Human Antibodies to VP4 Inhibit Replication of Enteroviruses Across Subgenotypes and Serotypes, and Enhance Host Innate Immunity.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and young children (<5 years). HFMD outbreaks occur frequently in the Asia-Pacific region, and these outbreaks are associated with enormous healthcare and socioeconomic burden. There is currently no specific antiviral agent to treat HFMD and/or the severe complications that are frequently associated with the enterovirus of serotype EV71. Therefore, the development of a broadly effective and safe anti-enterovirus agent is an existential necessity. In this study, human single-chain antibodies (HuscFvs) specific to the EV71-internal capsid protein (VP4) were generated using phage display technology. VP4 specific-HuscFvs were linked to cell penetrating peptides to make them cell penetrable HuscFvs (transbodies), and readily accessible to the intracellular target. The transbodies, as well as the original HuscFvs that were tested, entered the enterovirus-infected cells, bound to intracellular VP4, and inhibited replication of EV71 across subgenotypes A, B, and C, and coxsackieviruses CVA16 and CVA6. The antibodies also enhanced the antiviral response of the virus-infected cells. Computerized simulation, indirect and competitive ELISAs, and experiments on cells infected with EV71 particles to which the VP4 and VP1-N-terminus were surface-exposed (i.e., A-particles that don't require receptor binding for infection) indicated that the VP4 specific-antibodies inhibit virus replication by interfering with the VP4-N-terminus, which is important for membrane pore formation and virus genome release leading to less production of virus proteins, less infectious virions, and restoration of host innate immunity. The antibodies may inhibit polyprotein/intermediate protein processing and cause sterically strained configurations of the capsid pentamers, which impairs virus morphogenesis. These antibodies should be further investigated for application as a safe and broadly effective HFMD therapy.

Production and characterization of monoclonal antibodies against highly immunogenic Opisthorchis viverrini proteins and development of coproantigen detection.

Opisthorchis viverrini and other foodborne trematode infections are major health concerns in the Greater Mekong Subregion. Currently, the gold-standard diagnostic method for opisthorchiasis is conventional stool examination for the presence of parasite eggs. This method lacks sensitivity and needs an experienced technician. We therefore produced monoclonal antibodies to highly immunogenic O. viverrini proteins aiming at detecting specific antigens in the feces. In this study, BALB/C mice were immunized using semi-purified somatic antigens and spleen cells were fused with a Sp2/0 myeloma cell line. Four hybridomas (1A2, 1E12, 2C7 and 8D6) were selected and cloned due to their strong reaction against O. viverrini somatic protein, resulting in three IgM clones and one IgG2 clone. Immunohistochemistry showed that 1A2, 1E12, 2C7 and 8D6 stained the parenchyma cells, gut, tegument and muscles, respectively. Western-blot analysis revealed that only antibody 1A2 could detect coproantigen (approx. 73 kDa protein) in feces of hamsters infected with O. viverrini. The 1A2 monoclonal antibody may be of value in the diagnosis of opisthorchiasis by coproantigen detection.

Infection of the Jackal (Canis aureus) by Haplorchis taichui (Trematoda: Heterophyidae) in Southwestern Iran: A Clue for Potential Human Infection.

BACKGROUND: We detected eight trematodes in the small intestine of a road-killed jackal (Canis aureus) from Hamidiyeh District near the city of Ahvaz, Khuzestan Province in 2010. METHODS: Three worms were stained with carmine acid, mounted in Canada balsam on glass slides and examined under a light microscope at 1000X magnification. PCR and sequencing of a partial ITS2 sequence were used to approve the diagnosis. RESULTS: The flukes measured ≈1 mm in length with an elongated ovoid shape resembling the members of heterophyid, and only one testis, characteristics of the genus Haplorchis. Sequencing of a 481-bp fragment of the ITS2 locus from the worms revealed 97%-98% identity with the similar sequences of the H. taichui flukes previously identified in the fish, cat, and humans from Thailand, China, and Vietnam. CONCLUSION: Further studies with the application of reliable molecular tools to diagnose trematode infections in wildlife and humans can bring more insight into the epidemiology of fish-borne flukes including H. taichui in this area.

Identification of Hymenolepis diminuta Cysticercoid Larvae in Tribolium castaneum (Coleoptera: Tenebrionidae) Beetles from Iran.

BACKGROUND: Hymenolepis diminuta is a cestod of rodents and rarely infects humans. Infection in humans is via ingestion of infected insects. This study was aimed to detect H. diminuta cysticercoids in red flour beetles, Tribolium castaneum, and cockroaches originated from different regions of Iran. METHODS: The red flour beetles and cockroaches were collected from local bakeries in five cities including Tehran, Ahvaz, Kazerun, and Sabzevar during 2010-2011. Some beetles and cockroaches were colonized in insectary and adults from F1 generation were fed on H. diminuta eggs. Both laboratory-infected and field-collected samples were dissected and examined for cysticercoids. Detection of H. diminuta DNA in T. castaneum beetles was performed by targeting a partial sequence of Ribosomal gene. RESULTS: Except the beetles from Ahvaz, all specimens were negative for cysticercoid by microscopy. Of the four dissected beetles from Ahvaz, one harbored 12 cysticercoids. Also, 110 (52%) of laboratory-infected beetles showed infection with an average of 12-14 larvae. None of the cockroaches was infected. Two beetles from Ahvaz, including the remainder of the microscopic positive specimen, yielded the expected amplicon in PCR assay. The H. diminuta DNA sequences generated in this study were identical and matched 97-100% with similar sequences from GenBank database. CONCLUSION: Lack of infection in the majority of beetles may reflect a low rat infestation rate in those areas, alternatively, the examined specimens might not have been the representative samples of the T. castaneum populations.

Chicken IgY-based coproantigen capture ELISA for diagnosis of human opisthorchiasis.

Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions.

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles.

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials.

Immunodiagnosis of opisthorchiasis using parasite cathepsin F.

Opisthorchis viverrini, a food-borne trematode parasite endemic in the lower Mekong countries, is conventionally diagnosed by stool examination. However, parasitological stool-based diagnosis can be unreliable in light infections. The goal of this study was to develop the immunodiagnosis of opisthorchiasis using cathepsin F cysteine protease of O. viverrini in both indirect and sandwich ELISA assays. A recombinant O. viverrini cathepsin F (rOv-CF) of 40 kDa was expressed in E. coli strain BL21 (DE3), affinity purified, and deployed in ELISA assays. Human sera from 272 cases were investigated by indirect rOv-CF-based ELISA. Positive antibody response to rOv-CF was found in 137 out of 272 cases (50.37 %) using a cutoff OD (0.400) determined by ROC analysis. In comparison to parasitological stool examined for fluke eggs, the gold standard, the rOv-CF indirect ELISA showed a sensitivity and specificity of 62.1 and 84.05 %, respectively. Serum antibody levels correlated well with egg counts per gram feces (EPG) (P < 0.001). In addition, chicken IgY antibody raised against rOv-CF was tested in a sandwich ELISA for detection of coproantigen in the feces of experimentally infected hamsters. The sandwich ELISA using this chicken IgY in combination with rabbit antibody to O. viverrini somatic antigens showed sensitivity and specificity of 93.3 and 78.57 %, respectively. Together, these findings indicated the potential of rOv-CF for diagnosis of opisthorchiasis, including for uses with chicken IgY for detection of coproantigens of O. viverrini.

Heavy metal bioabsorption capacity of intestinal helminths in urban rats.

BACKGROUND: The aim of the present study was to evaluate the capability of helminths to absorb heavy metals in comparison with that of the host tissues. METHODS: We compared the concentration of cadmium (Cd) and chromium (Cr) in urban rats and in their harboring helminthes -Moniliformis moniliformis, Hymenolepis diminuta and larval stage of Taenia taenaeiformis (Cysticercus fasciolaris). The heavy metal absorption was evaluated in 1g wet weight of parasites and tissues digested in nitric acid, using Inductivity Coupled Plasma (ICP_OES). RESULTS: A higher concentration of heavy metals was revealed in the helminths than in the host tissues. Bioconcentration factor (BF= C in parasite/C in tissue) for both Cd and Cr absorption was more than 10-fold higher in M. moniliformis than in the three compared host tissues. The BF of Cd in M. moniliformis compared to the liver, kidney and muscle of the host was 9.16, 14.14 and 17.09, respectively. BF in Cr in the same parasite and the same host tissues ranged from 10.67, 7.06 and 4.6. High level of absorption in H. diminuta was significantly likewise; the individual BF of Cd and Cr in H. diminuta compared to the liver, kidney and muscle of the hosts was 4.95, 5.94 and 4.67 vs. 2.67, 11.56 and 5.59. The mean concentration of Cd and Cr in C. fasciolaris was also significantly higher than that in the rat livers (P<0.007 and P<0.004, respectively). CONCLUSION: This study claims that parasites of terrestrial animals exposed to heavy metals can be more accurate indicators than the host tissues as new environmental monitoring agents.