Promoting immunity with novel targeting antigen delivery vehicle based on bispecific nanobody.

As the most potent professional antigen presenting cells, dendritic cells (DCs) have been targeted in strategies to enhance vaccination efficacy. To date, targeted delivery has been mainly used for cancer therapy, with few studies focusing on vaccine antigens for animal epidemic diseases. In this study, we selected a series of mouse DC-specific nanobodies from a non-immunized camel. The four candidate nanobodies identified (Nb4, Nb13, Nb17, and Nb25), which showed efficient endocytosis of bone marrow-derived DCs, were evaluated as potential vaccine antigen targeted delivery vehicles. First, green fluorescent protein (GFP) was selected and four corresponding DCNb-GFP fusions were constructed for verification. Nb17-GFP was effective at promoting antibody production, inducing a cellular immune response, and increasing the IL-4 level. Second, foot-and-mouth disease virus (FMDV) and a FMDV-specific nanobody (Nb205) were selected and four bispecific nanobody DCNb-Nb205 fusions were generated to investigate the feasibility of a novel targeting antigen delivery vehicle. The resulting bispecific nanobody, Nb17-Nb205, could not only deliver FMDV particles instead of antigenic peptide, but also induced the production of specific antibodies, a cellular immune response, and IFN-γ and IL-4 levels upon immunization with a single subcutaneous injection. In conclusion, our results demonstrate the potential of bispecific nanobody as a novel and efficient DC-specific antigen delivery vehicle. This highlights the potential to expand targeted delivery to the field of animal epidemic diseases and provides a reference for the general application of nanotechnology in viral diseases.

Development of haemagglutination assay for titration of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) was one of the most economically important viral pathogens in all the swine-producing countries and often resulted in tremendous economic losses for the swine industry. As PCV2 could not cause cytopathogenic effects while propagated in infected cells, many complicated experiments should be performed to titrate its virus titer. In this study we developed a simple and effective hemagglutination assay for titration of virus titer of PCV2. To develop the hemagglutination assay, a recombinant bispecific nanobody (BsNb) against PCV2 and chicken red blood cells (cRBCs) was constructed based on two nanobodies (NbPCV11 and NbRBC48) which were selected from the non-immunized nanobody library, respectively. The hemagglutination assay was used to titrate the virus titer of PCV2 propagated in cell culture by simple naked-eye observation within 30 min, with the detection limit of 104.09 tissue culture infective dose 50 (TCID50)/mL, excellent specificity and reproducibility. Therefore, the hemagglutination assay had potential to be a rapid, reliable, cost-effective, user-friendly qualitative and semi-quantitative tool for titration of virus titer of PCV2 during the vaccine manufacturing process.

Development of GEM-PA-nanotrap for purification of foot-and-mouth disease virus.

Vaccination is the primary preventative measure against outbreaks of foot-and-mouth disease (FMD). The efficacy of inactivated FMD vaccines is mainly determined by the integrity of foot-and-mouth disease virus (FMDV) particles (referred to as 146S particles), and impurities in the inactivated vaccines could result in side effects. In this study, we developed an effective affinity purification method for the purification of FMDV from cellular lysates, referred to as GEM-PA-nanotrap. To develop the GEM-PA-nanotrap, a nanobody (Nb205) against FMDV vaccine strain O/MYA98/BY/2010 146S particles was selected from a non-immunized library and fused to a peptidoglycan-binding protein anchor (PA). The PA-Nb205 fusion protein was non-covalently coupled to the surface of Gram-positive enhancer matrix (GEM) particles, which were prepared from the non-living, non-genetically modified, Gram-positive, food-grade Lactococcus lactis bacteria. The GEM-PA-nanotrap was used to purify FMDV from cellular lysates through a simple incubation and centrifugation step. The FMDV recovery rate was more than 99%, the efficiency of nonviral protein removal was about 98.3%, and the purification process had almost no effect on the integrity and immunogenicity of 146S particles. Therefore, the GEM-PA-nanotrap has potential as an effective method for the recovery and purification of FMDV during the vaccine manufacturing process.

Novel monoclonal antibody-based immunochromatographic strip for detecting citrinin in fruit from Zhejiang province, China.

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site.

BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx mori.

PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemocytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa protein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva implied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.

TRANSCRIPTION FACTOR Bmsage PLAYS A CRUCIAL ROLE IN SILK GLAND GENERATION IN SILKWORM, Bombyx mori.

Salivary gland secretion is altered in Drosophila embryos with loss of function of the sage gene. Saliva has a reduced volume and an increased electron density according to transmission electron microscopy, resulting in regions of tube dilation and constriction with intermittent tube closure. However, the precise functions of Bmsage in silkworm (Bombyx mori) are unknown, although its sequence had been deposited in SilkDB. From this, Bmsage is inferred to be a transcription factor that regulates the synthesis of silk fibroin and interacts with another silk gland-specific transcription factor, namely, silk gland factor-1. In this study, we introduced a germline mutation of Bmsage using the Cas9/sgRNA system, a genome-editing technology, resulting in deletion of Bmsage from the genome of B. mori. Of the 15 tested samples, seven displayed alterations at the target site. The mutagenesis efficiency was about 46.7% and there were no obvious off-target effects. In the screened homozygous mutants, silk glands developed poorly and the middle and posterior silk glands (MSG and PSG) were absent, which was significantly different from the wild type. The offspring of G0 mosaic silkworms had indel mutations causing 2- or 9-bp deletions at the target site, but exhibited the same abnormal silk gland structure. Mutant larvae containing different open-reading frames of Bmsage had the same silk gland phenotype. This illustrated that the mutant phenotype was due to Bmsage knockout. We conclude that Bmsage participates in embryonic development of the silk gland.

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone.

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.

Characterization of adaptor protein complex-1 in the silkworm, Bombyx mori.

To investigate the function of adaptor protein complex-1 (AP-1) in the silkworm, we characterized AP-1 in the silkworm by RNAi technique and co-localization methods. As a result, AP-1 was found to exist as cytosolic form and membrane-bound form distinguished by phosphate status, showing molecular mass difference. There was relatively more cytosolic form of AP-1 than its membrane-bound counterpart in the silkworm. However, AP-1 distributed predominantly as cytosolic form in BmN cells. Interruption of AP-1 expression via DsRNA was more efficient in BmN cells than in the insect larval, which led to a tendency to dissociation between subcellular organelles like the Golgi apparatus and the mitochondria. Environmental condition changes like relatively higher temperature and treatment with dimethyl sulfoxide can lead to expression variance of AP-1 both in mRNA and protein level. In BmN cells, both the heavy chain γ and light chain σ could clearly co-localize with AP-1 ß, mostly forming pits in cytoplasm. Two isoforms of AP-1 σ corresponded to distinct subcellular distribution pattern, possibly due to C-terminal amino acids difference.

Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone.

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.