Lactoferrin enhanced efficacy of the BCG vaccine to generate host protective responses against challenge with virulent Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a disease with world wide consequences, affecting nearly a third of the world's population. The established vaccine for TB, an attenuated strain of Mycobacterium bovis Calmette Guerin (BCG), has existed since 1921. Lactoferrin, an iron-binding protein found in mucosal secretions and granules of neutrophils was hypothesized to be an ideal adjuvant to enhance the efficacy of the BCG vaccine, specifically because of previous reports of lactoferrin enhancement of IL-12 production from macrophages infected with BCG. Different vaccination protocols were investigated for generation of host protective responses against MTB infection using lactoferrin admixed to the BCG vaccine. Resulting effects demonstrate that BCG/lactoferrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology; significant reduction in tissue CFUs and pathology were observed post-challenge compared to those seen with BCG alone. Addition of lactoferrin to the vaccine led to reduced pathological damage upon subsequent infection with virulent MTB, with positive results demonstrated when admixed in oil-based vehicle (incomplete Freund's adjuvant, IFA) or when given with BCG in saline. The observed post-challenge results paralleled increasing production of IFN-gamma and IL-6, but only limited changes to proinflammatory mediators TNF-alpha or IL-1beta from BCG-stimulated splenocytes. Overall, these studies indicate that lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine, with potential to reduce related tissue damage and pulmonary histopathology.

Lactoferrin modulation of IL-12 and IL-10 response from activated murine leukocytes.

Lactoferrin possesses a wide range of immunomodulatory activities, including promotion of the delayed type hypersensitivity response (DTH) towards BCG (Bacillus Calmette Guerin) antigens. Addition of Lactoferrin as an adjuvant to the BCG vaccine was previously demonstrated to augment protection against subsequent mycobacterial challenge, with concomitant development of a strong T cell helper type 1 (TH1) immunity. Because generation of TH1 immunity is in large part dependent on the balance of monocytic pro- and anti-inflammatory cytokines, the effect of Lactoferrin on leukocytes was investigated. Lactoferrin enhanced proinflammatory responses in a dose-dependant manner from splenocyte and adherent (F4/80+) splenocyte populations, bone marrow derived monocytes (BMM), and J774A.1 cultured cells. In all scenarios tested, Lactoferrin induced a strong increase in the ratio of IL-12:IL-10 production from LPS stimulated cells. Examination of Lactoferrin effects on BCG infected J774A.1 cells and on BMM revealed similar immunomodulatory effects, with particularly strong increase in IL-12 production. Furthermore, immunization of mice with BCG admixed with Lactoferrin led to increased generation of CD4+ cells expressing IFN-gamma upon restimulation with BCG antigens. These results provide molecular evidence to support the role of Lactoferrin as an adjuvant candidate to augment development of DTH response to vaccine antigens.

Ontogeny of proteolytic immunity: IgM serine proteases.

We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically naïve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.

Toward selective covalent inactivation of pathogenic antibodies: a phosphate diester analog of vasoactive intestinal peptide that inactivates catalytic autoantibodies.

We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys(20) residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys(20) enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.

VIPase autoantibodies in Fas-defective mice and patients with autoimmune disease.

The immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) was cleaved by purified IgG from Fas-defective C3H/gld mice, lupus patients, and autoimmune thyroiditis patients. No VIPase activity was detected in IgG from control mice and humans. Kinetic analyses of VIPase IgG preparations suggested low-affinity recognition of VIP. Yet the VIPase activity was VIP selective, judged by lack of correlation with other protease activities expressed by the IgG and by noninterference of unrelated peptides in the activity. Recombinant Fv constructs selected from a human lupus phage show library displayed VIPase activity, confirming that the active site is located in the V domains. Inhibition of the VIPase activity by di-isopropylfluorophosphate suggested a serine protease-like mechanism of catalysis. Irreversible binding of a biotinyated phosphonate diester by the IgG and Fv preparations was observed, consistent with the presence of activated nucleophiles similar to those in enzymes capable of covalent catalysis. These observations show that VIP is a target for specific catalytic autoantibodies in autoimmune disease.

Roles of vasoactive intestinal peptide (VIP) in the expression of different immune phenotypes by wild-type mice and T cell-targeted type II VIP receptor transgenic mice.

Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1 and VPAC2, are quantitatively prominent and functionally critical in the immune system. Transgenic (T) mice constitutively expressing VPAC2 selectively in CD4 T cells, at levels higher than those found after maximal induction in CD4 T cells of wild-type (N) mice, have elevated blood concentrations of IgE, IgG1, and eosinophils; enhanced immediate-type hypersensitivity; and reduced delayed-type hypersensitivity. In contrast, VPAC2-null (K) mice manifest decreased immediate-type hypersensitivity and enhanced delayed-type hypersensitivity. The phenotypes are attributable to opposite skewing of the Th2/Th1 cytokine ratio, but no studies were conducted on the roles of T cell-derived VIP and altered expansion of the Th subsets. Dependence of the Th phenotype of T mice, but not of N or K mice, on T cell-derived VIP now is proven by showing that eliminating VIP from TCR-stimulated T cell cultures with VIPase IgG normalizes the elevated number of IL-4-secreting CD4 T cells, decreases the secretion of IL-4 and IL-10, and increases the secretion of IFN-gamma. Flexible responsiveness of CD4 T cells from N and K mice, but not T mice, to exogenous VIP in vitro and in vivo is shown by increased numbers of IL-4-secreting CD4 T cells, greater secretion of IL-4 and IL-10, and lesser secretion of IFN-gamma after TCR stimulation with VIP. The level of VIP recognized by CD4 T cells thus is a major determinant of the relative contributions of Th subsets to the immune effector phenotype.

New evidence for transmitter role of VIP in the airways: impaired relaxation by a catalytic antibody.

The identity of the transmitter(s) of nonadrenergic, noncholinergic airway smooth muscle relaxation has long been investigated. Recently, nitric oxide (NO) has been proposed as the main, if not the only transmitter. We earlier suggested vasoactive intestinal peptide (VIP) as a candidate transmitter and target for pathogenic catalytic autoantibodies (VIPases) found in certain humans. To re-examine the role of VIP, we studied the airway transport and effects of a model monoclonal antibody (Ab) capable of binding and cleaving VIP. In vitro receptor binding assays indicated the catalytic light chain subunit of the VIPase Ab to inhibit the saturable binding of (Tyr(10-125)I) VIP by guinea pig lung membranes, whereas a catalytically deficient mutant of the Ab light chain was without significant inhibitory activity. Systemically administered IgG preparations of the VIPase Ab accumulated in the airway lavage fluid of guinea pigs at levels close to those in blood, suggesting that the Ab reaches the airways freely. Electrical field stimulation (EFS)-induced relaxations of tracheal strips were weaker and shorter in VIPase-treated animals than in control nonimmune IgG-treated animals. The inhibitory effect of the VIPase was dose-dependent. VIPase-mediated inhibition of EFS-induced relaxation was evident both in the absence and presence of blockade of beta-adrenergic and cholinergic receptors. Thus, circulating VIP binding and cleaving antibodies can reach the airways and attenuate the neurogenic relaxation of guinea pig tracheal smooth muscle, probably by neutralizing endogenously released VIP. The findings support a role for VIP as a major mediator of neurogenic relaxation of guinea pig tracheal smooth muscle. Lack of complete abrogation of relaxation is consistent with a co-transmitter role for NO.

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice.

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren's syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.