Yasunobu Matsumoto

Prions prevent neuronal cell-line death

Prion diseases, such as scrapie and bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) in humans, are neurodegenerative conditions characterized by the accumulation of a post-transcriptionally modified, pathological form of a host-encoded glycoprotein, designated PrPSc. The physiological function of the normal cellular isoform, PrPC, is unknown, although studies of mice devoid of PrPC have indicated that it may be involved in normal synaptic function and survival of Purkinje cells, but findings have been inconsistent. We find that serum removal from the cell culture causes apoptosis in Prnp −/− cells (in which a disrupted form of the prion protein is produced) but not in Prnp +/+ (wild-type) cells. Transduction of PrP or the Bcl-2 gene suppressed apoptosis of Prnp −/− cells under serum-free conditions. We also found that Prnp −/− cells extended shorter neurites than Prnp +/+ cells, but expression of PrPC increased their length. These findings support the idea that the loss of function of PrPC may partly underlie the pathogenesis of prion diseases.

Nasal Immunization with a Malaria Transmission-Blocking Vaccine Candidate, Pfs25, Induces Complete Protective Immunity in Mice against Field Isolates of Plasmodium falciparum

ABSTRACT Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.

Mice Intranasally Immunized with a Recombinant 16-Kilodalton Antigen from Roundworm Ascaris Parasites Are Protected against Larval Migration of Ascaris suum

ABSTRACT Protective immunity to the pig roundworm, Ascaris suum, has been demonstrated by immunization of pigs with antigens derived from the parasites larval stages. We identified a protective antigen commonly expressed in the human and pig Ascaris infections as a 16-kDa protein (As16), which has no similarity at the amino acid level to mammalian proteins but has some similarity to those of the filarial parasites and Caenorhabditis elegans gene product. Localization analysis revealed that the native As16 was highly expressed in the adult worm intestine, hypodermis, and cuticles. In addition, As16 was detected in the parasite excretory and secretory products. Mice intranasally vaccinated with Escherichia coli-expressed recombinant As16 (rAs16), coupled with cholera toxin B subunit, generated a significant increase in the level of rAs16-specific immunoglobulin G (IgG) and IgE in serum. Mucosal IgA levels were also increased. The recombinant protein evoked a mixed (both Th1 and Th2) type of immune response characterized by elevated levels of gamma interferon and interleukin-10 in the culture supernatants of activated spleen cells. An increased level of IgG1 and IgG2a in serum was also observed. The vaccinated mice showed a reduction by 58% in the recovery of challenged larvae compared to a nonvaccinated control. These results suggest the possibility of developing a mucosal vaccine for human and pig ascariasis.

Immune Responses against a Single CD8+-T-Cell Epitope Induced by Virus Vector Vaccination Can Successfully Control Trypanosoma cruzi Infection

ABSTRACT In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFκB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.

Intranasal immunization with recombinant Ascaris suum 14-kilodalton antigen coupled with cholera toxin B subunit induces protective immunity to A. suum infection in mice

ABSTRACT Animals can be rendered immune to Ascaris parasites by immunization with infectious-stage larvae. The specific parasite gene products that mediate protective responses in ascariasis are unknown. We have identified a cDNA encoding Ascaris suum 14-kDa antigen (As14) and evaluated the vaccinal effect of theEscherichia coli-expressed recombinant protein (rAs14). GenBank analysis showed that As14 has low similarity at the amino acid level to a Caenorhabditis elegans gene product and to antigens of the filarial nematodes but not to other known proteins. In addition, As14 homologues were found to be expressed in human and dog roundworms. In mice that received intranasal administration of rAs14 coupled with cholera toxin B subunit (rAs14-CTB), there was a 64% reduction of recovery of larvae compared with that in the nontreated group. The vaccinated mice showed a significant increase in the total serum immunoglobulin G (IgG) levels and the mucosal IgA responses. Elevation of the rAs14-specific IgE response was also seen. Measurement of the IgG subclasses showed a higher level of IgG1 and a lower level of IgG2a antibody response in the sera of the immunized mice, suggesting that protection was associated with a type II immune response. As14 is the first protective antigen against A. suum infection to be identified. Our immunization trial results in laboratory animals suggest the possibility of developing a mucosal vaccine for parasitic diseases caused by ascarid nematodes.

Recombinant Ascaris 16-Kilodalton Protein-Induced Protection against Ascaris suum Larval Migration after Intranasal Vaccination in Pigs

We recently cloned a protective antigen that is commonly expressed in Ascaris species that infect humans and pigs. We evaluated the vaccinal effects of this 16-kilodalton protein (As16) in pigs, the natural host of Ascaris suum, by intranasal immunization. Pigs that received Escherichia coli-expressed recombinant As16 (rAs16) coupled with cholera toxin (CT) had significantly elevated levels of rAs16-specific serum immunoglobulin G (IgG) and mucosal-associated IgA antibodies. rAs16 evoked a type II immune response characterized by elevated levels of interleukin-4 and -10 in the culture supernatants of peripheral blood mononuclear cells of the vaccinated pigs. An increased level of rAs16-specific serum IgG1 was also detected. Pigs vaccinated with rAs16-CT were protected from migration of A. suum larvae through the lungs, as indicated by a 58% reduction in the recovery of lung-stage third-stage larvae (L3), compared with that in nonvaccinated controls. Purified immunoglobulin from rAs16-CT-vaccinated pigs inhibited survival of infective L3 and interrupted the molting of lung-stage L3. Immunofluorescence studies revealed that this immunoglobulin bound to the digestive tracts of L3, suggesting that it might inactivate functions of the gut tissues of Ascaris species. We conclude that rAs16 is a promising mucosal vaccine candidate for pig and human ascariasis.

Detection of enhancer repeats in the long terminal repeats of feline leukemia viruses from cats with spontaneous neoplastic and nonneoplastic diseases

Enhancer duplication in the long terminal repeat of feline leukemia virus (FeLV) was examined in primary cells from naturally FeLV-infected cats with various neoplastic and nonneoplastic diseases using the polymerase chain reaction. In all cases, a 170-bp band, corresponding to a standard exogenous FeLV with one copy of enhancer, was detected. Repeated enhancer sequences were found in all 8 cases of thymic-form lymphosarcoma, in some cases of lymphosarcoma of other forms (3/8) and myeloid tumors (2/3), and in only 1 of 6 cases with nonneoplastic diseases. The copy number of FeLV proviruses with a repeated enhancer seemed higher than that of those with one copy of enhancer in 3 cases of thymic form lymphosarcoma. In 5 cases of thymic form lymphosarcoma and in 1 case of erythroleukemia, coexistent FeLVs with double and triple enhancers of different sizes were found. Of the enhancer elements, only the SV40 core binding site was found in all the enhancer direct repeats of these FeLVs. All the provirus clones with single and duplicated enhancer sequences from a single tumor showed mutations or deletions characteristic to that tumor, indicating that enhancer repeats may arise in individual animals after infection with a single virus clone. The present findings indicate that FeLV with enhancer repeats generated in the cat is associated with the induction of neoplastic diseases in natural conditions.

Analysis of PrPc mRNA by in situ Hybridization in Brain, Placenta, Uterus and Testis of Rats

An amyloid-like isoform of a 33- to 34-kD glycoprotein, termed as the scrapie prion protein (PrPsc), plays a critical role in transmissible spongiform encephalopathies of animals and humans. It has even been suggested to present the responsible infectious agent. This protein is a posttranslationally modified form of the cellular isoform of prion protein (PrPc). Hitherto, little has been known about the functions of PrPc. In order to examine the localization of PrPc mRNA in rat tissues, the in situ hybridization technique was performed. In rat brain, PrPc mRNA was predominantly localized within pyramidal cells of the hippocampus, large neurons of the thalamus and neocortex, and Purkinje cells of the cerebellum. In the placenta, not only PrPc mRNA was localized to a subpopulation of decidual cells at the highest levels, it was also expressed in the amnion and mesodermal layer of the yolk sac. Furthermore, PrPc mRNA was also expressed in the myometrium of the uterus and seminiferous tubule in the testis. However, signals were not obtained in the lung, spleen, liver of prenatals and other fetus tissues. The distribution of rat PrPc mRNA portrayed the levels which were different among the various types of cells, suggesting that its expression may be regulated in a tissue-specific manner.

Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes.

Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control. To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT). Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response). Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes. The observed transmission-blocking effect was dependent on immune sera dilution. This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria.

Cloning and characterisation of a highly immunoreactive 37 kDa antigen with multi-immunoglobulin domains from the swine roundworm Ascaris suum

Antigens from larval stages of Ascaris suum have been shown to induce protection against challenge infection with infective A. suum eggs. We previously identified several antigens that reacted strongly with serum from pigs inoculated with infective eggs containing L3. In this study, we isolated an antigen with a molecular mass of 37 kDa and a pI of 4.8 (As37) from A. suum infective eggs using two-dimensional electrophoresis, and obtained a full-length cDNA by reverse transcription-polymerase chain reaction using primers designed based on the internal amino acid sequence of As37. The cDNA sequence consisted of 1,540 bp coding for a protein of 321 amino acids with a complex domain organisation. Simple modular architecture research tool (SMART) analysis indicated that As37 contains three immunoglobulin domains, indicating that it is a member of immunoglobulin superfamily (IgSF). A homology search of GenBank showed that As37 has significant similarity to Caenorhabditis elegans DIM-1 protein and has low similarity to part of the multi-repeat Ig domain from nematode twitchin and mammalian skeleton muscle titin, and to members of the IgSF at the amino acid sequence level. Localisation analysis revealed that antibodies to Escherichia coli-expressed recombinant As37 (rAs37) bound to muscle cells and the hypodermis. The antibodies identified a 37 kDa native antigen in human and dog roundworms, suggesting that there are As37 homologues in ascarid nematodes. Sera from mice, rabbits and pigs immunised with A. suum infective eggs reacted with rAs37 in immunoblot analyses. The potential use of rAs37 for protection against A. suum infection is discussed.