Immunoinformatics for Novel Multi-Epitope Vaccine Development in Canine Parvovirus Infections.

Canine parvovirus (CPV-2) is one of the most important pathogens of dogs of all ages, causing pandemic infections that are characterized by fatal hemorrhagic enteritis. The CPV-2 vaccine is recommended as a core vaccine for pet animals. Despite the intensive practice of active immunization, CPV-2 remains a global threat. In this study, a multi-epitope vaccine against CPV-2 was designed, targeting the highly conserved capsid protein (VP2) via in silico approaches. Several immunoinformatics methods, such as epitope screening, molecular docking, and simulation were used to design a potential vaccine construct. The partial protein sequences of the VP2 gene of CPV-2 and protein sequences retrieved from the NCBI were screened to predict highly antigenic proteins through antigenicity, trans-membrane-topology screening, an allergenicity assessment, and a toxicity analysis. Homologous VP2 protein sequences typically linked to the disease were identified using NCBI BLAST, in which four conserved regions were preferred. Overall, 10 epitopes, DPIGGKTGI, KEFDTDLKP, GTDPDDVQ, GGTNFGYIG, GTFYFDCKP, NRALGLPP, SGTPTN, LGLPPFLNSL, IGGKTG, and VPPVYPN, were selected from the conserved regions to design the vaccine construct. The molecular docking demonstrated the higher binding affinity of these epitopes with dog leukocyte antigen (DLA) molecules. The selected epitopes were linked with Salmonella enterica flagellin FliC adjuvants, along with the PADRE sequence, by GGS linkers to construct a vaccine candidate with 272 nucleotides. The codon adaptation and in silico cloning showed that the generated vaccine can be expressed by the E. coli strain, K12, and the sequence of the vaccine construct showed no similarities with dog protein. Our results suggest that the vaccine construct might be useful in preventing canine parvoviral enteritis (CPE) in dogs. Further in vitro and in vivo experiments are needed for the validation of the vaccine candidate.

Expression of the G protein-coupled receptor (GPR) 37 and GPR37L1 in the mouse digestive system.

G protein-coupled receptor (GPR) 37 and GPR37L1 are known to modulate the dopaminergic neuron activity, and recently, they are identified as candidate prosaposin receptors. Intercellular prosaposin is proteolytically processed into four saposins, each of which acts as a sphingolipid hydrolase activator in the lysosome. In contrast, extracellular prosaposin exerts a trophic effect on neurons via GPR37 and GPR37L1. In this study, the expression patterns of GPR37 and GPR37L1 in the mouse digestive system were examined immunohistochemically. The islets of Langerhans of the pancreas showed intense immunoreactivity for GPR37 and GPR37L1. Weak immunoreactivity for GPR37 and GPR37L1 was found in the nerve plexuses of the esophagus and small and large intestines. Colocalization of GPR37 and tyrosine hydroxylase immunoreactivity was observed in the neuron of the nerve plexus of the large intestine. This study suggests the possibility that prosaposin affects the function of islet-secreting cells. Also, the expression of GPR37 and GPR37L1 in the nerve plexus suggests that prosaposin exerts a trophic effect not only in the central nervous system, but also in the enteric nervous system.

Localization of AMPA, kainate, and NMDA receptor mRNAs in the pigeon cerebellum.

In the present study, we investigated the location of mRNAs for three types of ionotropic glutamate receptors (iGluRs) in the pigeon cerebellum and then compared the results with those of mammals. The following nine iGluRs subunits were analyzed by in situ hybridization: AMPA receptors (GluA1, GluA2, GluA3, and GluA4), kainate receptors (GluK1, GluK2, and GluK4), and NMDA receptors (GluN1 and GluN2A). Subunit hybridization revealed expression in different cell types of the cerebellar cortex: Purkinje cells expressed most subunits, including AMPA receptors (GluA1, GluA2, and GluA3), kainate receptors (GluK1 and GluK4), and NMDA receptors (GluN1); granule cells expressed four subunits of kainate (GluK1 and GluK2) and NMDA receptors (GluN1 and GluN2A); stellate and basket cells expressed GluK1, GluK2, and GluN1; Golgi cells expressed GluA1, GluA3, and GluN1; and Bergmann glial cells expressed only AMPA receptors (GluA2 and GluA4). Cerebellar nuclei showed no AMPA subunit signals, whereas kainate and NMDA receptors were observed in the five cerebellar nuclei divisions (CbL, CbMic, CbMim, CbMin, and CbMvm). The five divisions showed weak expression of GluK1, GluK2, and GluN2A; moderate to intense expression of GluK4; and intense expression of GluN1. These results demonstrate that in pigeons the cerebellar cortex expresses AMPA, kainate, and NMDA receptors, while the cerebellar nuclei express kainate and NMDA receptors. Taken together, these findings provide anatomical data for further analysis of the functions of iGluR-expressing neurons in glutamatergic circuits of the avian cerebellum.

Gene expression of AMPA, kainate, and NMDA receptor subunits in the pigeon spinal cord.

Glutamatergic neurons are widely distributed in gray matter across the four segments of the pigeon spinal cord. Postsynaptic neurons containing ionotropic glutamate receptors (iGluRs), however, have been elucidated in only some AMPA subunits. This study examined the localization of postsynaptic neurons having three types of iGluRs in the pigeon spinal cord. Nine mRNAs of iGluR subunits - namely GluA1, GluA2, GluA3, and GluA4 for AMPA receptors; GluK1, GluK2, and GluK4 for kainate receptors; and GluN1 and GluN2A for N-methyl-d-aspartate (NMDA) receptors - were analyzed by in situ hybridization. All three types of iGluRs were found in gray matter, with GluK4 and GluN1 subunits strongly expressed in laminae I-IX. GluA1 and GluA2 subunits were expressed in glial cells of white matter. In general, AMPA receptors were more weakly expressed than kainate and NMDA receptors. Among the four segments of the spinal cord, no significant differences were observed between the hybridization signals of the various iGluRs. Neurons with strong expression of GluK4 and GluN1 (but not the other subunits) were present in the marginal nucleus of cervicothoracic segments and in the accessory lobe of Lachi in lumbosacral segments, while GluA1, GluA2, and GluK2 were expressed in glycogen cells of the accessory lobe. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission in the avian spinal cord.

Prox1 mRNA expression in the medial cortex of the turtle (Pseudemys scripta elegans).

The medial cortex of the cerebrum in reptiles is thought to be homologous to the mammalian dentate gyrus, based on cytoarchitectures, fiber connections, and neurochemical profiles. To support this hypothesis, we examined the mRNA expression of vesicular glutamate transporter 1 (vGluT1), a glutamatergic gene marker, and Prox1, a selective gene marker for granule cells of the dentate gyrus, in the turtle medial cortex (zone 2). Reverse transcription-polymerase chain reaction revealed the presence of both mRNAs in the turtle cerebrum. In situ hybridization of zone 2, which is a layer of densely packed neurons in Nissl stains, intensely expressed vGluT1 and Prox1. In zone 3, which is a loosely packed layer, vGluT1 was intensely expressed, whereas Prox1 signals gradated from strong to negative toward zone 4. These findings demonstrate that zone 2 contains glutamatergic neurons and expresses Prox1 mRNA and suggest that zone 2 in the turtle cerebrum is homologous to the mammalian dentate gyrus.

Distribution of vesicular glutamate transporters in the brain of the turtle (Pseudemys scripta elegans).

The distribution of glutamatergic neurons has been extensively studied in mammalian and avian brains, but its distribution in a reptilian brain remains unknown. In the present study, the distribution of subpopulations of glutamatergic neurons in the turtle brain was examined by in situ hybridization using probes for vesicular glutamate transporter (VGLUT) 1-3. Strong VGLUT1 expression was observed in the telencephalic pallium; the mitral cells of the olfactory bulb, the medial, dorsomedial, dorsal, and lateral parts of the cerebral cortex, pallial thickening, and dorsal ventricular ridge; and also, in granule cells of the cerebellar cortex. Moderate to weak expression was found in the lateral and medial amygdaloid nuclei, the periventricular cellular layer of the optic tectum, and in some brainstem nuclei. VGLUT2 was weakly expressed in the telencephalon but was intensely expressed in the dorsal thalamic nuclei, magnocellular part of the isthmic nucleus, brainstem nuclei, and the rostral cervical segment of the spinal cord. The cerebellar cortex was devoid of VGLUT2 expression. The central amygdaloid nucleus did not express VGLUT1 or VGLUT2. VGLUT3 was localized in the parvocellular part of the isthmic nucleus, superior and inferior raphe nuclei, and cochlear nucleus. Our results indicate that the distribution of VGLUTs in the turtle brain is similar to that in the mammalian brain rather than that in the avian brain.

Differential projections of the densocellular and intermediate parts of the hyperpallium in the pigeon (Columba livia).

The visual Wulst in birds shows a four-layered structure: apical part of the hyperpallium (HA), interstitial part of HA (IHA), intercalated part of hyperpallium (HI), and densocellular part of hyperpallium (HD). HD also connects with the hippocampus and olfactory system. Because HD is subjacent to HI, the two have been treated as one structure in many studies, and the fiber connections of HD have been examined by afferents and efferents originating outside HD. However, to clarify the difference between these two layers, they need to be treated separately. In the present study, the fiber connections of HD and HI were analyzed with tract-tracing techniques using a combination of injections of cholera toxin subunit B (CTB) for retrograde tracing and biotinylated dextran amine (BDA) for anterograde tracing. When the two tracers were bilaterally injected in HD, a major reciprocal connection was seen with the dorsolateral subdivision (DL) of the hippocampal formation. When CTB and BDA were bilaterally injected in HI, strong reciprocal connections were found between HI and HA. Next, projection neurons in HD and HI were examined by double staining for CTB combined with vesicular glutamate transporter 2 (vGluT2) mRNA in situ hybridization. After CTB was injected in DL or HA, many neurons revealed CTB+/vGluT2+ in HD or HI, respectively. Furthermore, in situ hybridization showed that DL and HA contained neurons expressing various subunits of ionotropic glutamate receptors: AMPA, kainate, and NMDA types. These results suggest that glutamatergic neurons in HD and HI project primarily to DL and HA, respectively.

Proposed homology of the dorsomedial subdivision and V-shaped layer of the avian hippocampus to Ammon's horn and dentate gyrus, respectively.

The avian hippocampal formation differs considerably from that of mammals both in terms of position and cytoarchitecture. On the basis of fiber connections in pigeons, however, we previously proposed that the dorsomedial subdivision (DM) and the V-shaped layer of the hippocampal formation correspond to Ammon's horn and the dentate gyrus of mammals, respectively. In the present study, we provide evidence in support of this hypothesis by double staining hippocampal neurons using tract-tracing and gene expression. After cholera toxin subunit B (CTB) was injected into the lateral septal nucleus (SL), and vesicular glutamate transporter 2 (vGluT2) mRNA, a gene marker for glutamatergic neurons, was visualized in the same retrogradely labeled neurons with in situ hybridization, most CTB+/vGluT2+ neurons were concentrated in DM, but were rare in the V-shaped layer. The distribution pattern of CTB+/vGluT2+ neurons in the hippocampal formation did not change when CTB injection sites were shifted in a rostrocaudal direction in SL. SL expresses a variety of mRNAs for ionotropic glutamate receptor subunits (GluA1, GluA2, GluK2, GluK4, and GluN1). The findings indicate that DM neurons provide descending glutamatergic axons to SL. Additionally, the present study showed that Prox1 mRNA, a gene marker for the dentate gyrus in mammals, was intensely expressed in the V-shaped layer in the pigeon hippocampus. Together these results strengthen our original hypothesis that DM and the V-shaped layer in the pigeon hippocampus are homologous to Ammon's Horn and the dentate gyrus, respectively. © 2016 Wiley Periodicals, Inc.