An estimated 9 million new cases of tuberculosis (TB) and 2–3 million deaths are reported globally every year, making TB the leading cause of death from a single infectious pathogen . Immune effector mechanisms that lead to resistance or susceptibility to infection or clinical disease are not fully known. Bacille Calmette–Guérin (BCG) vaccines, developed a century ago, remain one of the most widely used vaccines globally. However, with the exception of tuberculous meningitis in children, the capacity of BCG to protect individuals against TB is controversial, because randomized trials have provided estimates ranging from 80% to no protection ; hence, the development of a more efficacious vaccine to replace BCG is a priority. However, past attempts to replace BCG have achieved moderate success, mainly because of the lack of complete knowledge of immunity against Mycobacterium tuberculosis (Mtb) infection.
It is generally accepted that cell-mediated immunity (CMI) plays a critical role in the protection against Mtb because it is an intracellular pathogen. However, antibody-mediated immunity has been disregarded based mainly on the results of early classical experiments (reviewed in ) and the T helper type 1 (Th1)/Th2 paradigm. According to this paradigm, Th1 cells protect the host from intracellular pathogens, whereas Th2 cells protect from extracellular pathogens. There is no doubt that immunity to Mtb is associated with Th1 cell activity [interferon (IFN)-γ and interleukin (IL)-12 in particular, and production of tumour-necrosis factor (TNF)] (reviewed in ), but the question is whether or not Th1 immunity alone is enough to protect the host from Mtb infection or development of disease, or dissemination of infection to different organs.
The contribution of humoral immunity to protection against Mtb infection or development of clinical disease has been controversial for more than a century. Successful development of serum therapy against some infectious diseases such as Streptococcus pneumoniae and Neisseria meningitides at the end of the 19th century inspired several scientists to study the beneficial role of serum therapy for TB, but the results varied from study to study. Many studies reported a protective or beneficial role, while others claimed a non-protective role or even a detrimental role for antibodies against Mtb infection. A detailed account of these early studies and their limitations can be found in a review by Glatmann-Freedman and Casadevall .
The second and probably the most important generalization, particularly during the last two decades, is based on the Th1/Th2 paradigm. The Th1/Th2 paradigm that emerged in the mid-1980s  stems from observations in mice of two types of T helper cells differing in cytokine secretion and other functions. This paradigm was adapted to human immunity, with Th1 and Th2 cells directing different immune response pathways. Th1 cells drive the type 1 pathway (cellular immunity) to fight viruses and other intracellular pathogens. Th2 cells drive the type-2 pathway (humoral immunity) and up-regulate antibody production to fight extracellular pathogens. During the last two decades, this paradigm has dominated not only our understanding of acquired immunity against infectious pathogens but also our approach to vaccine design.
Recently, however, major discrepancies in the Th1/Th2 model have been observed. There are new insights into our understanding of immunity to intracellular and extracellular pathogens. A substantial body of data suggest that protection against intracellular and extracellular pathogens is not limited strictly to either Th1 or Th2 responses. Data on the protective role of humoral immunity against intracellular pathogens (e.g. Plasmodium spp., Chlamydia, Cryptococcus neoformans and Mycobacteria) and that of Th1 responses against extracellular pathogens (e.g. schistosomiasis) are emerging.
This review focuses upon our current understanding of the Th1/Th2 model, recent findings on protective antibody responses against specific mycobacterial antigens, passive immunization studies using polyclonal and monoclonal antibodies in B cell knock-out and severe combined immunodeficient (SCID) mice, and vaccination studies that reinforce the notion that there is an essential interdependence between Th1 and Th2 responses against infectious pathogens, including mycobacteria. By taking malaria (intracellular parasites) and schistosomes (extracellular parasites) as an example, we will try to show recent progress in the development of candidate vaccines that induce mixed immune responses.
Limitations of the Th1/Th2 paradigm
In 1986, Mosmann and Coffman proposed a model wherein Th cells are divided into functional subsets on the basis of cytokine secretion, termed Th1 and Th2 . Th1 cells mediate CMI by secreting IFN-γ, thereby activating macrophages, natural killer (NK) cells and CD8+ T cells, whereas Th2 cells mediate humoral immunity by secreting antibodies. According to this model, Th1 and Th2 cells have a distinct division of labour in that Th1 cells protect against intracellular pathogens, whereas the role of Th2 cells is to fight extracellular pathogens. During the last two decades, it has been customary to categorize diseases into either of the categories, in many cases regardless of how poorly they actually fit these models . Vaccines were also designed to produce either CMI or antibody responses. However, it has been long recognized that there are major discrepancies with respect to this model [6–8], and that there are a host of new questions and research directions that need to be addressed. In the following section, some of these limitations will be presented briefly, with emphasis on immunity to intracellular and extracellular pathogens.
The Th1/Th2 paradigm maintains the notion that acquired immunity depends on the polarization of Th cells into Th1 or Th2. However, immunity against pathogens does not originate from Th polarization; instead Th polarization is a product of a chain of events that begin with antigen recognition, processing and presentation to T cells. Many factors regulate the polarization of newly activated naive T cells into mature Th1 or Th2 cells: the local cytokine milieu; the presence of immunologically active hormones; the dose and route of antigen administration; the type of antigen-presenting cells (APCs) stimulating the T cell; and ‘the strength of signal’, which is an ill-defined summation of the affinity of the T cell receptor (TCR) for the major histocompatibility complex (MHC)–antigen complex, combined with the timing and density of receptor ligation. Of these five factors, the most important is the cytokine milieu surrounding the newly activated T cells .
Naive CD4+ T helper cells are committed to a particular antigen by virtue of their specific TCR, but they are not committed to a particular cytokine-secretion profile. Rather, the invading microbe to which the T cell is specific, in combination with the APCs, differentiates the Th cell into an appropriate mature effector cell, which presumably then mediates an optimal immune response. When a naive CD4+ T cell is exposed to antigen in the context of IL-12 derived from macrophages or dendritic cells (DCs), it is driven to develop into a Th1 phenotype, characterized by the secretion of IFN-γ. Other cytokines, such as IL-2, lymphotoxin (TNF-β) and IL-10, have also been called Th1 cytokines, but none defines this lineage as clearly .
Several nutrients and hormones influence the Th1/Th2 balance measurably, including plant steroids/sterolins, melotonin, probiotics, progesterone and mineral selenium and zinc. Experimentally, Th1 polarization is transformed readily to Th2 dominance through depletion of glutathione and vice versa. Mercury depletes glutathione and polarizes Th cells into Th2-dominant cells .
Cytokine secretion by Th1 and Th2 cells
The model for the classification of Th1/Th2 cells on the basis of cytokine secretion emanates from studies carried out in mice. In humans the division of T lymphocytes based on cytokine production is not as stringent as in inbred mice. For instance, some human Th1 cells secrete IL-10 and IL-13 Th2 cytokines. Probably, the most important difference between Th1 and Th2 cells is that Th1 cells secrete IFN-γ but do not secrete IL-4, whereas Th2 cells secrete IL-4 but not IFN-γ. T cells secreting neither IFN-γ nor IL-4 are neither Th1 nor Th2 cells; the best example is Th17 .
Moreover, many diseases classified previously as Th1- or Th2-dominant fail to meet the set criteria . Th1 cells are highly proinflammatory and have been linked to the induction and progression of many autoimmune diseases such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis, whereas Th2 responses are implicated with allergy. However, loss of IFN-γ signalling in mice deficient in IFN-γ or IFN-γR does not confer resistance to autoimmunity. In contrast, such mice are more susceptible to autoimmunity (reviewed in ). These observations have led to new research directions and identification of Th17 cells, distinct from Th1 or Th2, that are capable of inducing tissue inflammation and autoimmunity [11,12]. Th17 cells produce IL-17A (IL-17) and IL-17F and, to a lesser extent, TNF and IL-6. IL-17 acts in vitro and in vivo as a potent proinflammatory cytokine. It has pleiotropic activities, one of which is to co-ordinate tissue inflammation by inducing the expression of proinflammatory cytokines such as IL-6 and TNF. Expression of IL-17 has been detected in sera and target tissues of patients with various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematous and asthma .
Antibody regulation of CMI
Numerous studies show that many cells other than Th cells and antibodies are involved in immune regulation. One of the initial indications of the pivotal role of antibodies in regulating the induction of T cell immunity against intracellular microbial pathogen was obtained from analysis of genital chlamydial infection in antibody-deficient mice. In the murine model system of chlamydial genital infection, CMI alone is sufficient for controlling chlamydial infection. However, CMI alone cannot prevent secondary chlamydial infection in this model. Detailed immunological analyses of primary and secondary genital chlamydial infection, using genetically engineered specific knock-out mice and antibody-mediated depletion of lymphocyte subsets and subpopulations, have revealed that re-exposure to Chlamydia in the absence of B cells resulted in an inefficient and delayed clearance of the pathogen and development of ascending infection (reviewed in ). Subsequent analysis of genital chlamydial infection in FcR gene knock-out mice revealed that the requirement of B cell function for efficient microbial clearance and prevention of complications during secondary infection was associated with FcR-dependent antibody enhancement of chlamydial uptake, processing and presentation of antigen for rapid T cell activation. Thus, FcR-dependent antibody-mediated enhancement of T cell activation appears to be an integral component of the acquired CMI (reviewed in [13,14]).
In addition to their role as professional APCs and antibody production, B cells regulate differentiation of T cells and the development of CMI. B cells can stimulate proliferation, survival and differentiation of T cells. Activated B cells can produce distinct arrays of cytokines. Some effector B (Be) 1 cells secrete cytokines associated typically with Th1 responses such as IFN-γ and IL-12, while Be 2 cells secrete the cytokine IL-4, characteristic of Th2 cells. In addition, activated B cells can secrete large amounts of IL-6 and IL-10 that play an instrumental role in T cell-mediated immunity. IL-6 is a crucial co-stimulator of T cell responses. In contrast, IL-10 suppresses immune reactions. IL-10 strongly inhibits DCs and macrophages. B cell-derived IL-10 also suppresses production of IL-6 and IL-12 by DCs, which can inhibit differentiation of Th1 and Th17 cells, respectively (reviewed in ).
B cells induce inflammatory diseases and suppress autoimmune diseases through the production of IL-10 . The regulatory role of B cells is not limited to autoimmune disorders. For instance, chronic infection with Schistosoma mansoni induces IL-10 production by B cells, which can suppress anaphylaxis. Similarly, some viruses have developed mechanisms to stimulate IL-10-producing B cells and thereby subvert the immune system .
Location of pathogens in relation to the host cell
One of the main reasons for the notion that antibodies are not protective against intracellular pathogens is because antibodies cannot enter the cytoplasm of cells and affect pathogens. However, facultative intracellular pathogens such Mtb have both intracellular and extracellular phases in their infectious cycle. Mtb enters the host through mucosal surfaces, after which the bacilli are taken up into the phagosomes of resident alveolar macrophages. By escaping phagosome–lysosome fusion, the intracellular bacilli are able to avoid being killed and continue to multiply, leading eventually to lysis of the infected cells. The extracellular bacilli are taken up by other macrophages and by blood monocytes that are attracted to the focus and then develop into immature macrophages. The latter cells ingest bacilli readily, but are incapable of killing virulent Mtb or inhibiting their growth. Thus the bacillary multiplication cycle is repeated within immature macrophages. The mycobacteria are transported to draining lymph nodes, where they multiply and continue the life cycle .
Mixed immune responses
According to the Th1/Th2 model, Th1 and Th2 cells have a division of labour in their protective role against infectious pathogens. Th1 immunity protects the host against intracellular pathogens, whereas Th2 immunity protects against extracellular pathogens. Thus, most candidate vaccines that are in development induce immune responses that are dominated either by cellular or humoral immunity. There is convincing evidence that intracellular microbial pathogens elicit a mixed specific immune response comprising humoral and T cell-mediated immunity. The protective efficacy of such mixed responses have been demonstrated for Chlamydia, Plasmodium, Cryptococcus neoformans and Schistosoma infections (reviewed in [13,16,17]).
Malaria and schistosomiasis are two important parasitic diseases in the tropics in terms of morbidity and mortality. Both parasites have a complex life cycle and release multi-stage antigens. Vaccine candidates that have shown promising results for malaria and schistosomiasis induce mixed CMI and humoral immunity.
Analysis of natural immunity in adults to P. falciparum infection has demonstrated that immune responses to malaria are acquired with age. As immunity is acquired with age, children remain more susceptible to infection and development of clinical disease than adults. Early vaccine studies demonstrated that immunization with irradiated sporozoites can induce sterile immunity, implying that a vaccine targeted to the pre-erythrocytic stage is a viable strategy to pursue .
The key antigens of focus for malaria vaccine include the circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP), involved in sporozoite motility and invasion of liver cells, and liver-stage antigen (LSA-1). Recently, a combination of a subunit protein vaccine that induces high antibody responses and a DNA vaccine that induces strong CMI have demonstrated that a combination of the two vaccines induces stronger humoral and cellular compared with either vaccine alone (reviewed in ). Two different approaches have been taken to achieve this goal; to develop an anti-sporozoite vaccine that, through strong antibody response, eliminates the parasite before it infects the liver, or to create a cytotoxic T lymphocyte (CTL)-inducing vaccine that clears parasite-infected hepatocytes and prevents development of the blood-stage of the disease .
Another good example where vaccine candidates that induce mixed Th1/Th2 responses have shown efficacy comes from studies of schistosomiasis. Similar to malaria, epidemiological data from Schistosoma-endemic communities show invariably that adolescents and adults are more resistant to infection than children. The widely held view for this age-related difference in resistance/susceptibility is that acquired immunity develops through years of exposure to parasite antigens in adults. In humans, results of sero-epidemiological studies have implicated immunoglobulin (Ig)E, IgA, IgM, IgG1 and IgG3 for protection against infection (reviewed in ). Encouraged by the above results, more than 100 candidate vaccines (some patented) have been identified by several laboratories. However, none of these candidate vaccines yielded the stated goal of consistent induction of 40% protection when tested by independent laboratories as recommended by the United Nations Development Programme (UNDP)/World Bank/Research and Training in Tropical Diseases (TDR)/World Health Organization (WHO) Committee .
However, important clues to our understanding of immunity to Schistosoma infection come from two sources. One vaccine that has consistently provided consistent protection against challenge infection is the irradiated cercariae of S. mansoni. Mice exposed to attenuated S. mansoni parasites develop partial immunity to infection by producing IFN-γ. Immunity is reduced by a deficiency of CD4+ cells, B cells and IFN-γ or IL-12, which strongly suggests the involvement of Th1 immunity .
Secondly, studies in Brazil have revealed the presence of endemic normal subjects also known as ‘putative resistants’ (PR), which display naturally acquired immunity without chemotherapy or vaccination. It has been shown that these PR are resistant to Schistosoma infection despite years of exposure to infection, and produce vigorous cellular and humoral immune responses to crude schistosome antigens, compared to those who developed clinical disease. In response to stimulation with these antigens, peripheral blood mononuclear cells from PR individual secrete both Th1- and Th2-type cytokines, whereas chronically infected individuals make a Th2 type response. It is the Th1 response (particularly IFN-γ) to schistosomulum antigens that is thought to be the key to resistance to schistosomiasis in these subjects .
Using these PR individuals, two new vaccine antigens that are expressed in the tegument membrane of S. mansoni, namely tetraspanin (SmTSP-2) and 29-kDa protein (Sm29), have been identified. Both proteins are recognized preferentially by sera from PR individuals as opposed to sera from chronically infected patients, supporting the potential of the PR immune response to guide discovery of tegument plasma membrane proteins as recombinant vaccines (reviewed in ).
Antibody-mediated protection against Mtb
Studies on the protective and non-protective roles of antibody responses against Mtb infection from as early as 1888 until the mid-1990s have been reviewed comprehensively by Glatmann-Freedman and Casadevall . Thanks to advances in molecular biological and immunological tools, our understanding of host–pathogen interaction and immune responses has changed significantly in recent years. Experimental studies in B cell-deficient mice, SCID mice, immunization using monoclonal and polyclonal antibodies and mucosal vaccination trials demonstrate convincingly the essential interdependence and synergy between CMI and humoral immunity.
Most of our understanding regarding the role of antibodies or B cells during mycobacterial infections comes from the mouse model. Studies of Mtb infections in gene knock-out mice incapable of making B cells are regarded as providing definitive evidence for the role played by antibodies against mycobacterial infections. These mice can be generated by disruption of one of the IgM µ-chain transmembrane region exon and therefore possess no mature B cells and are unable to produce antibodies. Using these µMT mice, Voldermeier et al. evaluated the role of B cells in the course of Mtb infection. The results showed that Mtb-infected µMT mice were found to have three- to eightfold elevated counts of viable bacilli compared with normal littermates at 3–6 weeks post-infection.
Upon aerosol infection with 100 colony-forming units (CFU) of Mtb Erdman, B cell-deficient mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils . Infected B cell-deficient mice show increased production of IL-10 in the lungs, whereas IFN-γ, TNF-α and IL-10R remain unchanged in wild-type mice. B cell-deficient mice have enhanced susceptibility to infection when challenged aerogenically with 300 CFU of M. tuberculosis, corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell-deficient mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus.
Guirado et al. investigated the protective role of immune of sera against reactivation of Mtb infection in SCID mice and found that passive immunization with sera obtained from mice treated with detoxified Mtb extracts (delivered in liposomes in a composition known as RUTI) exerted significant protection. The SCID mouse model consisted of aerosol infection by Mtb, followed by 3–8 weeks of chemotherapy with isoniazid and rifampicin, 25 and 10 mg/kg, respectively. After infection and antibiotic administration, two groups of mice were treated for up to 10 weeks with intraperitoneal passive immunization using hyper serum (HS) obtained from mice infected with Mtb, treated with chemotherapy [NIH, rifampin (RIF)] for 8 weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant difference were found between the HS and CT groups in the number of bacilli in the lungs, the extent of pulmonary granulomatous infiltration and the percentage of animals without pulmonary abscesses. These data suggest strongly a protective role of specific antibodies against lung dissemination of Mtb infection. The study also showed a strong antibody response against a wide antigenic range, with an IgG2a–IgG2b > IgG1 > IgG3 predominance .
Very recently, Lopez and colleagues evaluated the efficacy of two mAbs, TBA61 against the 16-kDa antigen (acr antigen) and TBA48, against the 38-kDa antigen in the control of pulmonary infection . Using an intratracheal model of pulmonary infection, they evaluated bacterial load and morphometric and histological changes in the lungs of infected treated mice. The results showed reduction of bacterial load and milder morphometric and histopathological changes in mice treated with mAb TBA61 against the 16-kDa protein of Mtb at 21 days post-infection with Mtb 37Rv compared to those treated with mAb TBA84 against the 38-kDa protein and control mice .
M. bovis BCG cells coated with IgG2 and IgG3 anti-heparin-binding haemagglutinin adhesion (HBHA) mAbs before intranasal inoculation limited extrapulmonary dissemination and reduced the number of bacilli in the spleen , whereas M. bovis cells coated with IgG2b anti-MBP83 mAbs before intravenous inoculation increased long-term survival but did not reduce the bacterial load . Furthermore, intraperitoneal injection of a large volume of a standard preparation of human gamma globulin from normal donors, either 16 or 44 days post-infection, reduced bacterial loads in the spleen and lungs of intravenously infected mice .
Monoclonal IgA anti-α-crystallin given intranasally impaired short-term protection against aerosol lung infection and granuloma formation in BALB/c mice . Mtb cells coated with mAb IgG3 against arabinomannan (AM) enhanced granulomatous formation and survival of intratracheally inoculated mice . Arabinomannan is an oligosaccharide component of lipoarabinomannan (LAM), a major structural component of the mycobacterial capsule.
Passive immunization of BALB/c mice with mAb (SMITB14) of IgG1 subclass to LAM and its corresponding F(ab') have been shown to give protection against Mtb infection in BALB/c mice, as determined by dose-dependent reduction in bacterial load in lungs and spleens, reduced weight loss and increased long-term survival . In addition to protective mAbs, a conjugate vaccine candidate derived from AM and Ag85B that elicits both cellular and humoral immune responses has been shown to be protective against challenge Mtb infection in mice, rabbits and guinea pigs. Using different routes of immunization (intravenous, subcutaneous, intramuscular) and different adjuvants (alum, tetanus toxoid, Eurocine™ L3 adjuvant), it was shown that the conjugate vaccine candidate was highly immunogenic and protective as estimated by long-term survival and reduced pathology in lungs and spleens of animals challenged with the virulent Mtb strain Harlingen .
In one study, Glatman-Freedman and colleagues  examined the |prevalence and diversity of AM among Mtb strains using four AM-binding mAbs, including the mAb known as 9d8, which binds AM specifically. Using whole cell enzyme-linked immunosorbent assay (ELISA), AM was recognized by 9d8 mAb on the surface of nine of 11 strains. However, the AM recognized by mAb 9d8 was found in the culture supernatants of all 11 Mtb strains tested, as demonstrated by capture ELISA. Other AM binding mAbs reacted both with the surfaces and with the culture supernatants of 11 strains . The above results indicate that, although AM was detected in all Mtb strains, the distribution of epitopes on the surfaces of these strains was different, a finding which may have important implications in developing vaccines and serodiagnostic tests. Some of the specific mycobacterial antigens (HBHA, a-crystallin, Ag85 complex) that induce protective antibody responses [32–39] are given in Table 1.
|Antigen||Formulation||Adjuvant||Administration route||Experimental animal||Immunogenicity||Effect/protection||Reference|
|MPT83||DNA/RNA||None||Intramuscular||BALB/c mice||IgG, IFN-γ, IL-12||Protection against challenge||Xue et al.|
|Ag85A||Native||DDA‡||Intranasal||BALB/c mice||IFN-γ, IL-12, IL-4, IgA, IgG||Protection against challenge||Giri et al.|
|Ag85B†||DNA||None||Intramuscular (4×)||BALB/c mice||IgG1, IgG2a, IFN-γ, TNF, CD8+ but not IL-4||Reduction in bacterial load||Teixeira et al.|
|Ag85B||DNA||¶LTK63||Intramuscular||C57BL/6 mice||IgG2A, reduction in CD4+ cells||Protection by reducing IFN-γ||Palma et al.|
|Ag85B–ESAT-6||rA85B-ESAT-6||None||Intramuscular||BALB/c mice||IgG1, IgG2A IFN-γ, IL-12||Reduction in CFU, pathology||Chang-Hong et al.|
|CFP-10–CFP-21||DNA||None||Intramuscular||C57BL/6||IFN-γ, IL-12 (p40), CTL, IgG1, IgG2A||Reduction in CFU||Grover et al.|
|HBHA||Native||DDA‡||Intravenous||C57BL/6||Anti-HBHA§ antibody, IFN-γ||Reduction in CFU||Parra et al.|
|HBHA||Recombinant||Cholera toxin||Mucosal||BALB/c male mice||CMI, antibody||Reduced CFU in spleen||Kohama et al.|
In addition to some of the encouraging results from earlier studies , Sanchez-Rodriguez et al., in a study in Mexico on the indigenous Indian population, have demonstrated that IgG against antigen 85-complex (Ag85) had beneficial effects. The study demonstrated that many serum samples were negative and the reactivity of the positive serum was limited to Ag85 and hspX. Two antigens, the 88-kDa and the 38-kDa (phosphoprotein), known for their reactivity to immune sera in many studies (reviewed in ), failed to be recognized by sera from the indigenous population.
Another interesting observation from this study is that positive antibody titres to the Ag85 were significantly more frequent in non-cavitary TB and than in patients who were cured with anti-tuberculosis therapy.
de Valliere et al. investigated the ability of human antibodies induced by BCG vaccination to protect against mycobacterial infection. Serum samples containing Mtb-specific antibodies were obtained from volunteers who had received two intradermal BCG vaccinations 6 months apart. Significant increases in LAM-specific IgG were detected after the primary and booster vaccinations. Internalization of BCG by phagocytic cells was shown to be enhanced significantly in post-vaccination serum samples. Furthermore, the inhibitory effects of neutrophils and monocytes/macrophages on mycobacterial growth were enhanced significantly by BCG-induced antibodies. The growth inhibitory effects of post-vaccination sera were reversed by pre-absorption of IgG with protein G. Finally, BCG-induced antibodies enhanced proliferation of IFN-γ production significantly in mycobacterium-specific CD4+ T cells and CD8+ T cells, as well as the proportion of proliferating CD8+ T cells. The study is unique in that it has demonstrated that mycobacterial antibodies enhance Th1 immune responses (IFN-γ), instead of down-regulating as thought previously (Th1/Th2 paradigm) .
Mechanisms of antibody protection
Supportive data for the role of antibody in host defence are provided by in vitro studies which demonstrate that specific antibody inhibits the replication of the pathogen, neutralizes toxin elaborated by the pathogen, promotes antibody-dependent cellular cytotoxicity, serves as an opsonin, triggers the complement cascade and prevents infection in tissue culture (reviewed in [13,43]).
One of the effector mechanisms of antibody responses believed to be important during Mtb infection is probably opsonization. As mentioned earlier, some studies [42,44] have shown increased internalization and increased killing of mycobacteria by neutrophils and macrophages in the presence of antibodies. In addition, mycobacteria coated with specific antibodies were processed more effectively and presented by DCs for stimulation of CD4+ and CD8+ responses [24,25,28].
The entry of Mtb into macrophages occurs via an array of different receptor molecules, including complement receptors (CRs), mannose receptors (MRs) and FcR. The ability of receptor molecules to internalize Mtb is probably related to the complex structure of the cell surface of Mtb. The precise receptor involved in phagocytic entry may have a major impact on the survival chances of Mtb once inside the macrophages. For instance, ingestion of particles through FcR results in respiratory burst and an inflammatory response in macrophages. In contrast, internalization via CR3 receptors prevents the activation of macrophages. In addition, CR3-mediated uptake of Mtb is strictly dependent upon the presence of host plasma membrane . It has been suggested that receptors specific for surface carbohydrate epitopes may allow mycobacteria to bypass the bactericidal activity of macrophages. For instance, LAM is known to mediate Mtb uptake by phagocytic cells, such as macrophages and dendritic cells, and has been reported to utilize at least two receptors, CD14 and murine MR, for entry into host phagocytes. In addition to facilitating Mtb entry into host phagocytes via CD14 and MR receptors, LAM has a significant effect on the hosts' immune responses, including inhibition of phagosome–lysosome fusion , suppression of T cell proliferation [46,47], inhibition of protein kinases  and inhibition of IFN-γ-mediated activation of macrophages . As suggested by Hamasur et al., the effects of anti-LAM antibodies and their F(ab')2 fragments could be interference with some of the biological effects of LAM on host immunity. Additional information on antibody-mediated immune protection mechanisms against intracellular pathogens can be found in review articles by Casadevall and Pirofski  and Casadevall .
Enhancement of the anti-mycobacterial activity of phagocytic cells by antibodies could be particularly important in the context of mucosal immunity. Antibodies of the IgG and IgA classes have been shown to be present in the mucosal secretion of the lower respiratory tract .