A randomized, double-blind, placebo-controlled phase I clinical trial of rotavirus inactivated vaccine (Vero cell) in a healthy adult population aged 18-49 years to assess safety and preliminary observation of immunogenicity.

We conducted a phase I, randomized, double-blind, placebo-controlled trial including healthy adults in Sui County, Henan Province, China. Ninety-six adults were randomly assigned to one of three groups (high-dose, medium-dose, and low-dose) at a 3:1 ratio to receive one vaccine dose or placebo. Adverse events up to 28 days after each dose and serious adverse events up to 6 months after all doses were reported. Geometric mean titers and seroconversion rates were measured for anti-rotavirus neutralizing antibodies using microneutralization tests. The rates of total adverse events in the placebo group, low-dose group, medium-dose group, and high-dose group were 29.17 % (12.62 %-51.09 %), 12.50 % (2.66 %-32.36 %), 50.00 % (29.12 %-70.88 %), and 41.67 % (22.11 %-63.36 %), respectively, with no significant difference in the experimental groups compared with the placebo group. The results of the neutralizing antibody assay showed that in the adult group, the neutralizing antibody geometric mean titer at 28 days after full immunization in the low-dose group was 583.01 (95 % confidence interval [CI]: 447.12-760.20), that in the medium-dose group was 899.34 (95 % CI: 601.73-1344.14), and that in the high-dose group was 1055.24 (95 % CI: 876.28-1270.75). The GMT of serum-specific IgG at 28 days after full immunization in the low-dose group was 3444.26 (95 % CI: 2292.35-5175.02), that in the medium-dose group was 6888.55 (95 % CI: 4426.67-10719.6), and that in the high-dose group was 7511.99 (95 % CI: 3988.27-14149.0). The GMT of serum-specific IgA at 28 days after full immunization in the low-dose group was 2332.14 (95 % CI: 1538.82-3534.45), that in the medium-dose group was 4800.98 (95 % CI: 2986.64-7717.50), and that in the high-dose group was 3204.30 (95 % CI: 2175.66-4719.27). In terms of safety, adverse events were mainly Grades 1 and 2, indicating that the safety of the vaccine is within the acceptable range in the healthy adult population. Considering the GMT and positive transfer rate of neutralizing antibodies for the main immunogenicity endpoints in the experimental groups, it was initially observed that the high-dose group had higher levels of neutralizing antibodies than the medium- and low-dose groups in adults aged 18-49 years. This novel inactivated rotavirus vaccine was generally well-tolerated in adults, and the vaccine was immunogenic in adults (ClinicalTrials.gov number, NCT04626856).

ML241 Antagonizes ERK 1/2 Activation and Inhibits Rotavirus Proliferation.

Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.

Safety, Immunogenicity, and Mechanism of a Rotavirus mRNA-LNP Vaccine in Mice.

Rotaviruses (RVs) are a major cause of diarrhea in young children worldwide. The currently available and licensed vaccines contain live attenuated RVs. Optimization of live attenuated RV vaccines or developing non-replicating RV (e.g., mRNA) vaccines is crucial for reducing the morbidity and mortality from RV infections. Herein, a nucleoside-modified mRNA vaccine encapsulated in lipid nanoparticles (LNP) and encoding the VP7 protein from the G1 type of RV was developed. The 5' untranslated region of an isolated human RV was utilized for the mRNA vaccine. After undergoing quality inspection, the VP7-mRNA vaccine was injected by subcutaneous or intramuscular routes into mice. Mice received three injections in 21 d intervals. IgG antibodies, neutralizing antibodies, cellular immunity, and gene expression from peripheral blood mononuclear cells were evaluated. Significant differences in levels of IgG antibodies were not observed in groups with adjuvant but were observed in groups without adjuvant. The vaccine without adjuvant induced the highest antibody titers after intramuscular injection. The vaccine elicited a potent antiviral immune response characterized by antiviral clusters of differentiation CD8+ T cells. VP7-mRNA induced interferon-γ secretion to mediate cellular immune responses. Chemokine-mediated signaling pathways and immune response were activated by VP7-mRNA vaccine injection. The mRNA LNP vaccine will require testing for protective efficacy, and it is an option for preventing rotavirus infection.

Immunogenicity of inactivated rotavirus in rhesus monkey, and assessment of immunologic mechanisms.

Rotavirus is one of the main pathogens causing severe diarrhea in infants and young children < 5 years of age. The development of the next-generation rotavirus vaccine is of great significance for preventing rotavirus infection and reducing severe mortality. The current study aimed to develop and evaluate the immunogenicity of inactivated rotavirus vaccine (IRV) in rhesus monkeys. Monkeys received two or three IRV injections intramuscularly at a 4-week interval. Neutralizing antibodies, cellular immunity, PBMC gene expression profiling, and immune persistence were evaluated. Three-dose immunization of IRV induced a higher level of neutralizing, IgG and IgA antibodies compared to two-dose immunization. IRV induced IFN-γ secretion to mediate cellular immune responses, including robust pro-inflammatory and antiviral responses. Chemokine-mediated signaling pathways and immune response were broadly activated by IRV injection. The IRV-induced neutralizing antibodies resulting from two doses returned to baseline levels 20 weeks after full immunization, while those resulting from three doses returned to baseline levels 44 weeks after full immunization. Increasing immunization dose and injection number will help to improve IRV immunogenicity and neutralizing antibody persistence.

Impact of maternal and pre-existing antibodies on immunogenicity of inactivated rotavirus vaccines.

Rotavirus (RV) is a major pathogen causing severe diarrhea in infants and children aged less than 5 years. Vaccination is an economically feasible and effective strategy to prevent rotavirus infections. However, immune efficacy of live vaccines could be interfered by maternal antibodies and pre-existing antibodies of children. To develop an inactivated rotavirus vaccine (IRV), we had previously isolated a wild-type human rotavirus strain ZTR-68-A (G1P[8]) from the fecal samples of infants having severe diarrhea in a region endemic for the presence of this pathogen. In our present study, we assessed whether the presence of maternal and pre-existing antibodies in newborn BALB/c mice affected the immunogenicity of IRV administered to these animals. Our results indicate that maternal antibodies, generated from either vaccine immunization or rotavirus infection, showed partial influence with the immune responses generated by two doses of IRV vaccination. Increasing the number of immunizations can significantly improve the titer of serum neutralizing antibody and a seroconversion rate of up to 100%. In newborn mice, single-virus infection did not elicit detectable levels of serum neutralizing antibodies. After an IRV vaccination, the immune responses of these mice remained unaffected, with no significant differences in titers compared with those of control-group mice. In summary, choosing a suitable immunization dose and dosing frequency is essential for the immune effectiveness of IRV. The results of this study will provide animal experimental support for the IRV clinical research in future.

Vaccination of pregnant rhesus monkeys with inactivated rotavirus as a model for achieving protection from rotavirus SA11 infection in the offspring.

Live-attenuated rotavirus vaccine has shown low protection in underdeveloped or developing countries. However, the inactivated rotavirus vaccine may have the potential to overcome some of these challenges. In the present study, the immunogenicity and protective efficacy of a bivalent inactivated rotavirus vaccine by parenteral administration were elevated in a neonatal rhesus monkey model. A bivalent inactivated rotavirus vaccine containing G1P[8] (ZTR-68 strain) and G9P[8] (ZTR-18 strain) was administered to pregnant rhesus monkeys twice at an interval of 14 days. Neutralizing antibodies against RV strains ZTR-68, ZTR-18, SA11, WA, UK, and Gottfried emerged in pregnant rhesus monkeys and were transplacentally transmitted to the offspring. In the vaccine group, clinical symptoms of diarrhea, viral load in the gut tissue and histopathological changes were significantly reduced in the neonatal rhesus monkeys following oral challenge with the SA11 strain.

MicroRNA-7 Inhibits Rotavirus Replication by Targeting Viral NSP5 In Vivo and In Vitro.

Rotavirus (RV) is the major causes of severe diarrhea in infants and young children under five years of age. There are no effective drugs for the treatment of rotavirus in addition to preventive live attenuated vaccine. Recent evidence demonstrates that microRNAs (miRNAs) can affect RNA virus replication. However, the antiviral effect of miRNAs during rotavirus replication are largely unknown. Here, we determined that miR-7 is upregulated during RV replication and that it targets the RV NSP5 (Nonstructural protein 5). Results suggested that miR-7 affected viroplasm formation and inhibited RV replication by down-regulating RV NSP5 expression. Up-regulation of miR-7 expression is a common regulation method of different G-type RV-infected host cells. Then, we further revealed the antiviral effect of miR-7 in diarrhea suckling mice model. MiR-7 is able to inhibit rotavirus replication in vitro and in vivo. These data provide that understanding the role of cellular miR-7 during rotaviral replication may help in the identification of novel therapeutic small RNA molecule drug for anti-rotavirus.

Immune and cytokine/chemokine responses of PBMCs in rotavirus-infected rhesus infants and their significance in viral pathogenesis.

The rotavirus (RV) is the most important causative agent of severe gastroenteritis in infants and children aged less than 5 years worldwide. However, the response and the roles of peripheral blood mononuclear cell (PBMC) in RV clearance have yet to be fully elucidated. In this study, we established the neonatal rhesus monkey model of RV infection with histopathological changes in the small intestine. Then, we investigated gene expression changes in PBMCs from the monkey model of RV infection. Similar pathways regulated in rhesus monkeys that received intragastric administration of the RV monkey SA11 strain (G3P[2]) and the human wild-type strain ZTR-68 (G1P[8]). Gene profiling showed differences in functional genes mainly associated with chemokine signaling pathways and cytokine-cytokine receptor interactions post RV infection. Transferrin and C-C motif chemokine ligand 23 (CCL23) gene expression were upregulated in PBMCs of monkeys when stimulated by simian and human RV strains. Monkeys infected with RV had an enhanced and prolonged inflammatory response that was associated with increased levels of CCL20, CCL23, and C-X-C motif chemokine ligand 1; while inhibition of major histocompatibility complex class I expression may be important for immune evasion by RV. The RV infection was also characterized by pathological changes in the small intestine with a cytokine and chemokine storm. This study identified the chemokine signaling pathway and immune response genes involved in RV infection in infant rhesus monkeys. The SA11 RV strain is more suitable for establishing a monkey diarrhea model than the ZTR-68 RV strain.

Neonatal rhesus monkeys as an animal model for rotavirus infection.

AIM: To establish a rotavirus (RV)-induced diarrhea model using RV SA11 in neonatal rhesus monkeys for the study of the pathogenic and immune mechanisms of RV infection and evaluation of candidate vaccines. METHODS: Neonatal rhesus monkeys with an average age of 15-20 d and an average weight of 500 g ± 150 g received intragastric administration of varying doses of SA11 RV ( 107 PFUs/mL, 106 PFUs/mL, or 105 PFUs/mL, 10 mL/animal) to determine whether the SA11 strain can effectively infect these animals by observing their clinical symptoms, fecal shedding of virus antigen by ELISA, distribution of RV antigen in the organs by immunofluorescence, variations of viral RNA load in the organs by qRT-PCR, histopathological changes in the small intestine by HE staining, and apoptosis of small intestinal epithelial cells by TUNEL assay. RESULTS: The RV monkey model showed typical clinical diarrhea symptoms in the 108 PFUs SA11 group, where we observed diarrhea 1-4 d post infection (dpi) and viral antigen shed in the feces from 1-7 dpi. RV was found in jejunal epithelial cells. We observed a viral load of approximately 5.85 × 103 copies per 100 mg in the jejunum at 2 dpi, which was increased to 1.09 × 105 copies per 100 mg at 3 dpi. A relatively high viral load was also seen in mesenteric lymph nodes at 2 dpi and 3 dpi. The following histopathological changes were observed in the small intestine following intragastric administration of SA11 RV: vacuolization, edema, and atrophy. Apoptosis in the jejunal villus epithelium was also detectable at 3 dpi. CONCLUSION: Our results indicate that we have successfully established a RV SA11 strain diarrhea model in neonatal rhesus monkeys. Future studies will elucidate the mechanisms underlying the pathogenesis of RV infection, and we will use the model to evaluate the protective effect of candidate vaccines.

Isolation and characterization of a new candidate human inactivated rotavirus vaccine strain from hospitalized children in Yunnan, China: 2010-2013.

AIM: To determine the distribution of rotavirus VP7 gene in hospitalized children in Yunnan, China. METHODS: A total of 366 stool specimens were collected from hospitalized children in hospitals in Yunnan Province from September 2010 to December 2013. The genomic RNA electropherotypes and the G genotypes of the rotaviruses were determined. A phylogenetic analysis of the VP7 gene was performed. Rotavirus isolation was performed, and characterized by plaque, minimum essential medium, and all genes sequence analysis. Quantification of antibodies for inactivated vaccine prepared with ZTR-68 was examined by enzyme-linked immunosorbent assay and microneutralization assay. RESULTS: Group A human rotavirus was detected in 177 of 366 (48.4%) stool samples using a colloidal gold device assay. The temporal distribution of rotavirus cases showed significant correlation with the mean air temperature. Rotaviruses were isolated from 13% of the rotavirus-positive samples. The predominant genotype was G1 (43.5%), followed by G3 (21.7%), G9 (17.4%), G2 (4.3%), G4 (8.7%), and mixed (4.3%) among a total of 23 rotavirus isolates. A rotavirus strain was isolated from a rotavirus-positive stool sample of a 4-month-old child in The First People's Hospital of Zhaotong (2010) for use as a candidate human inactivated rotavirus vaccine strain and for further research, and was designated ZTR-68. The genotype of 11 gene segments of strain ZTR-68 (RVA/Human-wt/CHN/ZTR-68/2010/G1P[8]) was characterized. The genotype constellation of strain ZTR-68 was identified as G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. The VP7 and VP4 genotypes of strain ZTR-68 were similar to Wa-like strains.CONCLUSIONSA high prevalence of the G1, G2, and G3 genotypes was detected from 2010 to 2012. However, a dominant prevalence of the G9 genotype was identified as the cause of gastroenteritis in children in Yunnan, China, in 2013. A candidate human inactivated rotavirus vaccine strain, designated ZTR-68 was isolated, characterized, and showed immunogenicity. Our data will be useful for the future formulation and development of a vaccine in China.

Rotavirus-encoded virus-like small RNA triggers autophagy by targeting IGF1R via the PI3K/Akt/mTOR pathway.

Rotaviruses are double-stranded RNA viruses that are a major cause of viral diarrhea in infants. Examining virus-host cell interaction is important for elucidating mechanisms of virus proliferation in host cells. Viruses can create an environment that promotes their survival and self-proliferation by encoding miRNAs or miRNA-like molecules that target various host cell. However, it remains unclear whether RNA viruses encode viral miRNAs, and their regulation mechanisms are largely unknown. We previously performed deep sequencing analysis to investigate rotavirus-encoded miRNAs, and identified the small RNA molecule Chr17_1755, which we named RV-vsRNA1755. In our present study, we determined that RV-vsRNA1755 is encoded by the rotavirus NSP4 gene and that it targets the host cell IGF1R, which is part of the PI3K/Akt pathway. We further explored the biological characteristics and functions of RV-vsRNA1755.Our results suggest that rotavirus adapts to manipulate PI3K/Akt signaling at early phases of infection. RV-vsRNA1755 targets IGF1R, blockading the PI3K/Akt pathway and triggering autophagy, but it ultimately inhibits autophagy maturation. A mechanism through which rotavirus encodes a virus-like small RNA (RV-vsRNA1755) that triggers autophagy by targeting the host cell IGF1R gene was revealed. These data provide a theoretical basis for therapeutic drug screening targeting RV-vsRNA1755.

Comparative analysis of the immunogenicity of monovalent and multivalent rotavirus immunogens.

The strategies for developing rotavirus (RV) vaccines have always been controversial. At present, both the monovalent RV vaccine and the multivalent RV vaccine have displayed excellent safety and efficacy against RV infection and shown cross-reactive immunity, which laid the question whether the multivalent RV vaccine could be replaced by the monovalent RV vaccine. In this study, we focused on comparing the immunogenicity (serum neutralization activity and protection against homotypic and heterotypic RVs' challenge) of individual standard RV strains (monovalent RV immunogens) and different combinations of them (multivalent RV immunogens). In result, RV immunogens showed general immunogenicity and heterotypic reaction but the multivalent RV immunogens exhibited greater serum neutralization activity and stronger heterotypic reaction than the monovalent RV immunogens (P<0.05). As to the protection, the multivalent RV immunogens also revealed more rapid and stronger protection against homotypic and heterotypic RVs' challenge than the monovalent RV immunogens. The results demonstrated that both the monovalent and multivalent RV immunogens exhibited high immunogenicity, but the monovalent RV immunogens could not provide enough neutralization antibodies to protect MA104 cells against the infection with heterotypic RV strains and timely protection against homotypic and heterotypic RVs, so the multivalent RV vaccine could not be replaced by the monovalent RV vaccine.

MicroRNA profile analysis of host cells before and after wild human rotavirus infection.

Rotavirus infection is an important cause of acute gastroenteritis in children, but the interaction between rotavirus and host cells is not completely understood. We isolated a wildtype (wt) rotavirus strain, ZTR-68(P [8] G1), which is derived from an infant with diarrhea in southwest China in 2010. In this study, we investigated host cellular miRNA expression profiles changes in response to ZTR-68 in early stage of infection to investigate the role of miRNAs upon rotavirus infection. Differentially expressed miRNAs were identified by deep sequencing and qRT-PCR and the function of their targets predicted by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. A total of 36 candidate miRNAs were identified. Comparative analysis indicated that 29 miRNAs were significantly down-regulated and 7 were up-regulated after infection. The data were provided contrasting the types of microRNAs in two different permissive cell lines (HT29 and MA104). The target assays results showed that mml-miR-7 and mml-miR-125a are involved in anti-rotavirus and virus-host interaction in host cells. These results offer clues for identifying potential candidates in vector-based antiviral strategies. J. Med. Virol. 88:1497-1510, 2016. © 2016 Wiley Periodicals, Inc.

Immunogenicity and efficacy in mice of an adenovirus-based bicistronic rotavirus vaccine expressing NSP4 and VP7.

NSP4 and VP7 are important functional proteins of rotavirus. Proper combination of viral gene expression is favorable to improving the protection effect of subunit vaccine. In the present study, We evaluated the immunogenicity and efficacy of the bicistronic recombinant adenovirus (rAd-NSP4-VP7) and two single-gene expressing adenoviruses (rAd-NSP4, rAd-VP7). The three adenovirus vaccines were used to immunize mice by intramuscular or intranasal administration. The data showed significant increases in serum antibodies, T lymphocyte subpopulations proliferation, and cytokine secretions of splenocyte in all immunized groups. However, the serum IgA and neutralizing antibody levels of the rAd-NSP4-VP7 or rAd-VP7 groups were significantly higher than those of the rAd-NSP4, while the splenocyte numbers of IFN-γ secretion in the rAd-NSP4-VP7 or rAd-NSP4 groups was greater than that of the rAd-VP7. Furthermore, the efficacy evaluation in a suckling mice model indicated that only rAd-NSP4-VP7 conferred significant protection against rotavirus shedding challenge. These results suggest that the co-expression of NSP4 and VP7 in an adenovirus vector induce both humoral and cell-mediated immune responses efficiently, and provide potential efficacy for protection against rotavirus disease. It is possible to represent an efficacious subunits vaccine strategy for control of rotavirus infection and transmission.

HIV-1 vaccines based on replication-competent Tiantan vaccinia protected Chinese rhesus macaques from simian HIV infection.

OBJECTIVE: To assess the efficacy of HIV vaccines constructed from replication-competent Tiantan vaccinia virus (rTV) alone or combined with DNA in protecting Chinese rhesus macaques from homologous Simian/Human Immunodeficiency Virus (SHIV)-CN97001 challenge. METHODS: The nef, gag, pol, and gp140 genes from strain CRF07_BC HIV-1 CN54 were selected to construct an HIV vaccine using the rTV or rTV/DNA vaccine. After vaccination, the vaccine and control groups were intravenously challenged with SHIV-CN97001 (32 MID50). HIV-specific antibodies and neutralizing antibodies, gp70 V1V2 binding antibodies, and cytotoxic T-lymphocyte responses were measured prospectively after vaccination with an ELISA, a virus infectivity assay in TZM-bl cells, and ELISPOT assays, respectively. Viral RNA was quantified after challenge with real-time reverse transcriptase-PCR (RT-PCR), and protection efficacy was determined with an analysis of CD8 lymphocyte depletion in vivo. RESULTS: Both rTV and DNA/rTV vaccine groups developed strong cellular and humoral responses against HIV-1 CN54 antigens, including Gag and Env, and also developed significant and persistent anti-Env antibodies and neutralizing antibodies after immunization. Both the rTV and DNA/rTV groups were significantly protected against SHIV-CN97001 or displayed lower viremia than the controls. After CD8 lymphocyte depletion, no viremia was detectable in the vaccinated monkeys, but rebounded rapidly in the control animals. Protection against infection correlated with vaccine-elicited neutralizing antibodies specific for homologous HIV-1 viruses. CONCLUSION: An rTV-based HIV-1 vaccine, with or without a DNA primer, provided protection from SHIV challenge in a macaque model. Replication-competent Tiantan vaccinia is a promising vector and should enable advances in HIV-1 vaccine development.

[Selection and Identification of the Biological Characteristics of a Cold-adapted Genotype G1P[8] ZTR-68 Rotavirus by Serial Cold-adapted Passaging].

We wished to select a cold-adapted genotype G1P[8] ZTR-68 rotavirus (China southwest strain) in MA104 cells for possible use as a live vaccine. ZTR-68 was recovered originally from children with diarrhea. The virus was cultivated at 37 degrees C at the first passage. Then, the cultivation temperature was decreased stepwise by 3 degrees C per eight passages. In total, the virus was passaged 32 times, and cultivation was terminated at 28 degrees C. Biological characteristics of the virus were analyzed during serial passages. There was no difference between the migration patterns of genomic dsRNA segments according to polyacrylamide gel electrophoresis of original and cold-adapted viruses. Infectious and red cell-agglutination titers of cold-adapted virus were lower than those of the parent virus. Also, the virus formed small-size plaques with irregular shapes at 31 degrees C and 28 degrees C. These results suggested that a genetically stable attenuated virus can be obtained through serial cold-adapted passages. Thus, an alternative strategy is provided by cold-adaption for development of attenuated live rotavirus vaccines.

Dopaminergic neuron-like cells derived from bone marrow mesenchymal stem cells by Lmx1α and neurturin overexpression for autologous cytotherapy in hemiparkinsonian rhesus monkeys.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopamine neurons mediated by the Lmx1a and neurturin in vitro: potential therapeutic application for Parkinson's disease in a rhesus monkey model.

hUC-MSCs hold great promise in vitro neuronal differentiation and therapy for neurodegenerative disorders including Parkinson's disease. Recent studies provided that Lmx1α play an important role in the midbrain dopamine cells differentiation. Neurturin is desired candidate gene for providing a neuroprotective to DA neurons. In this study, we investigated a novel neuronal differentiation strategy in vitro with Lmx1α and NTN. We transferred these two genes to hUC-MSCs by recombinant adenovirus combined with Lmx1α regulatory factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored by using behavioral test for six months after implantation. The result showed that cells isolated from the umbilical cord were negative for CD45, CD34 and HLA-DR, but were positive for CD44, CD49d, CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western blot. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. After hUC-MSCs were induced with endogenous and exogenous factors, the mature neurons specific gene TH, Pitx3 was transcripted and the neurons specific protein TH, ß-tubulinIII, NSE, Nestin, MAP-2 was expressed in those differentiated cells. In addition, the PD monkeys, transplanted with the induced cells demonstrated the animals' symptoms amelioration by the behavioral measures. Further more, pathological and immunohistochemistry data showed that there were neuronal-like cells survived in the right brain of those PD monkeys, which may play a role as dopaminergic neurons. The findings from this study may help us to better understand the inside mechanisms of PD pathogenesis and may also help developing effective therapy for Parkinson's disease.

Recovery of behavioral symptoms in hemi-parkinsonian rhesus monkeys through combined gene and stem cell therapy.

BACKGROUND AIMS: The use of adipose mesenchymal stromal cells (ASCs) in cellular and genic therapy has attracted considerable attention as a possible treatment for neurodegenerative disorders, including Parkinson disease. However, the effects of gene therapy combined with intracerebral cell transplantation have not been well defined. Recent studies have demonstrated the respective roles of LIM homeobox transcription factor 1, alpha (LMX1A) and Neurturin (NTN) in the commitment of embryonic stem cells (ESCs) to a midbrain dopaminergic neuronal fate and the commitment of mesenchymal stromal cells to cells supporting the nutrition and protection of neurons. METHODS: We investigated a novel in vitro neuronal differentiation strategy with the use of LMX1A and Neurturin. We were able to elicit a neural phenotype regarding cell morphology, specific gene/protein expression and physiological function. Neuronal-primed ASCs derived from rhesus monkey (rASCs) combined with adenovirus containing NTN and tyrosine hydroxylase (TH) (Ad-NTN-TH) were implanted into the striatum and substantia nigra of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored with the use of behavioral tests and health measures until the fourth month after implantation. RESULTS: The differentiated cells transcribed and expressed a variety of dopaminergic neuron-specific genes involved in the SHH/LMX1A pathway. Single-photon emission computed tomography analysis and postmortem analysis revealed that the grafting of rASCs combined with Ad-NTN-TH had neuroprotective effects compared with Ad-NTN-TH or rASCs alone. Behavioral measures demonstrated autograft survival and symptom amelioration. CONCLUSIONS: These findings may lead to cellular sources for autologous transplantation of Parkinson disease. Combined transplantation of Ad-NTN-TH and induced rASCs expressing LMX1A and NTN may be a better therapy candidate for the treatment of Parkinson disease.

Construction, expression and immunogenicity of a novel anti-hypertension angiotensin II vaccine based on hepatitis A virus-like particle.

Hypertension is a serious worldwide public health problem. The aim of this study is to design anti-hypertension angiotensin II (Ang II) vaccine using molecular biology and immunological method. This novel anti-hypertension vaccine, which is a chimeric protein named pHAV-4Ang IIs, presents four successive repeated Ang IIs as the functional epitope on the surface of the hepatitis A virus-like particle(HAVLP). In this study, pHAV-4Ang IIs was expressed using Bac-to-Bac Baculovirus Expression System. With the RT-PCR analysis, SDS-PAGE, western blot, IFA, electron microscope methods for identification of expression products, these results confirmed that stable expression of pHAV-4Ang IIs can be effectively achieved in infected sf9 cells. Spontaneous hypertensive rats (SHRs) were immunized with pHAV-4Ang IIs to test immunogenicity and pharmacodynamic action. The results showed that this anti-hypertension vaccine can induce high titer Ang II -specific IgG antibody for almost 10 weeks. When antibody titer reached the peak at 8th week, the mean systolic blood pressure (SBP) degraded approximately 23 mmHg compared with the PBS control group, and the mean diastolic blood pressure (DBP) degraded approximately 12 mmHg compared with the PBS control group. These results suggest that this anti-hypertension vaccine has good immunogenicity and good effect on reduction of blood pressure in SHRs, which provide reliable base for large-scale preparation of this hypertension vaccine in the future, and a new direction of exploration for the development of anti-hypertension therapeutic vaccine.