their variability, the results obtained with serum therapy were valuable, demonstrat‐ ing some beneficial effect of serum on the course of TB in humans, mainly in cases of early or localized TB [45]. Moreover, it was demonstrated that long periods of treatment were necessary to achieve a sustained effect [45].

#### *4.2.2. Polyclonal mouse antibodies*

A recent study re-examined the usefulness of immune serum in the context of a therapeutic vaccine against TB [32]. This vaccine, named RUTI, is generated from detoxified *M. tuberculo‐ sis* cell fragments that facilitate a balanced T helper response to a wide range of antigens along with intense antibody production [47]. Local accumulation of specific CD8+ T cells and a strong humoral response after immunization are characteristic features of RUTI that contribute to its protective properties. In that study, immune serum was generated by immunizing mice with RUTI [32]. Severe Combined Immunodeficiency (SCID) mice were inoculated with *M. tuberculosis* and treated with chemotherapy for 3–8 weeks. After chemotherapy they were treated for up to 10 weeks with intraperitoneal injections of the generated immune serum. Mice treated with immune serum from RUTI vaccinated animals showed significant decreases in lung CFU in addition to reduced extent of granulomatous response and abscess formation [47]. These results indicate that protective serum antibodies can be elicited by vaccination, and that antibodies may be usefully combined with chemotherapy [32, 47, 48].

#### *4.2.3. Human gammaglobulins*

#### *4.2.3.1. Specific human polyclonal antibodies*

Evidence for the stimulatory role of specific polyclonal antibodies on cellular immunity in experimental mycobacterial infections was reported by de Valliere and colleagues in 2005 [15]. In this study, serum samples containing specific antimycobacterial antibodies were obtained from volunteers vaccinated twice with BCG by the intradermal route. Significant titres of IgG antibodies against lipoarabinomannan (LAM) were detected in the sera. BCG internalization into phagocytic cells was significantly increased in the presence of these BCG induced antibodies as were the growth inhibitory effects of neutrophils and macrophages on myco‐ bacteria. Furthermore, these antibodies induced significant production of IFN-γ by CD4+ and CD8+ T cells [15].
