Display of multiple proteins on engineered canine parvovirus-like particles expressed in cultured silkworm cells and silkworm larvae.

Recent progress has been made dramatically in decorating virus-like particles (VLPs) on the surface or inside with functional molecules, such as antigens or nucleic acids. However, it is still challenging to display multiple antigens on the surface of VLP to meet the requirement as a practical vaccine candidate. Herein this study, we focus on the expression and engineering of the capsid protein VP2 of canine parvovirus for VLP display in the silkworm-expression system. The chemistry of the SpyTag/SpyCatcher (SpT/SpC) and SnoopTag/SnoopCatcher (SnT/SnC) are efficient protein covalent ligation systems to modify VP2 genetically, where SpyTag/SnoopTag are inserted into the N-terminus or two distinct loop regions (Lx and L2) of VP2. The SpC-EGFP and SnC-mCherry are employed as model proteins to evaluate their binding and display on six SnT/SnC-modified VP2 variants. From a series of protein binding assays between indicated protein partners, we showed that the VP2 variant with SpT inserted at the L2 region significantly enhanced VLP display to 80% compared to 5.4% from N-terminal SpT-fused VP2-derived VLPs. In contrast, the VP2 variant with SpT at the Lx region failed to form VLPs. Moreover, the SpT (Lx)/SnT (L2) double-engineered chimeric VP2 variants showed covalent conjugation capacity to both SpC/SnC protein partners. The orthogonal ligations between those binding partners were confirmed by both mixing purified proteins and co-infecting cultured silkworm cells or larvae with desired recombinant viruses. Our results indicate that a convenient VLP display platform was successfully developed for multiple antigen displays on demand. Further verifications can be performed to assess its capacity for displaying desirable antigens and inducing a robust immune response to targeted pathogens.

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate.

In this study, silkworm larvae were used for expression of porcine rotavirus A (KS14 strain) inner capsid protein, VP6, and outer capsid protein, VP7. Initially, VP6 was fused with Strep-tag II and FLAG-tag (T-VP6), and T-VP6 was fused further with the signal peptide of Bombyx mori 30k6G protein (30k-T-VP6). T-VP6 and 30 k-T-VP6 were then expressed in the fat body and hemolymph of silkworm larvae, respectively, with respective amounts of 330 µg and 50 µg per larva of purified protein. Unlike T-VP6, 30k-T-VP6 was N-glycosylated due to attached signal peptide. Also, VP7 was fused with PA-tag (VP7-PA). Additionally, VP7 was fused with Strep-tag II, FLAG-tag, and the signal peptide of Bombyx mori 30k6G protein (30k-T-ΔVP7). Both VP7-PA and 30k-T-ΔVP7 were expressed in the hemolymph of silkworm larvae, with respective amounts of 26 µg and 49 µg per larva of purified protein, respectively. The results from our study demonstrated that T-VP6 formed nanoparticles of greater diameter compared with the ones formed by 30k-T-VP6. Also, higher amount of VP6 expressed in silkworm larvae reveal that VP6 holds the potential for its use in vaccine development against porcine rotavirus with silkworm larvae as a promising host for the production of such multi-subunit vaccines.

Identification of secretion domain of Neospora caninum profilin.

Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported that Neospora caninum profilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41-163), ΔN1 (aa50-163), and ΔN2 (aa144-163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.

Histological and electrophysiological analysis of the corticospinal pathway to forelimb motoneurons in common marmosets.

Using histological and electrophysiological methods, we identified the neuroanatomical properties of the common marmoset corticospinal tract (CST), which underlies hand/arm motor control. Biotinylated dextran amine (BDA) was injected into the primary motor cortex to anterogradely label CST axons in the cervical segments, revealing that most CST axons descend in the contralateral dorsolateral funiculus (DLF; 85.0%), and some in the ipsilateral DLF (10.7%). Terminal buttons were mainly found in the contralateral lamina VII of the gray matter, but projection to lamina IX, where forelimb motoneurons are located, was rare. Bilateral projections were more abundant than found in the rat CST, resembling the CST organization of other primates. Intracellular recordings were made from 57 forelimb motoneurons on the contralateral side to stimulation, which revealed no monosynaptic excitatory postsynaptic potentials (EPSPs), but di- or polysynaptic EPSPs and inhibitory synaptic potentials were commonly found. Local field potentials showed monosynaptic excitation mainly in laminae VII, where abundant BDA-labeled CST terminals were observed. These results suggest that direct corticomotoneuronal projection is absent in common marmosets but di- or oligosynaptic effects would be mediated by spinal interneurons.

Three-dimensional motion analysis of arm-reaching movements in healthy and hemispinalized common marmosets.

Spinal cord injury (SCI) is a devastating neurological injury. At present, pharmacological, regenerative, and rehabilitative approaches are widely studied as therapeutic interventions for motor recovery after SCI. Preclinical research has been performed on model animals with experimental SCI, and those studies often evaluate hand and arm motor function using various indices, such as the success rate of the single pellet reaching test and the grip force. However, compensatory movement strategies, involuntary muscle contraction, and the subject's motivation could affect the scores, resulting in failure to assess direct recovery from impairment. Identifying appropriate assessments of motor impairment is thus important for understanding the mechanisms of motor recovery. In this study, we developed a motion capture system capable of reconstructing three-dimensional hand positions with millimeter and millisecond accuracy and evaluated hand kinematics during food retrieval movement in both healthy and hemispinalized common marmosets. As a result, the endpoint jerk, representing the accuracy of hand motor control, was asserted to be an appropriate index of upper limb motor impairment by eliminating the influence of the subject's motivation, involuntary muscle contraction, and compensatory strategies. The result also suggested that the kinematics of the limb more consistently reflects motor restoration from deficit due to spinal cord injury than the performance in the single pellet reaching test. Because of recent attention devoted to the common marmoset as a nonhuman primate model for human diseases, the present study, which clarified arm-reaching movements in spinalized marmosets, provides fundamental knowledge for future therapeutic studies.