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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 the Escherichia 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.

Protective immunity to the pig roundworm, Ascaris suum, has been demonstrated by immunization of pigs with antigens derived from the parasite's 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.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho‐phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli‐expressed recombinant enzyme has a specific activity of 937 µmol Pi·min−1·mg−1 protein corresponding to a kcat value of 638 s−1 at 55 °C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose‐dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble‐ and membrane‐bound H+‐PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.

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.

Bovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission.

How homochiral amino acids and sugars arose out of a presumably prochiral prebiotic environment is a puzzling question, [1] to which many answers have been proposed: for example, absolute asymmetric synthesis with circularly polarized light, photoreactions in chiral crystals, and asymmetric automultiplication with asymmetric autocatalysis, as exemplified by the recent work of Soai et al. [2] There are reports describing a connection between the chirality of amino acids and sugars: Amplification of ee was observed in the aaminoxylation of an aldehyde using proline as catalyst to generate a key intermediate of sugars, [3] while amino acids [5] Herein, we demonstrate experimentally a connection between an amino acid with low ee (10 % ee) and a chiral sugar intermediate of high enantiomeric purity (96 % ee).Amino acids promote several organic transformations, and proline, a widely distributed amino acid, is one of the most effective organic catalysts.[6] In 1971, Hajos and Parrish, as well as Eder et al., reported an intramolecular aldol reaction catalyzed by proline. [7] Following the observation of the intermolecular version of this reaction by List, Lerner, and Barbas in 2000, [8] other highly enantioselective catalytic reactions using proline have been developed, including aldol [9] and Mannich [10] reactions, and a-amination [11] and aaminoxylation [12] of carbonyl compounds. We have observed that a solution of proline with high ee can be obtained from solid proline of low optical purity during the dissolution process. As proline is only very sparingly soluble in pure CHCl 3 , and as the addition of EtOH increases its solubility therein, we employed CHCl 3 containing 1 % EtOH as solvent. CHCl 3 stabilized with amylene (not with EtOH) was distilled from CaH 2 before use, and then EtOH was added (1 %). At first, reproducibility of the solubility was poor. After several experiments, both the surface and the particle size of the solid proline were found to be important. Proline that had been recrystallized from EtOH and ground with a mortar under an Ar atmosphere was employed.The experiment using 10 % ee l-rich proline was performed as follows: CHCl 3 (20 mL) containing 1 % EtOH was added to a mixture of l-proline (550 mg) and d-proline (450 mg; 10 % ee combined) at 0 8C, and the suspension was stirred for 24 h at this temperature under an Ar atmosphere. After filtration of the insoluble proline, a solution (17-19 mL) containing proline (40-65 mg) was obtained, the ee value of which was very high (85-99 % ee). It is particularly unusual that a proline solution with very high ee was obtained from proline of low ee; this phenomenon is not observed in other solvents such as EtOH and dimethyl sulfoxide.A solution of proline with very high ee (97-99 % ee) was also obtained from proline with even lower ee (1.0 % ee). Even by using nearly racemic proline (< 0.4 % ee), which was prepared from l-proline (250 AE 1 mg) and d-proline (250 AE 1 mg), the optical purity of proline in solution was found to be very high. With the e...

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.