Marijke Holtrop

Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Musosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: Induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered uni elated antigens and is therefore of potential inter-est for development of mucosal vaccines. However; despite the great demand for such mucosal vaccines, the use of LT holotoxin us an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity and a pentamer of identical B subunits, which mediates binding to ganglioside G(M1), the cellular receptor for the toxin. In this paper; we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself; retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However; supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT rLTB in particular; represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases

Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat‐labile enterotoxin and its B subunit

Escherichia coli (E. coli ) heat‐labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co‐administered antigens. LT is composed of one copy of the A subunit, which has ADP‐ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP‐ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP‐ribosylation activity, GM1‐binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1‐binding activity is essential for effective induction of anti‐LTB antibodies. On the other hand, an LT mutant lacking ADP‐ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1‐binding activity, adjuvanticity of LT was found to be independent of GM1‐binding affinity. Moreover, a mutant lacking both GM1‐binding and ADP‐ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP‐ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP‐ribosylation‐independent adjuvant effect of the A subunit.

Genetic immunization against cervical carcinoma

Infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPVs depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6 and E7 is selectively maintained in premalignant and malignant cervical lesions these proteins are attractive candidates for immunotherapeutic and prophylactic strategies. This report describes the construction, characterization and the in vivo immunotherapeutic potential of recombinant Semliki Forest virus (SFV) expressing the HPV16 E6 and E7 proteins (SFV-E6E7). Western blot analysis and immunofluorescence staining demonstrated expression of E6 and E7 in BHK cells infected with SFV-E6E7. Immunization of mice with SFV-E6E7 resulted in an efficient in vivo priming of HPV-specific CTL activity. The induced CTL lysed murine tumor cells transformed with the HPV16 genome and EL4 cells loaded with an immunodominant class I-binding HPV E7 peptide. CTLs could reproducably be induced by immunization with three injections of as few as 105 infectious units of SFV-E6E7. Protection from tumor challenge was studied using the tumor cell line TC-1. Immunization with 5 × 106 SFV-E6E7 particles protected 40% of the mice from tumor challenge. These results indicate that E6E7 expression by the efficient and safe recombinant SFV system represents a promising strategy for immunotherapy or immunoprophylaxis of cervical carcinoma.

Immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus 16 E6 and E7

We are developing immunization strategies against cervical carcinoma and premalignant disease, based on the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPV). Thus far, protein-based, as well as genetic immunization studies have demonstrated low to moderate cellular immune responses against E6 and E7. To improve these responses, we modified the structure and expression level of the E6 and E7 proteins produced by the SFV vector. Specifically, a construct was generated encoding a fusion protein of E6 and E7, while furthermore a translational enhancer was included (enhE6,7). Infection of cells with recombinant SFV-enhE6,7 resulted in the production of large amounts of the E6,7 fusion protein. The fusion protein was more stable than either one of the separate proteins. Immunization of mice with SFV-enhE6,7 resulted in strong, long-lasting HPV-specific cytotoxic T lymphocyte responses. Tumor challenge experiments in mice demonstrated that immunization with SFV-enhE6,7 resulted in prevention of tumor outgrowth and subsequent protection against tumor re-challenge.

Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering RNAs

Correction to: Gene Therapy (2006) 13, 400–411. doi:10.1038/sj.gt.3302673 In the Materials and methods section, the paper states erroneously that an amount of 0.51 μmol cationic lipid was used for the preparation of virosomes from 1.5 μmol viral phospholipid. The correct amount is 0.77 μmol cationiclipid corresponding to 34 mol% in the final lipid mixture.

Regulation of the expression of mitochondrial proteins : relationship between mtDNA copy number and cytochrome-c oxidase activity in human cells and tissues

The relationship between the relative amounts of nuclear and mitochondrial genes for cytochrome-c oxidase subunits and their transcripts and cytochrome-c oxidase activity was investigated in several human tissues and cell lines to get more insight into the regulation of the expression of this mitochondrial enzyme complex. The results show: (1) a wide range of mtDNA copy numbers; (2) constant ratios between the steady-state levels of the transcripts for the various cytochrome-c oxidase subunits, and (3) large variations in cytochrome-c oxidase activity in different tissues and cell lines that could not be related to the differences in mtDNA copy number. We conclude that the transcription of genes for both mitochondrial and nuclear cytochrome-c oxidase subunits is regulated coordinatedly, but also that the mtDNA copy number plays a minor role in determining differences in cytochrome-c oxidase activity between different cell and tissue types.

The mitochondrial genetic system as a target for chemotherapy: Tetracyclines as cytostatics

The mitochondrial genetic system is indispensable for the biosynthesis of the enzyme complexes involved in aerobic energy generation. Tetracyclines inhibit the expression of only the mitochondrial genes because they specifically block mitochondrial protein synthesis. A salient feature is that this inhibition occurs at the low concentration required for anti-bacterial treatment, provided that this concentration is maintained continuously. Evidence is presented that the growth of carcinogen-induced tumors can be inhibited by tetracyclines. It is further shown that the development in the cheek pouch of the Syrian hamster of a transplantable hypernephroma from human origin can be strongly retarded by tetracyclines as well. Therefore, the mitochondrial genetic system has to be reckoned as a target for chemotherapy and tetracyclines as cytostatic agents.

Mutational analysis of the role of ADP-ribosylation activity and GM1-binding activity in the adjuvant properties of the Escherichia coli heat-labile enterotoxin towards intranasally administered keyhole limpet hemocyanin

The Escherichia coli heat‐labile enterotoxin (LT) is known for its potent mucosal immunoadjuvant activity towards co‐administered antigens. LT is composed of one A subunit, which has ADP‐ribosylation activity, and a homopentameric B subunit, which has high affinity for the toxin receptor, ganglioside GM1 . In previous studies, we have investigated the role of the LTA and LTB subunits in the adjuvanticity of LT towards influenza virus hemagglutinin (HA), administered intranasally to mice. We now studied the adjuvant properties of LT and LT variants towards keyhole limpet hemocyanin (KLH), which, in contrast to HA, does not bind specifically to mucosal surfaces. It is demonstrated that LT mutants without ADP‐ribosylation activity, as well as LTB, retain mucosal immunoadjuvant activity when administered intranasally to mice in conjunction with KLH. As with influenza HA, adjuvanticity of LTB required GM1 ‐binding activity, whereas GM1 ‐binding was not essential for adjuvant activity of LT. Furthermore, we found that also recombinant LTA alone acts as a potent mucosal adjuvant, and that this adjuvanticity is independent of ADP‐ribosylation activity. It is concluded that binding of the antigen to mucosal surfaces does not play an essential role in the immunostimulation by LT and LT variants, and that both recombinant LTA and LTB represent powerful nontoxic mucosal adjuvants.

The restriction fragment map of rat-liver mitochondrial DNA: a reconsideration.

Abstract 1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments of Eco RI, Hind III, Bam HI and Hap II is constructed on the basis of: (a) the analysis of partially restricted fragments; (b) analysis of the double digests of total mtDNA; (c) the digestion of isolated restriction fragments with other restriction endonucleases; (d) the identification of fragments of complete single and double digestions and of partially digested fragments containing the base sequences complementary to the 12-S and 16-S RNAs of rat-liver mitochondrial ribosomes. 3. The genes for the ribosomal RNAs are shown to be closely linked. This result differs from data previously reported (Saccone, C., Pepe, G., Cantatore, P., Terpstra, P. and Kroon, A.M. (1976) in The Genetic Function of Mitochondrial DNA, pp. 27–36, Elsevier/North-Holland Biomedical Press, Amsterdam). 4. The origin of replication (D-loop) is localized in the vicinity of the small ribosomal RNA gene and the direction of replication is distant from this gene. 5. The mitochondrial tRNA genes are scattered over the genome as in other animal mtDNAs. The approximate minimal number of tRNA genes is 16–20. 6. We concluded previously that the Eco RI restriction fragments A and D are not adjacent and failed to show the overlap of the 16 S rRNA gene for the Eco RI fragment D and Hind III fragment A. This misinterpretation was due to the fact that the two smallest Eco RI fragments could not be detected with the methods applied and to the lower specific radioactivity of the ribosomal RNAs used in the first series of hybridization experiments.