A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis. Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.

Unique model of dormant infection for tuberculosis vaccine development.

Most individuals exposed to Mycobacterium tuberculosis become infected but hinder the infectious process in dormant foci, known as latent tuberculosis. This limited infection usually stimulates strong T-cell responses, which provide lifelong resistance to tuberculosis. However, latent tuberculosis is still poorly understood, particularly because of the lack of a reliable animal model of dormant infection. Here we show that inoculation of mice with a unique streptomycin-auxotrophic mutant of Mycobacterium tuberculosis recapitulates dormant infection. The mutant grows unimpaired in the presence of streptomycin and no longer grows but remains viable for long periods of time after substrate removal, shifting from the log growth phase to the latent stage, as indicated by augmented production of alpha-crystallin. Mice challenged with the mutant and inoculated with streptomycin for approximately 3 weeks developed a limited infection characterized by a low bacteriological burden and the presence of typical granulomas. After substrate withdrawal, the infection was hindered but few microorganisms remained viable (dormant) in the animals' tissues for at least 6 months. In addition, the animals developed both potent T-cell responses to M. tuberculosis antigens, such as early culture filtrate, Ag85B, and ESAT-6, and resistance to reinfection with virulent M. tuberculosis. Therefore, infection of mice or other animals (e.g., guinea pigs) with M. tuberculosis strain 18b constitutes a simple and attractive animal model for evaluation of antituberculosis vaccines in the context of an M. tuberculosis-presensitized host, a prevailing condition among humans in need of a vaccine.

Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I antigen-processing pathway.

CD8+ T cells play an important role in the host response to infection with Mycobacterium tuberculosis (Mtb). Mtb resides in an arrested phagosome that is phenotypically similar to an early endosome. The mechanisms by which Mtb-derived Ags gain access to the HLA-I-processing pathway are incompletely characterized. Studies with CD8+ T cell lines have suggested that Mtb Ags gain access to the HLA-I pathway in an alternate vacuolar pathway that is both brefeldin A (BFA) and TAP independent. To define the requirements of entry of Ag into the HLA-I pathway, we have used human CD8+ T cell clones specific for the secreted Mtb Ag CFP10. Human monocyte-derived dendritic cells were pulsed with CFP10 expressed in a recombinant adenovirus, surface adsorbed to microspheres, or in its native form by Mtb. When delivered by adenovirus, processing and presentation of CFP10 were blocked by both BFA and the proteasomal blocker lactacystin. In contrast, processing of CFP10 adsorbed to the surface of microspheres was not affected by either of these Ag-processing inhibitors. BFA, lactacystin, and TAP inhibition blocked the recognition of Mtb-infected dendritic cells, suggesting that processing was via a cytosolic pathway for this secreted protein Ag. We conclude that secreted proteins from Mtb can be processed in a BFA- and proteasome-dependent manner, consistent with egress of Ag into the cytosol and subsequent loading of proteasomally derived peptides.

Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41.

MTB41 is a Mycobacterium antigen that is recognized by CD4+ T cells early after experimental infection of mice with Mycobacterium tuberculosis and by PBMC from healthy PPD positive individuals. Immunization of mice with plasmid DNA encoding the MTB41 gene sequence results in the development of antigen-specific CD4+ and CD8+ T cells, and protection against challenge with virulent M. tuberculosis. In the present studies, in contrast to DNA immunization, we show, that a strong MTB41-specific CD4+ T cell response, but no MHC class I restricted cytotoxic T lymphocyte (CTL) activity is detected in the spleen cells of infected mice. Therefore, this data suggests that the induction of CD8+ T cell response to MTB41 epitopes by DNA immunization may not be relevant to protection because these epitopes are not recognized during the infectious process. We also compared the repertoire of rMTB41 epitope recognition by CD4+ T cells of M. tuberculosis-infected mice with the recognition repertoire of mice immunized with the recombinant rMTB41 protein. Both regimens of sensitization lead to the recognition of the same molecular epitope. Coincidentally, immunization with the soluble recombinant protein plus adjuvant, a regimen known to generate primarily CD4+ T cells, resulted in induction of protection comparable to BCG in two well-established animal models of tuberculosis (mice and guinea pigs).

Skin test performed with highly purified Mycobacterium tuberculosis recombinant protein triggers tuberculin shock in infected guinea pigs.

Tuberculin shock due to inoculation of Mycobacterium tuberculosis antigens in patients with tuberculosis is a serious syndrome originally described over 100 years ago by Robert Koch. Here, we present experimental evidence that a single M. tuberculosis recombinant protein, CFP-10, triggers this syndrome. Intradermal inoculation of CFP-10 elicits in M. tuberculosis-infected mice high levels of serum tumor necrosis factor alpha and causes tuberculin shock in infected guinea pigs characterized by hypothermia and death within 6 to 48 h after the antigen inoculation. Autopsies of these animals revealed intense polycythemia and hemorrhagic patches in the lung parenchyma, a pathological observation consistent with tuberculin shock. These results point to the possible occurrence of tuberculin shock in sensitive individuals inoculated with highly purified M. tuberculosis recombinant proteins as vaccine candidates or skin test reagents.

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein.

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.

Cloning and characterization of a gene encoding an immunoglobulin-binding receptor on the cell surface of some members of the family Trypanosomatidae.

Several members of the Trypanosomatidae family, when freshly isolated from their mammalian hosts, have immunoglobulins adsorbed to their cell surfaces. However, a significant portion of these antibody molecules is not parasite specific, i.e., the immunoglobulins are bound to the parasite's cell surface molecules via noncognitive interactions. It has been proposed that this noncognitive adsorption of immunoglobulins to the parasite is mediated by an Fc-like receptor present in several members of the Trypanosomatidae family. However, the molecular identification of this receptor has never been defined. Here, we describe the cloning of a gene encoding a protein that might represent this molecule. The gene, named Lmsp1, was cloned by screening a Leishmania major cDNA expression library using a rabbit antiserum. Lmsp1 is present in both Leishmania and Trypanosoma and is expressed in all developmental stages of these parasites. The predicted protein has a molecular mass of 16.6 kDa and contains an RGD sequence starting at residue 104 and three cysteine residues at positions 55, 74, and 116. The purified recombinant protein strongly binds to normal immunoglobulins of various animal species (humans, rabbits, sheep, goats, guinea pigs, donkeys, rats, and mice) and the binding to human immunoglobulins appears to be immunoglobulin G (IgG) and IgM isotype specific. Moreover, Lmsp1 binds to both purified Fc and Fab fragments of IgG from both humans and rabbits. The mapping of the Lmsp1 epitopes that bind human IgG revealed that different sequences of the molecule bind to Fc or Fab. In addition, fluorescence-activated cell sorter analyses with a specific rabbit anti-Lmsp1 antiserum showed that Lmsp1 is associated with the parasite's cell surface. Finally, inhibition experiments point to an active role of this molecule in the immunoglobulin-mediated attachment and penetration of Trypanosoma cruzi in its macrophage host cells, thus suggesting that Lmsp1 is a putative Trypanosomatidae immunoglobulin receptor.

LeIF A recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile

We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major. Infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-ã (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-ã. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-ã by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1-226) maintained the ability to stimulate IFN-ã from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags