Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

Targeting the C-terminus of galectin-9 induces mesothelioma apoptosis and M2 macrophage depletion.

Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors.

Serological evidence of Ebola virus exposure in dogs from affected communities in Liberia: A preliminary report.

Filoviruses such as Ebola virus (EBOV) cause outbreaks of viral hemorrhagic fevers for which no FDA-approved vaccines or drugs are available. The 2014-2016 EBOV outbreak in West Africa infected approximately 30,000 people, killing more than 11,000 and affecting thousands more in areas still suffering from the effects of civil wars. Sierra Leone and Liberia reported EBOV cases in every county demonstrating the efficient spread of this highly contagious virus in the well-connected societies of West Africa. In communities, canines are often in contact with people while scavenging for food, which may include sickly bush animals or, as reported from the outbreak, EBOV infected human bodies and excrement. Therefore, dogs may serve as sentinel animals for seroprevalence studies of emerging infectious viruses. Further, due to their proximity to humans, they may have important One Health implications while offering specimens, which may be easier to obtain than human serum samples. Previous reports on detecting EBOV exposure in canines have been limited. Herein we describe a pilot project to detect IgG-responses directed against multiple filovirus and Lassa virus (LASV) antigens in dogs from EBOV affected communities in Liberia. We used a multiplex Luminex-based microsphere immunoassay (MIA) to detect dog IgG binding to recombinant filovirus antigens or LASV glycoprotein (GP) in serum from dogs that were old enough to be present during the EBOV outbreak. We identified 47 (73%) of 64 dog serum samples as potentially exposed to filoviruses and up to 100% of the dogs from some communities were found to have elevated levels of EBOV antigen-binding IgG titers. The multiplex MIA described in this study provides evidence for EBOV IgG antibodies present in dogs potentially exposed to the virus during the 2014-16 outbreak in Liberia. These data support the feasibility of canines as EBOV sentinels and provides evidence that seroprevalence studies in dogs can be conducted using suitable assays even under challenging field conditions. Further studies are warranted to collect data and to define the role canines may play in transmission or detection of emerging infectious diseases.

Green Turtles (Chelonia mydas) Have Novel Asymmetrical Antibodies.

Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain-only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.

Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

Monoclonal antibodies against the connexin43-interacting protein CIP85.

The connexin43 (Cx43)-interacting protein of 85 kDa CIP85 has been identified as an interacting partner for the cytoplasmically located, carboxyl-terminal tail of Cx43. Further characterization has shown that the interaction between Cx43 and CIP85 is associated with increased turnover of Cx43 that may be lysosome-mediated. This suggests that CIP85 may regulate the endocytic trafficking of Cx43 from the plasma membrane and its degradation, and thus, indirectly influence gap junction function. This study reports the first successful production of monoclonal antibodies (MAbs) against CIP85. These antibodies are useful in detecting CIP85 expressed in several species in immunoblotting, immunoprecipitation, and immunofluorescence microscopy experiments. These MAbs will assist in defining the functional roles of CIP85, including its influence on Cx43 trafficking and intercellular communication through Cx43-containing gap junctions.

Generation and characterization of mouse monoclonal antibodies against CIP75, an UbL-UBA domain-containing protein.

CIP75 is a member of the UbL(ubiquitin-like)-UBA (ubiquitin-associated) domain containing protein family, which has a variety of functions. One specific role described for several members of the UbL-UBA family is the involvement in the proteasomal degradation of target proteins. We have reported that CIP75 interacts with the gap junction protein, connexin43 (Cx43), and that CIP75 may modulate the proteasomal degradation of Cx43. Thus, CIP75 may have a critical role in regulating Cx43 levels, and thus intercellular gap junctional communication. This study reports the development of monoclonal antibodies (MAbs) against CIP75 and the characterization of these antibodies through immunoblotting, immunoprecipitation, and immunofluorescence microscopy analyses. These MAbs will be useful tools in future studies to elucidate the role of CIP75 in Cx43 proteasomal degradation as well as other potential activities.

Survey of methicillin-resistant Staphylococcus aureus (MRSA) carriage in healthy college students, Hawai'i.

Currently, the carriage rate for Community-Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) is unknown in Hawai'i. This survey focuses on a healthy population of 95 college students and 5 faculty who completed a survey related to possible risk factors for colonization of Staphylococcus aureus and were sampled for S. aureus from their anterior nares. Thirty-three (33%) subjects were carrying Staphylococcus aureus and of those, 3 (3%) carried MRSA. There was no significant association between Staphylococcus aureus carriage and ethnicity, gender exposure to seawater, prior Staphylococcus aureus infections, recent antibiotic use, or pets. Additional testing of a larger group of healthy individuals would be beneficial in assessing factors associated with CA-MRSA and Methicillin-susceptible Staphylococcus aureus (MSSA) carriage in Hawai'i.

Pilibacter termitis gen. nov., sp. nov., a lactic acid bacterium from the hindgut of the Formosan subterranean termite (Coptotermes formosanus).

A previously undescribed anaerobic, heterofermentative, non-spore-forming, Gram-positive rod was isolated from the hindgut of the Formosan subterranean termite Coptotermes formosanus Shiraki. The DNA G+C content of this bacterium was 37.8 mol%. Sequence analysis of the 16S rRNA gene revealed that this organism is related to, but distinct from, several genera of lactic acid bacteria, principally several species of the genus Enterococcus. Phenotypic traits that serve to separate this organism from related genera include high levels of the cellular fatty acid C18 : 1omega9c and the production of ethanol along with lactic acid as fermentation products. Based on the collected phylogenetic and phenotypic evidence, it is proposed that the unknown organism represents a novel species in a new genus, Pilibacter termitis gen. nov., sp. nov. The type strain is TI-1T (= ATCC BAA-1030T = CCUG 49613T).