Cholera Toxin B Subunit Research Articles

Neuroanatomical Comparative Studies on the Motor and Sensory Neurons Associated with Cheonji(PC1) in the Rats

This study was performed to comparative investigate the distribution of primary sensory and motor neurons associated with Daereung(PC7) acupoints by using neural tracing technique. A total 16 SD rats were used in the present study. After anesthesia, the rats received microinjection of 6 ㎕ of cholera toxin B subunit(CTB) into the corresponding sites of the acupoints Daereung(PC7), in the human body for observing the distribution of the related primary sensory neurons in dorsal root ganglia(DRGs) and motor neurons in the spinal cord(C3∼T4) and sympathetic ganglia. Three days after the microinjection, the rats were anesthetized and transcardially perfused saline and 4% paraformaldehyde, followed by routine section of the DRGs, sympathetic chain ganglia(SCGs) and spinal cord. Labeled neurons and nerve fibers were detected by immunohistochemical method and observed by light microscope equipped with a digital camera. The labeled neurons were recorded and counted. From this research, the distribution of primary sensory and motor neurons associated with Daereung(PC7) acupoints were concluded as follows. Muscle meridian related Daereung(PC7) controlled by spinal segments of C5∼T1, C6∼T4, respectively.

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).

Prevailing Sydney like Norovirus GII.4 VLPs induce systemic and mucosal immune responses in mice.

The newly emerged Norovirus (NoV) Sydney 2012 strain has been sweeping all over the world, causing acute non-bacterial gastroenteritis in adults and children. Due to a lack of cell culture system, virus like particles (VLPs) has been assembled and used as vaccine candidates in preclinical and clinical studies. Expression of the major capsid protein of NoVs using recombinant baculovirus expression system in Sf9 cells leads to formation of VLPs that are morphologically and antigenically similar to true virions. In this study, VLPs were successfully produced using the VP1 of Sydney-2012-like strain and its immunogenicity was evaluated by different routes and its capability in inducing mucosal immune responses in the presence and absence of adjuvants in BALB/c mice. Administration of NoV VLPs in the presence of Al(OH)3 or monophosphoryl lipid A (MPL-A) led to high titers of VLP-specific IgG antibodies. Administration of VLPs orally in the presence of cholera toxin subunit B (CTB) didn't enhance mucosal immune response as less fecal IgA positive mice were observed when compared with those given VLPs only. Our study represents the first immunogenicity study of VLPs derived from current pandemic Sydney 2012 strain and which might have implications in the development of NoVs vaccine in china.

Area-specific analysis of the distribution of hypothalamic neurons projecting to the rat ventral tegmental area, with special reference to the GABAergic and glutamatergic efferents

The ventral tegmental area (VTA) is a main regulator of reward and integrates a wide scale of hormonal and neuronal information. Feeding-, energy expenditure-, stress, adaptation- and reproduction-related hypothalamic signals are processed in the VTA and influence the reward processes. However, the neuroanatomical origin and chemical phenotype of neurons mediating these signals to the VTA have not been fully characterized. In this study we have systematically mapped hypothalamic neurons that project to the VTA using the retrograde tracer Choleratoxin B subunit (CTB) and analyzed their putative gamma-aminobutyric acid (GABA) and/or glutamate character with in situ hybridization in male rats. 23.93 ± 3.91% of hypothalamic neurons projecting to the VTA was found in preoptic and 76.27 ± 4.88% in anterior, tuberal and mammillary hypothalamic regions. Nearly half of the retrogradely-labeled neurons in the preoptic, and more than one third in the anterior, tuberal and mammillary hypothalamus appeared in medially located regions. The analyses of vesicular glutamate transporter 2 (VGLUT2) and glutamate decarboxylase 65 (GAD65) mRNA expression revealed both amino acid markers in different subsets of retrogradely-labeled hypothalamic neurons, typically with the predominance of the glutamatergic marker VGLUT2. About one tenth of CTB-IR neurons were GAD65-positive even in hypothalamic nuclei expressing primarily VGLUT2. Some regions were populated mostly by GAD65 mRNA-containing retrogradely-labeled neurons. These included the perifornical part of the lateral hypothalamus where 58.63 ± 19.04% of CTB-IR neurons were GABAergic. These results indicate that both the medial and lateral nuclear compartments of the hypothalamus provide substantial input to the VTA. Furthermore, colocalization studies revealed that these projections not only use glutamate but also GABA for neurotransmission. These GABAergic afferents may underlie important inhibitory mechanism to fine-tune the reward value of specific signals in the VTA.

Connectivity and neurochemistry of the commissura anterior of the pigeon (Columba livia).

The anterior commissure (AC) and the much smaller hippocampal commissure constitute the only interhemispheric pathways at the telencephalic level in birds. Since the degeneration study from Zeier and Karten (1973), no detailed description of the topographic organization of the AC has been performed. This information is not only necessary for a better understanding of interhemispheric transfer in birds, but also for a comparative analysis of the evolution of commissural systems in the vertebrate classes. We therefore examined the fiber connections of the AC by using choleratoxin subunit B (CTB) and biotinylated dextran amine (BDA). Injections into subareas of the arcopallium and posterior amygdala (PoA) demonstrated contralateral projection fields within the anterior arcopallium (AA), intermediate arcopallium (AI), PoA, lateral, caudolateral and central nidopallium, dorsal and ventral mesopallium, and medial striatum (MSt). Interestingly, only arcopallial and amygdaloid projections were reciprocally organized, and all AC projections originated within a rather small area of the arcopallium and the PoA. The commissural neurons were not GABA‐positive, and thus possibly not of an inhibitory nature. In sum, our neuroanatomical study demonstrates that a small group of arcopallial and amygdaloid neurons constitute a wide range of contralateral projections to sensorimotor and limbic structures. Different from mammals, in birds the neurons that project via the AC constitute mostly heterotopically organized and unidirectional connections. In addition, the great majority of pallial areas do not participate by themselves in interhemispheric exchange in birds. Instead, commissural exchange rests on a rather small arcopallial and amygdaloid cluster of neurons. J. Comp. Neurol. 524:343–361, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Antibody Production and Th1-biased Response Induced by an Epitope Vaccine Composed of Cholera Toxin B Unit and Helicobacter pylori Lpp20 Epitopes.

The epitope vaccine is an attractive potential for prophylactic and therapeutic vaccination against Helicobacter pylori (H.pylori) infection. Lpp20 is one of major protective antigens which trigger immune response after H.pylori invades host and has been considered as an excellent vaccine candidate for the control of H.pylori infection. In our previous study, one B-cell epitope and two CD4(+) T-cell epitopes of Lpp20 were identified. In this study, an epitope vaccine composed of mucosal adjuvant cholera toxin B subunit (CTB) and these three identified Lpp20 epitopes were constructed to investigate the efficacy of this epitope vaccine in mice. The epitope vaccine including CTB, one B-cell, and two CD4(+) T-cell epitopes of Lpp20 was constructed and named CTB-Lpp20, which was then expressed in Escherichia coli and used for intraperitoneal immunization in BALB/c mice. The immunogenicity, specificity, and ability to induce antibodies against Lpp20 and cytokine secretion were evaluated. After that, CTB-Lpp20 was intragastrically immunized to investigate the prophylactic and therapeutic efficacy in infected mice. The results indicated that the epitope vaccine CTB-Lpp20 possessed good immunogenicity and immunoreactivity and could elicit specific high level of antibodies against Lpp20 and the cytokine of IFN-γ and IL-17. Additionally, CTB-Lpp20 significantly decreased H.pylori colonization in H.pylori challenging mice, and the protection was correlated with IgG, IgA, and sIgA antibody and Th1-type cytokines. This study will be better for understanding the protective immunity of epitope vaccine, and CTB-Lpp20 may be an alternative strategy for combating H.pylori invasion.

A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles.

Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

Role of plasma-membrane-bound sialidase NEU3 in clathrin-mediated endocytosis.

Gangliosides are sialic acid-containing glycosphingolipids mainly expressed at the outer leaflet of the plasma membrane. Sialidase NEU3 is a key enzyme in the catabolism of gangliosides with its up-regulation having been observed in human cancer cells. In the case of CME (clathrin-mediated endocytosis), although this has been widely studied, the role of NEU3 and gangliosides in this cellular process has not yet been established. In the present study, we found an increased internalization of Tf (transferrin), the archetypical cargo for CME, in cells expressing complex gangliosides with high levels of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting the participation of gangliosides in this process. However, the reduction in Tf endocytosis caused by NEU3 was still observed in glycosphingolipid-depleted cells, indicating that NEU3 could operate in a way that is independent of its action on gangliosides. Additionally, internalization of α2-macroglobulin and low-density lipoprotein, other typical ligands in CME, was also decreased in NEU3-expressing cells. In contrast, internalization of cholera toxin β-subunit, which is endocytosed by both clathrin-dependent and clathrin-independent mechanisms, remained unaltered. Kinetic assays revealed that NEU3 caused a reduction in the sorting of endocytosed Tf to early and recycling endosomes, with the Tf binding at the cell surface being also reduced. NEU3-expressing cells showed an altered subcellular distribution of clathrin adaptor AP-2 (adaptor protein 2), but did not reveal any changes in the membrane distribution of clathrin, PtdIns(4,5)P2 or caveolin-1. Overall, these results suggest a specific and novel role of NEU3in CME.

Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in both neonatal and weaned pigs. Because the cholera toxin B subunit (CTB) has a high level of amino acid identity to the ETEC heat-labile toxin (LT) B-subunit (LTB), we selected MucoRice-CTB as a vaccine candidate against ETEC-induced pig diarrhea. When pregnant sows were orally immunized with MucoRice-CTB, increased amounts of antigen-specific IgG and IgA were produced in their sera. CTB-specific IgG was secreted in the colostrum and transferred passively to the sera of suckling piglets. IgA antibodies in the colostrum and milk remained high with a booster dose after farrowing. Additionally, when weaned minipigs were orally immunized with MucoRice-CTB, production of CTB-specific intestinal SIgA, as well as systemic IgG and IgA, was induced. To evaluate the cross-protective effect of MucoRice-CTB against ETEC diarrhea, intestinal loop assay with ETEC was conducted. The fluid volume accumulated in the loops of minipigs immunized with MucoRice-CTB was significantly lower than that in control minipigs, indicating that MucoRice-CTB-induced cross-reactive immunity could protect weaned pigs from diarrhea caused by ETEC. MucoRice-CTB could be a candidate oral vaccine for inducing both passive and active immunity to protect both suckling and weaned piglets from ETEC diarrhea.

Cholera Toxin Subunit B as Adjuvant--An Accelerator in Protective Immunity and a Break in Autoimmunity.

Cholera toxin subunit B (CTB) is the nontoxic portion of cholera toxin. Its affinity to the monosialotetrahexosylganglioside (GM1) that is broadly distributed in a variety of cell types including epithelial cells of the gut and antigen presenting cells, macrophages, dendritic cells, and B cells, allows its optimal access to the immune system. CTB can easily be expressed on its own in a variety of organisms, and several approaches can be used to couple it to antigens, either by genetic fusion or by chemical manipulation, leading to strongly enhanced immune responses to the antigens. In autoimmune diseases, CTB has the capacity to evoke regulatory responses and to thereby dampen autoimmune responses, in several but not all animal models. It remains to be seen whether the latter approach translates to success in the clinic, however, the versatility of CTB to manipulate immune responses in either direction makes this protein a promising adjuvant for vaccine development.

The epitope analysis of an antibody specifically against Vibrio cholerae O1 Ogawa by phage library study

To prevent epidemic and pandemic cholera disease, an indispensible approach is to develop cholera vaccines based on comprehensive epitope information of this pathogen. This study aimed to utilize our previously raised monoclonal antibody IXiao3G6, which can recognize an epitope in lipopolysaccharide (LPS) sites of Ogawa, to identify mimetic peptides, which may represent Ogawa LPS's epitope information. A phage display library screening using IXiao3G6 antibody resulted in identification of a mimic peptide (MP) with high avidity. A recombinant protein, containing one cholera toxin subunit B (CTB) and two MP repeats (CTB-(MP)2), was subsequently constructed and investigated for its immunological characteristics. The findings collectively demonstrated that the MP presenting phages and CTB-(MP)2 recombinant protein were both capable of inhibiting the interaction between IXiao3G6 and Ogawa/Ogawa LPS specifically in a dose-dependent manner.

New Advantageous Method for the Production of Purified Cholera Toxin B-Subunit and Monoclonal Antibodies to It

Put forward is an efficient method for manufacturing cholera toxin B-subunit. Its advantages are relative simplicity and economy feasibility, as well as maximum output of the purified B-subunit, absolutely free from toxic A-subunit contaminant. All this is due to the deployment of cholera vibrio recombinant strain producing only cholera toxin B-subunit instead of cholera toxin as it is, which results in lack of residual preparation toxicity. Applied has been gel-penetration column chromatography, providing for stable native state and maximum antigen output. The method under discussion is verified experimentally. Sample purity has been analyzed after each phase of chromatographic investigation on TSK gel HW-60, using disc electrophoresis. It is established that three steps of purification are ample for the obtainment of cholera toxin B-subunit preparation free from admixtures. Immunological activity of the purified B-subunit is validated by monoclonal antibody obtainment. Designed preparation of cholera toxin B-subunit and monoclonal antibodies to it can serve as a basis for the development of various immune-diagnostic test-systems alternatives.

Spatial receptive fields in the retina and dorsal lateral geniculate nucleus of mice lacking rods and cones.

In advanced retinal degeneration loss of rods and cones leaves melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the only source of visual information. ipRGCs drive non-image-forming responses (e.g., circadian photoentrainment) under such conditions but, despite projecting to the primary visual thalamus [dorsal lateral geniculate nucleus (dLGN)], do not support form vision. We wished to determine what precludes ipRGCs supporting spatial discrimination after photoreceptor loss, using a mouse model (rd/rd cl) lacking rods and cones. Using multielectrode arrays, we found that both RGCs and neurons in the dLGN of this animal have clearly delineated spatial receptive fields. In the retina, they are typically symmetrical, lack inhibitory surrounds, and have diameters in the range of 10-30° of visual space. Receptive fields in the dLGN were larger (diameters typically 30-70°) but matched the retinotopic map of the mouse dLGN. Injections of a neuroanatomical tracer (cholera toxin β-subunit) into the dLGN confirmed that retinotopic order of ganglion cell projections to the dLGN and thalamic projections to the cortex is at least superficially intact in rd/rd cl mice. However, as previously reported for deafferented ipRGCs, onset and offset of light responses have long latencies in the rd/rd cl retina and dLGN. Accordingly, dLGN neurons failed to track dynamic changes in light intensity in this animal. Our data reveal that ipRGCs can convey spatial information in advanced retinal degeneration and identify their poor temporal fidelity as the major limitation in their ability to provide information about spatial patterns under natural viewing conditions.

Immunogenicity characterization of the multi-epitope vaccine CTB-UE with chitosan-CpG as combination adjuvants against Helicobacter pylori

Urease is considered as an excellent vaccine candidate antigen against Helicobacter pylori (H. pylori) infection. Our previous study reported a novel multi-epitope vaccine CTB-UE which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and five cell epitopes from urease subunits. Murine experiments indicated that it could induce cellular and humoral immune responses intensively and attenuate H. pylori infection effectively in mice model. However, the body expression and lack of suitable adjuvant of this epitope vaccine restricted its application. In this study, new recombinant Escherichia coli strains was established to increase the solubility by fusing thioredoxin (Trx) and the combination adjuvants which composed of the chitosan and CpG were adopted to enhance the immunogenicity of CTB-UE for oral immunization. The experimental results indicated that the levels of IgG2a, IgG1 and IgA in the serum and the levels of sIgA in stomach, intestine and feces were significantly higher in the vaccinated group compared with the model control group. Additionally, chitosan-CpG combination adjuvants changed the ratio of IgG2a/IgG1 and conferred Th1/Th17-mediated protective immune responses. These results demonstrate that the oral vaccine with chitosan-CpG as combination adjuvants may be a promising vaccine candidate against H. pylori infection.

Protection against Helicobacter pylori infection in BALB/c mice by oral administration of multi-epitope vaccine of CTB-UreI-UreB.

Chronic gastric infection by the Gram-negative bacterium Helicobacter pylori (H. pylori) is strongly associated with gastritis, gastric ulcer and the development of distal gastric carcinoma and gastric mucosal lymphoma in humans. Antibiotic treatment of H. pylori is becoming less effective because of increasing antibiotic resistance; other treatment approaches such as specifically targeted methods, etc. to destroy this organism would be beneficial. An epitope vaccine is a promising option for protection against H. pylori infection. In this study, a multi-epitope vaccine was constructed by linking cholera toxin B subunit (CTB), two antigenic fragments of H. pylori urease I subunit (UreI20-29, UreI98-107) and four antigenic fragments of H. pylori urease B subunit (UreB12-23, UreB229-251, UreB327-400, UreB515-561), resulting in the recombinant CTB-UreI-UreB (BIB). Its protective effect against H. pylori infection was evaluated in BALB/c mice. Significant protection against H. pylori challenge was achieved in BALB/c mice immunized with BIB (15/18, 83.3%), rIB plus rCTB (6/18, 33.3%) and rIB (2/18, 11.1%) separately, while no protective effect was found in the mice immunized with either adjuvant rCTB alone or PBS. The induction of significant protection against H. pylori is possibly mediated by specific serum IgA and mucosal sIgA antibodies, and a mixed Th1/Th2/Th17 cells response. This multi-epitope vaccine might be a promising vaccine candidate that helps to control H. pylori infection.

Leptin Acts in the Hypothalamic Paraventricular Nucleus to Increase Sympathetic Nerve Activity: Potential Role of Astroglia

The hypothalamic paraventricular nucleus (PVN) is a key site of sympathetic regulation. Activation of neurons in the PVN increases sympathetic nerve activity (SNA) and arterial pressure (AP). However, whether leptin can act in the PVN to increase SNA and AP is unclear. Nanoinjection of leptin in the PVN dose dependently increased (p<0.05) LSNA (vehicle: 108 ± 1 %, 15ng: 140 ± 7%, 30ng: 165 ± 10%, 60ng: 192 ± 12%, 120min after injection), MAP (-3 ± 3, 11 ± 8, 16 ± 4 and 22 ± 4, respectively) and HR (13 ± 16, 40 ± 2, 51 ± 14 and 87 ± 17, respectively). Nanoinjections outside of the PVN were ineffective. To determine the cellular site of action of leptin in the PVN, immunohistochemical detection of the leptin receptor, GFAP (a marker of astrocytes), Iba-1 (a marker for microglia) and cholera toxin subunit b (CTb) retrogradely transported from the rostral ventrolateral medulla (RVLM) was performed. In the PVN few neurons expressed leptin receptor, and no retrogradely-labeled neurons and no Iba-1-positive cells were labeled for the leptin receptor. In contrast, nearly all of the GFAP-labeled neurons were positive for leptin receptor immunostaining and some of these astrocytes were in close apposition to RVLM-projecting neurons. In conclusion, activation of leptin receptors likely on astrocytes within the PVN increases LSNA; the mechanism(s) by which this astrocytic signaling affects neuronal activity within the PVN to increase sympathetic outflow is unknown. Supported by: NIH grant HL088552 and an American Heart Association Grant-in-aid (VLB) and an American Heart Association Grant-in-aid (CJM).

Effects of ocular hypertension in the visual system of pigmented mice.

To study the effects of ocular hypertension (OHT) on the visual system of C57BL/6 pigmented mice, the limbal and episcleral veins of the left eye were laser photocoagulated (LP). LP increased the intraocular pressure during the first five days (d), reaching basal values at 7d. To investigate the effect of OHT on the retinal ganglion cell (RGC) retrograde axonal transport, hydroxistilbamidine methanesulfonate (OHSt) was applied to both superior colliculi (SCi) and the retinas were dissected 2 or 4 weeks after LP. To determine RGC survival, these same retinas were immunoreacted against Brn3a (general RGC population) and melanopsin (intrinsically photosensitive RGCs, m+RGCs). To study whether OHT affected non-RGC neurons in the ganglion cell layer (GCL), RGCs were immunodetected with Brn3a and all GCL nuclei counterstained with DAPI in a group of animals examined 4 weeks post-LP. Innervation of the SCi was examined at 10 days, 8 or 14 weeks after LP with the orthogradely transported cholera toxin subunit-B. OHT resulted in diffuse and sectorial loss of OHSt+RGCs (50% at 2 weeks and 62% at 4 weeks) and in a comparable loss of Brn3a+RGCs at the same time intervals. m+RGCs decreased to 59% at 2 weeks and to 46% at 4 weeks, such loss was diffuse, did not parallel the sectorial loss of the general RGC population and was more severe in the superior-temporal retina. In the GCL, cell loss is selective for RGCs and does not affect other non-RGC neurons. The retinotectal innervation appeared significantly reduced at 10 days (55.7%) and did not progress further up to 14 weeks (46.6%). Thus, LP-induced OHT results in retrograde degeneration of RGCs and m+RGCs, as well as in the loss of CTB-labelled retinotectal terminals.

The Cholera Toxin B Subunit (CTB) Fused to the Porcine Arterivirus Matrix M and GP5 Envelope Proteins Fails to Enhance the GP5-Specific Antibody Response in Pigs Immunized with Adenovectors.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of the Arteriviridae family. As the current commercial vaccines are incompletely protective effective against PRRSV infection, we developed a vaccine strategy using replicating but non-disseminating adenovectors (rAdVs) expressing the PRRSV M matrix protein in fusion with the neutralizing major epitope-carrying GP5 envelope protein (Roques et al. in Vet Res 44:17, 2013). Although production of GP5-specific antibodies (Abs) was observed, no PRRSV-specific neutralizing Abs (NAbs) were induced in pigs given the rAdVs expressing M-GP5 or M-GP5m (GP5m being a mutant form of GP5). Nevertheless, partial protection was observed in the M-GP5m-rAdV-inoculated pigs experimentally infected with PRRSV. Here, we determined the impact of the cholera toxin B subunit (CTB, known for its adjuvant effect) in fusion with the C-terminus of M-GP5m on the Ab response to PRRSV. Three-week-old pigs were immunized twice both intramuscularly and intranasally at 3-week intervals with rAdV-expressing the green fluorescent protein (rAdV-GFP), rAdV-M-GP5m, or rAdV-M-GP5m-CTB. Pigs immunized with rAdV-M-GP5m showed a high level of serum GP5-specific Abs (as determined by an indirect ELISA). In contrast, CTB in fusion with M-GP5m had an unexpected severe negative impact on GP5-specific Ab production. PRRSV-specific NAbs could not be detected in any pigs of all groups.

Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice

No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT-CCMp24 was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT-CCMp24. And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus.

Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3–5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.