Joaquín Sánchez

Polymorphonuclear neutrophils are necessary for the recruitment of CD8+ T cells in the liver in a pregnant mouse model of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection

ABSTRACT The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+cell populations decreased, particularly the CD8+ T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response.

Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

ABSTRACT A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminate Chlamydophila abortusinfections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12−/− and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/− mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/− mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/−infected mice. Both WT and IL-12−/− mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge withC. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/−mice. The lack of IL-12 resulted in few infiltrating CD4+ T cells in the liver relative to the number in WT mice, although the number of CD8+ T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion

Two different lengths of the gene encoding Escherichia coli heat‐stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat‐labile toxin A‐subunit (LTA). The hybrid genes directed expression of chimeric LTA‐STa proteins. Association of these chimeras with native heat‐labile toxin B‐subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.

New approaches to improve a peptide vaccine against porcine Taenia solium cysticercosis.

Cysticercosis caused by Taenia solium frequently affects human health and rustic porciculture. Cysticerci may localize in the central nervous system of humans causing neurocysticercosis, a major health problem in undeveloped countries. Prevalence and intensity of this disease in pigs and humans are related to social factors (poor personal hygiene, low sanitary conditions, rustic rearing of pigs, open fecalism) and possibly to biological factors such as immunity, genetic background, and gender. The indispensable role of pigs as an obligatory intermediate host in the life cycle offers the possibility of interfering with transmission through vaccination of pigs. An effective vaccine based on three synthetic peptides against pig cysticercosis has been successfully developed and proved effective in experimental and field conditions. The well-defined peptides that constitute the cysticercosis vaccine offer the possibility to explore alternative forms of antigen production and delivery systems that may improve the cost/benefit of this and other vaccines. Encouraging results were obtained in attempts to produce large amounts of these peptides and increased its immunogenicity by expression in recombinant filamentous phage (M13), in transgenic plants (carrots and papaya), and associated to bacterial immunogenic carrier proteins.

Lipophosphopeptidoglycan of Entamoeba histolytica Induces an Antiinflammatory Innate Immune Response and Downregulation of Toll-Like Receptor 2 (TLR-2) Gene Expression in Human Monocytes

Introduction Bacterial infection results in activation of the innate im-mune system. The recognition of a pathogen is mediated bya set of germline-encoded receptors that are referred to aspattern-recognition receptors (PRRs) that can directly rec-ognize invariant molecular structures known as pathogen-associated molecular patterns (PAMPs), which are sharedby large groups of microorganisms. One PAMP recognizedby PRRs is lipopolysaccharide (LPS), a mayor componentof the outer membrane of Gram-negative bacteria. LPSstimulates host cells and makes them produce various proin-flammatory cytokines, such as TNF- a , IL-1 a , and IL-6.LPS is captured by LPS-binding protein (LBP), which de-livers to CD14 on the cell surface. Because CD14 lacks anintracellular signaling domain, a transmembrane coreceptorhas been postulated in LPS-responsive cells that transducesthe LPS signaling into the cytoplasm. In Drosophila , theToll receptor participates in establishing the dorsoventralpatterns in the embryo and inducing antifungal immune re-sponse in the adult fly. Toll is a type I transmembrane pro-tein with a cytoplasmic domain with sequence homology tothe interleukin-1 receptor (IL-1R). Activation of Toll leadsto induction of genes through the transcription factor NF-kBpathway (1). Recently, several Toll-like receptors (TLRs)have been identified in macrophages and monocytes, basedon homology to Drosophila protein. Other studies havevariably suggested that TLR-2 and TLR-4 serve as the mainmediators of responses to LPS (2). In addition, TLR-2 mayalso act as a signaling receptor for other common bacterialstructures: peptidoglycan (PGN) and lipoteichoic acid(LTA) from Gram-positive bacteria and lipoarabinomannan(LAM) from Mycobacterium. Lipophosphopeptidoglycan (LPPG) antigens from Ent-amoeba histolytica were described for the first time by Isi-basi et al., and were later used to study its expression underdifferent culture conditions and in different E. histolytica strains using monoclonal antibodies. One antibody againstLPPG was able to inhibit adhesion of amebas and cytotoxic-ity to target cells. Recently, the expression of LPPG wascorrelated to amebic virulence. In addition, the protectionagainst invasive amebiasis by a single monoclonal antibodydirected against LPPG has been studied (3). We studiedLPPG, and found that, due to its polysaccharide nature, thisameba surface molecule behaves like a PAMP. LPPG isprobably recognized by TLR-2, but instead of inducing apro-inflammatory response, it induced production of inter-leukin-10 and downregulation of tlr -2 gene expression. Materials and Methods Isolation of LPPG. Trophozoites from E. histolytica strainHM-1 were cultivated under axenic conditions, and wereused to extract LPPG using the phenol–water method de-scribed by Westphal and Jann and modified by Isibasi et al.Contaminating LPS in LPPG preparations were determinedusing quantitative Limulus lysate assay (BioWhittaker). Inall preparations, 0.020 ng of LPS/ m g of carbohydrate wasdetected. Monocyte isolation and LPPG/LPS stimulation assay. Periph-eral blood mononuclear cells were isolated on Ficoll-paquegradients (Pharmacia LKB Biotecnology) and cultured (5 3 10

B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection

ABSTRACT The resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection is dependent on gamma interferon and CD8+ T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains (μMT). In the absence of B cells, infection with C. abortus leads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. μMT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in μMT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both μMT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor β in the sera of μMT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events of C. abortus primary infection that can protect mice against an exaggerated inflammatory response.

Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin.

Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea. We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA [Sanchez et al.: FEBS Lett. 208 (1986) 194-198]. Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis. Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB). We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits. The resulting LTA::STa/LTB complexes were found in the E. coli periplasm. This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT. The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.

Detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (STa-related) peptides to the catalytic subunit. A potential new strategy to generate immunostimulants for vaccination.

Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives. Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed. Using a two-plasmid system these derivatives were co-expressed inVibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs). Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT. The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA. This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations. Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees. Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity. These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT. The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.

SYNTHESIS IN VIBRIO CHOLERAE AND SECRETION OF HEPATITIS B VIRUS ANTIGENS FUSED TO ESCHERICHIA COLI HEAT-LABILE ENTEROTOXIN SUBUNIT B

A simple and effective electroporation method for the transformation of Vibrio cholerae with nonmobilizable plasmids is described. Expression plasmids directing the synthesis of fusion proteins with the subunit B of Escherichia coli heat-labile enterotoxin B (LT-B) were transformed into nontoxinogenic V. cholerae vaccine strains. A protein consisting of two overlapping immunodominant antibody-binding sites of the hepatitis B virus (HBV) middle surface antigen fused to the C terminus of full-length LT-B was secreted into the supernatant of V. cholerae cultures, whereas two other LT-B/HBV fusion proteins were mostly retained within the cells or rapidly degraded in the culture supernatant. While the secretion of fusion proteins with cholera toxin subunit B (CT-B) from V. cholerae has been described, this is to our knowledge the first report describing extracellular secretion of defined foreign epitopes fused to LT-B in V. cholerae. The fusion of guest epitopes to LT-B or CT-B and secretion in V. cholerae could be an interesting system to rapidly produce pure fusion proteins for immunisation, functional studies or diagnostic procedures. An LT-B/pre-S2 fusion protein purified from the supernatant of recombinant V. cholerae induced serum IgG antibodies against LT-B and against the HBV middle surface antigen in mice after parenteral and oral immunisation.

Expression of Cholera Toxin under Non-AKI Conditions in Vibrio cholerae El Tor Induced by Increasing the Exposed Surface of Cultures

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.