Hector J. Hernandez

Human Monoclonal Antibodies Directed against Toxins A and B Prevent Clostridium difficile-Induced Mortality in Hamsters

ABSTRACT Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.

The immunobiology of Th1 polarization in high‐pathology schistosomiasis

Summary:  Schistosomiasis is a serious global helminthic disease, in which the main immunopathology consists of a granulomatous and fibrosing reaction against tissue‐trapped parasite eggs. The severity of this inflammatory process, the product of a CD4+ T‐cell‐mediated immune response against parasite egg antigens, is, however, markedly uneven, both in human patients and among mouse strains in an experimental model. Severe schistosomiasis is associated with persistently elevated pro‐inflammatory T‐helper‐1 (Th1)‐type cytokines, whereas milder pathology is present when Th2 cytokines dominate. This scenario is supported by the pronounced pathology resulting from the obliteration of pathways that facilitate Th2 differentiation and by the development of more intense lesions in mouse strains that fail to downregulate the Th1 response. Genetically prone high‐pathology mice have a higher proportion of CD4+ T cells in lymph nodes and granulomas, in which the Th1 phenotype is driven by interleukin‐12; they also develop a dominant repertoire against peptide 234–246 of the major Sm‐p40 egg antigen, utilizing a strikingly restricted T‐cell receptor structure that involves Vα11.3β8. In turn, low‐pathology mice exhibit enhanced CD4+ T‐cell apoptosis, which contributes to limit pathology. The definition of distinctive immune profiles associated with polar forms of schistosomiasis opens opportunities for targeted immuno‐intervention in individuals suffering from or at risk of severe disease.

Th1-polarizing immunization with egg antigens correlates with severe exacerbation of immunopathology and death in schistosome infection

In schistosomiasis mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammatory process, which is mediated by, and dependent on, MHC class II-restricted CD4 T helper (Th) lymphocytes specific for schistosome egg antigens (SEA). In the mouse model of the disease, CBA mice develop large granulomas, whereas in C57BL/6 (BL/6) mice these granulomas are significantly smaller. To further investigate how the prevailing cytokine environment influences the development of the egg-induced immunopathology, we immunized the low-pathology BL/6 mice with SEA in complete Freunds adjuvant (CFA) once before, and once again during, the course of a 7-week infection. This immunization caused a pronounced Th1 shift in the SEA-specific CD4 T cell response, which was detected in the mesenteric lymph nodes (MLNs) and spleens, as well as in the granulomatous lesions themselves. The immunized mice displayed a dramatic enhancement of hepatic egg-induced immunopathology manifested by a marked increase in granuloma size and parenchymal inflammation, leading to early death. Control mice immunized with equivalent amounts of SEA or CFA alone displayed the smaller hepatic lesions in a Th2-dominant environment typically seen in the unimmunized BL/6 mice. Analysis of granuloma and MLN lymphocytes from the SEA/CFA-immunized mice revealed that the proportion of CD4 T cells was unchanged in comparison with the control BL/6 groups and remained significantly lower than that seen in the normally high-pathology CBA strain. These results suggest that the shift toward Th1-type cytokine production by a numerically stable population of CD4 T cells correlates with severe exacerbation of immunopathology in schistosomiasis.

The immune response and immunopathology in infection with Schistosoma mansoni: a key role of major egg antigen Sm-p40

The immune response and related granulomatous inflammation in infection with Schistosoma mansoni are ultimately dependent on SEA‐sensitized CD4+ Th cells and comprise multiple pathways variously involving the activation and recruitment of different cell populations and the production of different inflammatory cytokines, all under the influence of regulatory genetic factors. The spontaneous down‐regulation of granuloma formation (immunomodulation), in turn, is a well‐known phenomenon, but the full extent of its precipitating factors is still uncertain. This review describes a pathway leading to immunomodulation that features at its centre the down‐regulatory cytokine IL‐10. This mechanism is attractive because it offers a cogent correlation between findings in the laboratory and those displayed by patients affected with the disease. The Sm‐p40 antigen, a major component of schistosome eggs, elicits a strong CD4+ Th cell response in H‐2k mice that correlates with intense granuloma formation; in contrast, its immunogenicity is relatively minor during infection of other mouse strains that develop smaller granulomas. Of great interest is that the Sm‐p40 antigen only elicits a Th‐1 type cytokine response, a phenotype that remains constant even as the overall response to SEA shifts to a Th‐2 type. The Sm‐p40 molecule has a dominant epitope that is the target of CD4+ Th cells from infected H‐2k mice; indeed, a minimal peptide that bears the epitope binds to I‐Ak. The importance of pursuing a systematic elucidation of the major egg antigens, resides in the exciting possibility of specifically desensitizing the CD4+ Th cells that mediate granuloma formation, which may achieve meaningful prevention or amelioration of clinical disease.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis.

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the hosts genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

Enhanced Egg-Induced Immunopathology Correlates With High IFN-γ in Murine Schistosomiasis: Identification of Two Epistatic Genetic Intervals

The genetic basis of dissimilar immunopathology development among mouse strains infected with Schistosoma mansoni is not known. We performed a multipoint parametric linkage analysis on a cohort of F2 mice, offspring of brother-sister mating between (high pathology CBA × low pathology BL/6)F1 mice, to examine whether the observed differences in the type of immune response or the extent of hepatic immunopathology are linked to any particular genomic intervals. The F2 mice exhibited cytokine responses and immunopathologies that revealed a statistically significant correlation between prominent egg Ag-stimulated IFN-γ production by mesenteric lymph node cells and hepatic egg granuloma size. Increased IFN-γ production showed suggestive linkage to a dominant CBA locus on chromosome 1 and a recessive CBA locus on chromosome 5; significantly, there was an epistatic interaction between the two IFN-γ loci. An additional locus with suggestive linkage to granuloma formation and a CBA-recessive mode of inheritance was mapped to centromeric chromosome 13. Our analysis identified the first three genetic regions that appear to influence the immunopathology in murine schistosomiasis; however, further congenic dissection studies will furnish a more precise understanding of the genetic control of this disease.

Nasal Administration of Schistosoma mansoni Egg Antigen-Cholera B Subunit Conjugate Suppresses Hepatic Granuloma Formation and Reduces Mortality in S. mansoni-Infected Mice

Granulomatous inflammation in schistosomiasis is a delayed‐type hypersensitivity reaction mediated by CD4+ T cells specific for parasite egg antigens (Ags). In an attempt to control T‐cell responses leading to excessive harmful inflammation and granuloma formation, especially in the liver, BALB/c mice were intranasally (i.n.) treated with soluble Schistosoma mansoni egg Ags (SEA) conjugated to cholera toxin B subunit (CTB), a mucosa‐binding protein with demonstrated capacity to suppress inflammatory T‐cell functions after mucosal administration. Treatment with CTB–SEA significantly conjugate a reduced liver granuloma formation in infected mice associated with decreased SEA specific Th1‐ and Th2‐type immune responses by liver leukocytes. Importantly, treatment with CTB–SEA conjugate also significantly reduced the mortality in chronically infected mice. In S. mansoni‐infected large‐granuloma forming CBA mice, i.n. treatment with purified Sm‐p40, the major egg antigen, conjugated to CTB likewise significantly inhibited hepatic egg granuloma formation. A reduction of SEA‐driven lymphoproliferation and of interferon (IFN)‐γ, interleukin (IL)‐4 and IL‐5 production, together with an increase in transforming growth factor (TGF)‐β1 production, were observed in splenic cells from CTB‐Sm‐p40‐treated SEA‐sensitized mice, as well as in liver leukocytes from CTB‐Sm‐p40‐treated schistosome‐infected mice. These results indicate that mucosal administration of SEA or purified Sm‐p40 antigen in conjunction with CTB is highly effective in curtailing immunopathologic manifestations of schistosomiasis in vivo in infected hosts.

Diminished immunopathology in Schistosoma mansoni infection following intranasal administration of cholera toxin B-immunodominant peptide conjugate correlates with enhanced transforming growth factor-β production by CD4 T cells

In Schistosoma mansoni infection, CD4 T cells specific for parasite egg antigens mediate perioval granuloma formation in the liver and intestines. Mice of the CBA strain develop a severe form of disease and a significant proportion of their CD4 T cell response is directed against the major egg antigen Sm‐p40 and its immunodominant T cell epitope peptide 234–246. Here, we show that intranasal (i.n.) treatment of infected CBA mice with a fusion protein of the cholera toxin B subunit (CTB) with peptide 234–246 (CTB::pep) results in significant down‐modulation of hepatic granulomatous inflammation and fibrosis. Moreover, egg antigen‐stimulated dispersed hepatic granuloma cells, as well as mesenteric lymph node CD4 T cells from the CTB::pep‐treated mice, produced significantly more transforming growth factor (TGF)‐β than that produced by treated or untreated controls. The data demonstrate that i.n. administration of a single immunodominant peptide conjugated to CTB can lead to down‐regulation of the hepatic immunopathology associated with schistosomiasis, and that this down‐regulation is, at least in part, mediated by TGF‐β.

Expansion of CD4 T cells expressing a highly restricted TCR structure specific for a single parasite epitope correlates with high pathology in murine schistosomiasis.

The hepatic immunopathology in schistosomiasis mansoni is mediated by CD4 T cells specific for egg antigens and varies considerably among mouse strains. Previous studies in high pathology C3H mice suggested that a strong T cell response was due to the recognition of an immunodominant epitope within the major egg antigen Sm‐p40 (Sm‐p40234–246). Using a panel of T cell hybridomas, we have now examined the egg antigen‐specific TCR repertoire in two high pathology strains, C3H and CBA. We found that nearly half of the hybridomas responded to the Sm‐p40234–246 epitope and, of these, nearly all expressed Vα11.3 associated with Vβ8. Furthermore, in response to egg antigen stimulation, transcript levels of Vα11.3J36 (the most prevalent rearrangement expressed by Sm‐p40234–246‐specific hybridomas), increased in high pathology (CBA) but not in low pathology (BALB/c) strains. Our findings suggest that exacerbated schistosome egg‐induced immunopathology can be driven by T cells expressing a highly restricted TCR structure specific for a single parasite epitope.