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Biomedical subjects

M Hofnung

Publications and source records attributed to M Hofnung.

At least 73 records · Page 4Linked to original sources
PubMed1994

Molecular and cellular parameters controlling the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

Extensive work is being performed to develop live recombinant bacterial vaccines. The use of non-pathogenic bacteria or attenuated strains derived from pathogens may allow protection against the pathogen and at the same time induce immunity against one or several foreign antigens expressed by the recombinant micro-organism. Several bacteria such as attenuated Salmonella or BCG have been used successfully in several experimental models to induce protective immune responses against several pathogens. However, the presentation to the immune system of a foreign antigen in a context different from the natural one may greatly modify the characteristics of elicited immune responses. It is therefore of the utmost importance to establish rules concerning the influence of the vector on the immunogenicity of recombinant antigens. Using a bacterial system that allows the expression of genetically engineered hybrid proteins, we have analysed: 1) the role of the molecular environment on the immunogenicity of foreign B- or T-cell epitopes; 2) the influence of the cellular location of a foreign B-cell epitope on the induction of specific immune responses; 3) the role of the bacterial vector (E. coli or Salmonella typhimurium) on the isotypic characteristic of antibody responses induced against the recombinant antigen. These studies revealed the complexity of the mechanisms which control the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

ATP-Binding Cassette Transporters↗
PubMed1993

Location of tolerated insertions/deletions in the structure of the maltose binding protein.

In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the maltose binding protein for which the maltose binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within alpha helices allowed an interpretation regarding protein structure accommodation to such modifications.

ATP-Binding Cassette Transporters↗
PubMed1993

Induction of T cell responses by chimeric bacterial proteins expressing several copies of a viral T cell epitope.

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120-132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132-145) were able to induce strong peptide-specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen-presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide-specific T cell response with a lower dose of priming antigen.

ATP-Binding Cassette Transporters↗
PubMed1993

Immunodominance of a recombinant T-cell epitope depends on its molecular environment.

In the present study, we have investigated the influence of the molecular environment of a T-cell epitope on its immunogenicity. We genetically inserted into different sites of two bacterial recipient proteins, LamB or MalE, an immunodominant T-cell epitope: the 120-132 T-cell epitope from the PreS2 region of HBV. The T-cell epitope was introduced, either alone (PreS:T) or with an adjacent B-cell epitope (PreS:TB). After purification, the hybrid proteins were injected into mice and we studied the immunogenicity of recombinant T-cell epitopes by analyzing the in vitro proliferative responses of LN cells from these mice to the inserted peptides. The immunization of mice with recombinant MalE protein containing the PreS:T or PreS:TB peptides at two different sites induced strong peptide-specific proliferative responses, indicating that the insertion sites did not affect the immunodominance of the inserted T-cell epitope. A strong T-cell proliferative response was also obtained after immunization of mice with hybrid LamB protein containing the PreS:TB epitope at position 153. In contrast, the recombinant proteins which contained only the PreS:T epitope at positions 153 or 374 failed to stimulate T-cell responses. Therefore, this study demonstrates that the immunogenicity of recombinant T-cell epitopes may be strongly affected both by the insertion site and by inserted adjacent residues.

Amino Acid Sequence↗
PubMed1993

The SOS chromotest: a review.

The SOS chromotest is reviewed through over 100 publications corresponding to the testing of 751 chemicals. 404 (54%) of these chemicals present a genotoxic activity detectable in the SOS chromotest. Their SOS inducing potencies span more than 8 orders of magnitude. For 452 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 373 (82%) of these compounds give similar responses in both tests (236 positive and 137 negative responses). Thus the discrepancies between both tests concern 79 compounds (18%). A case by case analysis shows that many of these compounds are at the same time very weak SOS inducers and very weak mutagens. Thus we think that, most of the time, the discrepancies between the two tests may be accounted for by differences in the interpretation of the results rather than by the experimental results themselves. However, there are some compounds which are clearly SOS inducers but devoid of mutagenic activity in the Ames test (such as quinoline-1-oxide) and to a larger extent, clearly mutagenic compounds which do not induce the SOS response in the SOS chromotest (such as benzidine, cyclophosphamide, acridines, ethidium bromide). We also analyzed the correlation between SOS induction, mutagenesis and carcinogenesis according to the classification of Lewis. For 65 confirmed carcinogens (class 1), the sensitivity, i.e., the capacity to identify carcinogens, was 62% with the SOS chromotest and 77% with the Ames test. For 44 suspected carcinogens (class 2), the sensitivity was 66% with the SOS chromotest and 68% with the Ames test. Thus, we confirmed previous observations made on 83 compounds that there is a close correlation between the results given by both bacterial tests. The capacity of the Ames test to identify carcinogens is higher than that of the SOS chromotest. However, because the number of false positive compounds was lower in the SOS chromotest, the specificity, i.e., the capacity to discriminate between carcinogens and non-carcinogens of the SOS chromotest, appeared higher than that of the Ames test. Thus, the results of the SOS chromotest and of the Ames test can complement each other. The SOS chromotest is one of the most rapid and simple short-term test for genotoxins and is easily adaptable to various conditions, so that it could be used as an early--perhaps the earliest--test in a battery.

Animals↗
PubMed1993

Immune responses to hybrid maltose-binding proteins.

The Escherichia coli maltose-binding protein is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at 'permissive sites' within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303. The hybrid proteins are easily purifiable and immunogenic, inducing peptide-specific B- and T-cell responses. When delivered by live bacteria (E. coli K12 and aroA Salmonella typhimurium) antibody responses can be induced against both the MalE carrier and the inserted B-cell epitope. We discuss the induction of T-cell responses by bacterial delivery systems.

ATP-Binding Cassette Transporters↗
PubMed1993

Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins.

A peptide comprising residues glu293 to ser334 from the principal neutralization determinant (V3 loop) of the envelope of human immunodeficiency virus type 1 (HIV1 LAVBRU isolate) has been inserted within internal permissive sites of either LamB or MalE, two envelope proteins from Escherichia coli K12. The MalE hybrid protein (MalE133-V3 loop) was stably expressed in the periplasm of Escherichia coli K12, and the V3 loop peptide was detectable on the surface of the native protein by an anti-gp160 monoclonal antibody (mAb 110-A). The disulfide bridge between the two cysteines of the loop was formed. In contrast, genetic coupling to the outer membrane protein LamB did not allow the expression of a stable hybrid protein, and major proteolytic cleavage products of the LamB153-V3 loop were detected by mAb 110-A. The two plasmid-encoded hybrid genes were transferred to an aroA mutant of Salmonella typhimurium. Constitutive expression of the MalE133-V3 loop had no detectable effect on cell growth and on the survival in vivo of the recipient strain. The LamB153-V3 loop was not stably expressed in Salmonella, either in vitro or in vivo. Live recombinant salmonellas expressing MalE-V3 and LamB-V3 loop hybrids were used to immunize mice. The MalE-V3 loop hybrid induced anti-HIV1 envelope antibodies detectable by Western blot and ELISA, while the anti-HIV1 envelope antibodies induced by the LamB-V3 loop hybrid were only detectable by Western blot. In addition, purified MalE-V3 loop hybrid protein was able to stimulate in vitro and induce in vivo a V3 loop-specific T-cell proliferative response.

AIDS Vaccines↗
PubMed1993

Determination of target nucleotides involved in 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000)-DNA adduct formation.

The characterization of target nucleotides involved in the binding to DNA of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic nitrofuran derivative, was investigated. Since R7000 undergoes metabolic activation prior to interacting with DNA, plasmids containing AT-rich and GC-rich sequences were devised and treated by R7000 in bacterial cells presenting nitroreductase activity. The nucleotide modifications to these homogeneous fragments that resulted from R7000 treatment were analyzed using the 'postlabeling' method. A preferential binding to the GC segment was demonstrated. Using a modification of the Maxam-Gilbert sequencing technique, it was demonstrated that activated R7000 creates alkali-labile phosphodiester bonds at the positions of guanines. In addition, the analysis of DNA replication-blocking properties of R7000 lesions was performed using avian myeloblastosis virus (AMV) reverse transcriptase as DNA polymerase. The termination of DNA replication occurred preferentially at the sites of guanine residues in the template strand, indicating that one nucleotide was inserted opposite a lesion. All these results indicate that guanine residues are the preferential sites of formation of R7000-DNA adducts.

Adenosine Triphosphate↗
PubMed1993

Immunological relatedness of the LamB proteins among members of Enterobacteriaceae.

We have studied the immunological relatedness of LamB proteins from a wide range of enterobacterial species, using antibodies directed against denatured Escherichia coli K12 and Klebsiella pneumoniae LamB proteins (LamBE.c. and LamBK.p., respectively), and anti-peptide antibodies directed against 10 distinct loops of LamB from E. coli K12 predicted to protrude either side of the outer membrane. We have shown that a protein immunologically related to LamBE.c. and LamBK.p. was present in all members of Enterobacteriaceae tested. A protein recognized by several anti-peptide antibodies was identified in E. coli, Shigella sonnei, Salmonella typhimurium and Kleb. pneumoniae, as well as in two Citrobacter species, two Enterobacter species and Kluyvera ascorbata. The recognition patterns obtained with the anti-peptide antibodies were in agreement with the LamB protein sequence data available. They indicated that the cell surface and also the periplasmic loops of LamB are subject to great antigenic variability.

Amino Acid Sequence↗
PubMed1993

Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella.

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.

Base Sequence↗
PubMed1992

Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes.

We previously determined a number of 'permissive' sites in the periplasmic maltose-binding protein (MalE) from Escherichia coli. These sites accept the insertion of heterologous peptides without major deleterious consequences for the activities, structure and cellular location of the protein. This study explores the versatility of two such permissive sites for the synthesis of foreign peptides, and examines the antigenicity and the immunogenicity of the inserts. One site is located after amino acid 133 (aa133) of MalE, and the other after aa303. Both sites tolerate inserts of up to at least 70 aa and accept sequences of different natures. Hydrophobic aa sequences are accepted, although strongly hydrophobic sequences, such as the Sendai virus F protein membrane anchor, affected export. We compared the antigenic and the immunogenic properties of peptides derived from the coat proteins of HBV and poliovirus which contain well defined B-cell epitopes. Specific monoclonal antibodies show that the antigenic properties of the inserted B-cell epitopes were different at the two sites. Despite these differences, the inserted peptides elicited strong and comparable antibody responses in mice against the corresponding synthetic peptides. In this case, and with these criteria, the molecular context of the peptides did not affect the immunogenicity of B-cell epitopes. We show for the first time that when a foreign peptide carrying a T-cell epitope was inserted in MalE, the hybrid proteins can elicit a T-cell response against the foreign peptide both in vivo and in vitro. Furthermore, the MalE hybrid was as efficient as free peptide in stimulating T-cell hybridomas in vitro. The MalE vectors provide a powerful genetic system to study how the position and the conformation of a peptide within a protein affect the B-cell and T-cell responses.

ATP-Binding Cassette Transporters↗
PubMed1992

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145-167, 173-187, 197-226, 237-300, 311-329, and 367-387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.

ATP-Binding Cassette Transporters↗
PubMed1991

Palindromic units are part of a new bacterial interspersed mosaic element (BIME).

Palindromic Units (PU or REP) were defined as DNA sequences of 40 nucleotides highly repeated on the genome of Escherichia coli and other Enterobacteriaceae. PU are found in clusters of up to six occurrences always localized in extragenic regions. By sorting the DNA sequences of the known PU containing regions into different classes, we show here for the first time that, besides the PU themselves, each PU clusters contains a number of other conserved sequence motifs. Seven such motifs were identified with the present list of PU regions. Remarkably, each PU cluster is exclusively composed of a mosaic combination of PU and of these other sequence motifs. We demonstrate directly by hybridization experiments that one of these motifs (called L) is indeed present at a large number of copies on the Escherichia coli chromosome and that its distribution follows the same species specificity as PU sequences themselves. We propose that the mosaic pattern of motif combination in PU clusters reveals a new type of bacterial genetic element which we propose to call BIME for Bacterial Interspersed Mosaic Element. The Escherichia coli genome contains about 500 BIME.

Base Sequence↗
PubMed1991

First characterization of the mutagenic specificity of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) in Salmonella typhimurium.

In order to investigate the mutational events induced by the very potent genotoxic agent 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), we evaluated its mutagenic potency in a battery of his- mutants of Salmonella typhimurium designed to study mutagenic specificity (Levin and Ames, 1986). Using this system we could show that R7000 is able to induce base-pair substitutions, mainly GC----TA transversions and, perhaps to a lower extent, AT----TA transversions. In addition, our results suggest that this chemical may be a very efficient inducer of base-pair deletions/insertions resulting in frameshifts. Whereas the induction of base-pair substitutions by R7000 is dependent on the presence of plasmid pKM101 carrying the mucA,B operon, the induction of base-pair deletions/insertions is independent of the presence of the plasmid.

Alanine↗