The B subunits of cholera and Escherichia coli heat-labile toxins enhance the immune responses in mice orally immunised with a recombinant live P-fimbrial vaccine for avian pathogenic E. coli

This study aimed to investigate the adjuvant effect of recombinant attenuated Salmonella expressing cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) for the P-fimbriae subunit-based vaccine of avian pathogenic E. coli (APEC) in a murine model. The PapA-specific sIgA and IgG responses were significantly enhanced after immunisation with the Salmonella-PapA vaccine in the presence of CTB or LTB. The group immunised with the Salmonella-LTB strain promoted Th1-type immunity, whereas that immunised with the Salmonella-CTB strain produced Th2-type immunity. We concluded that both Salmonella-CTB and -LTB strains can enhance the immune response to PapA, and that the LTB strain may be a more effective adjuvant for APEC vaccination, which requires higher Th1-type immunity for protection. Thus, our findings provide evidence that immunisation with an adjuvant, LTB, is one of the strategies of developing effective vaccines against P-fimbriated APEC

Therapeutic Effects of Anti-CD115 Monoclonal Antibody in Mouse Cancer Models through Dual Inhibition of Tumor-Associated Macrophages and Osteoclasts

Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+) CD163(+) M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+)CD163(+) M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition

Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus

A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermophilus (Streptococcus salivarius subsp. thermophilus). pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned. Random Sau3A fragments from the S. thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E. coli as the host. The pool of recombinant plasmids were introduced into S. thermophilus and Lactococcus lactis subsp. lactis in order to search for promoter activity in these hosts. For S. thermophilus, it was necessary to first select erythromycin-resistant transformants and then to screen for chloramphenicol resistance among these. Direct selection of chloramphenicol-resistant clones was, however, possible in L. lactis subsp. lactis. Six fragments exhibiting promoter activity were characterized in S. thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity. Three of the promoter-carrying fragments were sequenced. The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases. Upstream from these potential transcription start points, sequences homologous to the E. coli sigma 70 and the Bacillus subtilis vegetative sigma 43 (or sigma A) consensus promoters were identified.</jats:p

Conjugative mobilization as an alternative vector delivery system for lactic streptococci

Due to the current variability in applying polyethylene glycol-mediated protoplast transformation to lactic streptococci, a study was undertaken to assess the feasibility of conjugative mobilization as an alternative method for vector delivery. By using the broad-host-range conjugative plasmid pVA797, the partially homologous cloning vector pVA838 was successfully introduced into various strains of Streptococcus lactis, Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, Streptococcus thermophilus, and Streptococcus faecalis. Frequencies ranged from 10(-2) to 10(-6) transconjugants per recipient. Both pVA797 and pVA838 were acquired intact, without alteration in functionality. Also, the shuttle vector pSA3, which shares partial homology with pVA797, was mobilized via conjugation. The use of S. lactis LM2301 as the intermediate donor allowed the use of physiologic and metabolic characteristics for recipient differentiation. The construction of a vector containing a "DNA cassette" conferring mobilization and the resolution, segregation, and stability of the cointegrates, pVA797, pVA838, and pSA3, are also reported.</jats:p

Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826.

Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an alpha-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg l-1

Gene-based vaccines and immunotherapeutics

DNA vaccines, comprised of plasmid DNA encoding proteins from pathogens, allergens, and tumors, are being evaluated as prophylactic vaccines and therapeutic treatments for infectious diseases, allergies, and cancer; plasmids encoding normal human proteins are likewise being tested as vaccines and treatments for autoimmune diseases. Examples of in vivo prophylaxis and immunotherapy, based on different types of immune responses (humoral and cellular), in a variety of disease models and under evaluation in early phase human clinical trials are presented. Viral vectors continue to show better levels of expression than those achieved by DNA plasmid vectors. We have focused our clinical efforts, at this time, on the use of recombinant viral vectors for both vaccine as well as cytokine gene transfer studies. We currently have four clinical programs in cancer immunotherapy. Two nonspecific immunotherapy programs are underway that apply adenoviral vectors for the transfer of cytokine genes into tumors in situ. An adenovirus-IFNγ construct (TG1042) is currently being tested in phase II clinical trials in cutaneous lymphoma. A similar construct, adenovirus-IL2 (TG1024), also injected directly into solid tumors, is currently being tested in patients with solid tumors (about one-half of which are melanoma). Encouraging results are seen in both programs. Two cancer vaccine immunotherapy programs focus on two cancer-associated antigens: human papilloma virus E6 and E7 proteins and the epithelial cancer-associated antigen MUC1. Both are encoded by a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] and both are coexpressed with IL-2. Encouraging results seen in both of these programs are described

Production of cholera toxin B subunit in Lactobacillus

The intracellular expression of the B subunit of cholera toxin (CTB) was first achieved in Lactobacillus paracasei LbTGS1.4 with an expression cassette including the P25 promoter of Streptococcus thermophilus combined with the translation initiation region from the strongly expressed L. pentosus D-lactate dehydrogenase gene (ldhD). Secretion of CTB was next attempted in L. paracasei LbTGS1.4 and L. plantarum NCIMB8826 with four different signal sequences from exported proteins of lactic acid bacteria (Lactococcus lactis Usp45 and PrtP, Enterococcus faecalis unknown protein and S. pyogenes M6 protein). Host-dependent secretion of CTB was clearly observed: whereas none of the secretion cassettes led to detectable CTB in the extracellular fraction of L. paracasei LbTGS1.4, secretion of CTB molecules was clearly achieved with three of the selected signal sequences in L. plantarum NCIMB8826. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved