E Agsteribbe

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.

Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides

Several studies have shown that immunization with heat shock proteins (HSPs) purified from tumors of virus-infected cells induces specific cytotoxic T-cell (CTL) activity. This immune response is directed against peptides bound to the HSPs rather than against the HSPs themselves. The peptides are derived from tumor- or virus-specific proteins which are degraded in the course of normal protein turnover and processing for presentation by MHC class I molecules. The HSPs appear to function as carriers for the antigenic peptides. Upon immunization they ensure their uptake by specialized macrophages and their introduction into the MHC class I presentation route which is otherwise accessible only for intracellular proteins. Using influenza virus nucleoprotein (NP) as a model antigen, we have tested whether an HSP-based vaccine can be preduced by overexpressing an antigen in cultured cells prior to purification of the HSPs. The transfection system based on the Semliki Forest virus (SFV) replicon was employed to achieve high expression of NP. Since SFV-mediated transfection of murine cells was inefficient we used the hamster-derived cell line BHK21, which can be transfected with 100% efficiency, as a source for NP peptide-loaded gp96. The protein was purified from transfected cells and used for first vaccination studies. The hamster gp96 preparation was well tolerated in mice, an antibody response against the foreign protein was not observed. Preliminary results suggest that a cellular immune response against NP was indeed induced. SFV transfection is applicable for any known antigen and is therefore considered to be an elegant means for the production of HSP-based vaccines capable of inducing a cellular immune response.

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.

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.

Circular DNA from mitochondria of Neurospora crassa.

Abstract Isolated mitochondria from two wild-type strains of Neurospora crassa were disrupted by osmotic shock in order to release mitochondrial DNA. In the electron microscope this DNA appeared to consist for a large part of circular molecules, most of which showed the typical supertwisted aspect, characteristic for closed circular duplex DNA. Strain 5256 contained circular molecules of 19 μm, one circle of 5.7 μm was found. Strain 5297 contained circular molecules of 19 μm as well as many small circles ranging in size from 0.5 to 7 μm. One circular molecule of 39 μm was found. The number of crossovers for molecules of both strains is about 7 per μm.

The role of ADP-ribosylation and G M1 -binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin

The mucosal route of vaccination has attracted a great deal of attention recently. Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S‐IgA antibodies in addition to systemic IgG antibodies. These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces. Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof. For the induction of a mucosal immune response, the vaccine needs to be applied locally. However, local vaccination with non‐replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included. The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat‐labile enterotoxin (LT). Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations. In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune‐stimulatory properties.

Decreased synthesis and inefficient mitochondrial import of hsp60 in a patient with a mitochondrial encephalomyopathy

In a recent paper (Agsteribbe et al. (1993) Biochem. Biophys. Res. Commun. 193, 146-154) we suggested deficiency of heat shock protein 60 (hsp60) as the possible cause of a systemic mitochondrial encephalomyopathy with multiple deficiency of mitochondrial enzymes. In this paper we present new data which strongly support this hypothesis. Hsp60 deficiency appeared to be not a common side effect of impaired mitochondrial metabolism as eight out of ten fibroblast cultures from patients with systemic mitochondrial myopathy contained normal quantities of the protein. The low steady state amount of hsp60 in the fibroblasts of our patient is caused by decreased synthesis of the protein and not by its enhanced degradation indicating that the hsp60 deficiency is indeed a primary defect. Processing of hsp60 but not of other mitochondrial proteins is markedly retarded in the patient cells. Other functional properties of the patient hsp60 like the assembly of hsp60 monomers to the native 14mer complex and the affinity of this complex to denatured protein are not impaired. Our results underline that a primary defect in hsp60 synthesis and/or processing causing a low steady state amount of hsp60 is the molecular basis of this mitochondrial disorder. The presented data provide for the first time substantial evidence that deficiency of a heat shock protein can give rise to pathological conditions in man.

Mucosal immunogenicity of the Escherichia coli heat-labile enterotoxin: role of the A subunit

The Escherichia coli heat-labile enterotoxin (LT) is a potent mucosal immunogen, inducing high secretory as well as systemic antibody responses upon oral or intranasal (i.n.) administration. In addition, LT has the capacity to act as an adjuvant in antibody responses against coadministered other antigens. To investigate the role of the individual subunits of LT in the mucosal immunogenicity and adjuvanticity of LT, the LT holotoxin and the non-toxic B subunit (LTB) were cloned separately and purified from overproducing E. coli cultures. Mice were immunized i.n. with the recombinant LT, LTB and combinations of the two and the induction of LTB-specific serum IgG and IgA as well as mucosal S-IgA was monitored. Intranasal administration of 2 micrograms LTB by itself induced a moderate systemic and a low mucosal antibody response, the latter being restricted to the site of immunization. However, addition of a trace amount (50 ng) of LT holotoxin to LTB strongly stimulated both serum antibody and mucosal S-IgA responses to LTB: the antibody levels induced by 2 micrograms LTB supplemented with 50 ng LT were similar to those seen after immunization with 2.9 micrograms of the LT holotoxin alone (representing an amount of 2 micrograms LTB). Furthermore, immunization with LT-supplemented LTB or with LT holotoxin alone, but not immunization with LTB alone, induced an S-IgA response in distant mucosal tissues including the lung, intestine and the urogenital system. Nicking of the LTA chain with trypsin did not enhance the immunogenicity of LT. These results indicate that, although the LTA chain plays an important role in the mucosal immunogenicity of LT including priming of the common mucosal immune system, extremely low amounts of the LT holotoxin suffice for the induction of high antibody responses to LTB, the trace LT and LTB acting in a synergistic fashion.

Morphology of the mitochondria in heat shock protein 60 deficient fibroblasts from mitochondrial myopathy patients. Effects of stress conditions.

We have described two mitochondrial (mt) myopathy patients with reduced activities of various mt enzymes associated with significantly decreased amounts of heat shock protein 60 (hsp60). Experimental evidence suggested that the lack of hsp60 was the primary defect. Since hsp60 is essential for the proper folding of enzyme subunits in the mt matrix a partial deficiency of this protein can explain the observed defects of the mitochondria. Here we report on morphological studies aimed at obtaining more insight into the relation between lack of hsp60 and pathological changes of the mitochondria. Under standard culture conditions mitochondria in the partially hsp60 deficient fibroblasts showed profound morphological aberrations. In contrast, the mitochondria in fibroblasts from a MELAS patient and a cytochrome c oxidase-deficient patient appeared normal. Under stress conditions the integrity of the hsp60 deficient mitochondria declined even further: heat shock induced a temporary collapse of the electrochemical potential across the inner mt membrane, but did not affect the ultrastructure of the mitochondria; prolonged growth in confluent cultures resulted in decrease in mt number. The altered mt morphology in the hsp60 deficient cells is probably indicative of the severely impaired mt metabolism whereas the decreased stress tolerance is likely to be a direct result of paucity of the heat shock protein. Both variables are potentially useful in the diagnosis and molecular characterization of mt disorders with systemic manifestation and multiple enzyme deficiency.