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1.
Virus Genes ; 55(4): 550-556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161411

RESUMO

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

2.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541860

RESUMO

Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

3.
Int Immunopharmacol ; 64: 217-222, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30199846

RESUMO

BACKGROUND: Rift Valley fever virus (RVFV) is an emerging arbovirus in Africa and the Arabian Peninsula, in which infection with RVFV poses a serious threat to humans and livestock globally. Approved treatments for RVFV infection, especially for use in humans, have not yet been developed. There is an urgent need for effective drugs to prevent RVFV disease. METHODS: In previous study, we developed RVFV virus like particles (VLPs) expressing the surface glycoproteins Gn and Gc. The morphology was shown to be similar to live RVFV under electron microscopy. In this study, we immunized horses with RVFV VLPs, prepared the immunoglobulin F(ab')2 fragments, and characterized its in vitro neutralization and in vivo efficacy in mice. RESULTS: F(ab')2 was found to potently neutralize RVFV in VeroE6 cells, and passive transfer of immunoglobulin F(ab')2 fragments resulting in reduced mortality in RVFV infected mice. CONCLUSION: Our results show that passive immunotherapy with equine immunoglobulin F(ab')2 fragments is a promising strategy to treat RVFV infections.

4.
J Vet Sci ; 19(2): 200-206, 2018 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28693302

RESUMO

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV). RVF is mainly prevalent on the Arabian Peninsula, the African continent, and several islands in the Indian Ocean near southeast Africa. RVFV has been classified by the World Organisation for Animal Health (OIE) as a category A pathogen. To avoid biological safety concerns associated with use of the pathogen in RVFV neutralization assays, the present study investigated and established an RVFV pseudovirus-based neutralization assay. This study used the human immunodeficiency virus (HIV) lentiviral packaging system and RVFV structural proteins to successfully construct RVFV pseudoviruses. Electron microscopy observation and western blotting indicated that the size, structure, and shape of the packaged pseudoviruses were notably similar to those of HIV lentiviral vectors. Infection inhibition assay results showed that an antibody against RVFV inhibited the infective ability of the RVFV pseudoviruses, and an antibody neutralization assay for RVFV detection was then established. This study has successfully established a neutralization assay based on RVFV pseudoviruses and demonstrated that this method can be used to effectively evaluate antibody neutralization.


Assuntos
Testes de Neutralização/métodos , Vírus da Febre do Vale do Rift , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Western Blotting , Microscopia Eletrônica , Proteínas Recombinantes/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Vale do Rift/ultraestrutura
5.
Oncotarget ; 8(53): 91505-91515, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29207661

RESUMO

Several studies have shown that interleukin-18 (IL-18) plays an important role in both innate and adaptive immune responses. In this study, we investigated the pathogenicity and immunogenicity of recombinant rabies virus expressing IL-18 (rHEP-IL18). Experimental results showed that Institute of Cancer Research (ICR) mice that received a single intramuscular immunization with rHEP-IL18 elicited the highest titers of serum neutralizing antibodies and the strongest cell-mediated immune responses to prevent the development of rabies disease, compared with immunization with the parent virus HEP-Flury. Mice inoculated with rHEP-IL18 developed significantly higher IFN-γ responses, increased percentages of CD4+ and CD8+ T-lymphocytes compared to HEP-Flury. Flow cytometry results show that rHEP-IL18 recruited more activated T- and B-cells in lymph nodes or peripheral blood, which is beneficial for virus clearance in the early stages of infection. A higher percentage of mice immunized with rHEP-IL18 survived wild-type rabies virus (RABV) challenge, compared to HEP-Flury mice. Our results show that rHEP-IL18 is promising as a novel vaccine for RABV prevention and control.

6.
Virol J ; 14(1): 204, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070075

RESUMO

BACKGROUND: Marburg virus (MARV) causes severe haemorrhagic fever in humans and nonhuman primates and has a high mortality rate. However, effective drugs or licensed vaccines are not currently available to control the outbreak and spread of this disease. METHODS: In this study, we generated MARV virus-like particles (VLPs) by co-expressing the glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. MARV VLPs and three adjuvants, Poria cocos polysaccharide (PCP-II), poly(I:C) and aluminium hydroxide, were evaluated after intramuscular vaccination in mice. RESULTS: Murine studies demonstrated that vaccination with the MARV VLPs induce neutralizing antibodies and cellar immune responses. MARV VLPs and the PCP-II adjuvant group resulted in high titres of MARV-specific antibodies, activated relatively higher numbers of B cells and T cells in peripheral blood mononuclear cells (PBMCs), and induced greater cytokine secretion from splenocytes than the other adjuvants. CONCLUSION: MARV VLPs with the PCP-II adjuvant may constitute an effective vaccination and PCP-II should be further investigated as a novel adjuvant.


Assuntos
Glicoproteínas/imunologia , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas da Matriz Viral/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linfócitos B/imunologia , Citocinas/sangue , Citocinas/metabolismo , Expressão Gênica , Glicoproteínas/genética , Imunidade Celular , Marburgvirus/genética , Camundongos , Células Sf9 , Linfócitos T/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Proteínas da Matriz Viral/genética , Vacinas Virais/genética
7.
Vaccine ; 35(16): 2069-2075, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28314561

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 104) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4+ and CD8+ T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
8.
Antiviral Res ; 140: 55-61, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28040513

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation.


Assuntos
Infecções por Coronavirus/prevenção & controle , Imunidade Celular , Imunidade Humoral , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Quimera , Citocinas/imunologia , Citocinas/metabolismo , Eritrócitos/virologia , Camundongos , Parvovirus Canino/genética , Ligação Proteica , Suínos , Células Th1/imunologia , Células Th2/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/administração & dosagem
9.
Oncotarget ; 8(8): 12686-12694, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-27050368

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans with a case fatality rate of over 39%, and poses a considerable threat to public health. A lack of approved vaccine or drugs currently constitutes a roadblock in controlling disease outbreak and spread. In this study, we generated MERS-CoV VLPs using the baculovirus expression system. Electron microscopy and immunoelectron microscopy results demonstrate that MERS-CoV VLPs are structurally similar to the native virus. Rhesus macaques inoculated with MERS-CoV VLPs and Alum adjuvant induced virus-neutralizing antibodies titers up to 1:40 and induced specific IgG antibodies against the receptor binding domain (RBD), with endpoint titers reaching 1:1,280. MERS-CoV VLPs also elicited T-helper 1 cell (Th1)-mediated immunity, as measured by ELISpot. These data demonstrate that MERS-CoV VLPs have excellent immunogenicity in rhesus macaques, and represent a promising vaccine candidate.


Assuntos
Infecções por Coronavirus/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Western Blotting , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Insetos , Macaca mulatta , Microscopia Eletrônica de Transmissão
10.
Antiviral Res ; 137: 125-130, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27890674

RESUMO

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab')2 from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab')2 efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab')2 for potential use in the prophylactic or therapeutic treatment of exposed or infected patients.


Assuntos
Anticorpos Antivirais/uso terapêutico , Infecções por Coronavirus/terapia , Imunização Passiva , Fragmentos de Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Cavalos/imunologia , Humanos , Fragmentos de Imunoglobulinas/isolamento & purificação , Camundongos , Receptores Imunológicos , Infecções Respiratórias/terapia , Infecções Respiratórias/virologia
11.
Sci Rep ; 6: 24179, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27067649

RESUMO

Recent successes with monoclonal antibody cocktails ZMapp(TM) and MIL77 against Ebola virus (EBOV) infections have reignited interest in antibody-based therapeutics. Since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. We produced purified equine antisera from horses hyperimmunized with EBOV virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. BALB/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. To decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate F(ab')2 fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. Full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other Fc-dependent antibody mechanisms may also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab')2 at or starting at 1 or 2 dpi were also fully protected from EBOV infection. These results justify future efficacy studies for purified equine products in NHPs.


Assuntos
Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Fragmentos de Imunoglobulinas/administração & dosagem , Fatores Imunológicos/administração & dosagem , Profilaxia Pós-Exposição/métodos , Animais , Modelos Animais de Doenças , Cobaias , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Análise de Sobrevida , Resultado do Tratamento
12.
Int J Biol Macromol ; 87: 7-15, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26875535

RESUMO

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.


Assuntos
Adjuvantes Imunológicos/farmacologia , Isatis/química , Raízes de Plantas/química , Polissacarídeos/farmacologia , Vacinas Antirrábicas/imunologia , Animais , Anticorpos Neutralizantes/sangue , Antígenos Virais/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Citocinas/metabolismo , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Raiva/efeitos dos fármacos , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas de Produtos Inativados/imunologia
13.
Arch Virol ; 161(5): 1125-33, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26831931

RESUMO

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Ebolavirus/genética , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificação , Transcrição Reversa , Sensibilidade e Especificidade , Alinhamento de Sequência
14.
Protein Expr Purif ; 104: 7-13, 2014 12.
Artigo em Inglês | MEDLINE | ID: mdl-25218147

RESUMO

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.


Assuntos
Glicoproteínas/genética , Fragmentos de Peptídeos/genética , Vírus da Raiva/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Sobrevivência Celular , DNA/administração & dosagem , Humanos , Camundongos , Plasmídeos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/toxicidade
15.
Virus Genes ; 48(3): 411-20, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24535572

RESUMO

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Raiva/prevenção & controle , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Raiva/virologia , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vírus da Raiva/genética , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética
16.
Wei Sheng Wu Xue Bao ; 53(4): 409-15, 2013 Apr 04.
Artigo em Chinês | MEDLINE | ID: mdl-23858717

RESUMO

OBJECTIVE: To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism, virulence genes and antigenic sites. METHODS: We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR, and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11. We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers. We amplified the full-length cDNA of CVS-11 by RT-PCR. We obtained four fragments and cloned into pcDNA3. 1. We named the full-length cDNA plasmid pcDNA3. 1-CVS-11. We also cloned helper plasmids pcDNA3.1-N, P, L and G expressing N, P, L and G protein of CVS-11 strain, respectively. We co-transfected NA cells with the full-length plasmid and four helper plasmids. We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully. RESULTS: Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides. The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses. We successfully constructed a reverse genetic system of CVS-11, namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N, P, L, G and rescued the rCVS-11 from a full-length infectious cDNA clone. CONCLUSION: The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus.


Assuntos
DNA Complementar/genética , Genoma Viral , Vírus da Raiva/genética , Linhagem Celular , Clonagem Molecular , Vetores Genéticos/genética , Plasmídeos/genética , Análise de Sequência de DNA/métodos , Transfecção/métodos
17.
Virus Res ; 173(2): 398-403, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23333291

RESUMO

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.


Assuntos
Antivirais/administração & dosagem , Aptâmeros de Nucleotídeos/administração & dosagem , Vírus da Raiva/efeitos dos fármacos , Raiva/prevenção & controle , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Modelos Animais de Doenças , Vírus da Cinomose Canina/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Parvovirus Canino/efeitos dos fármacos , Vírus da Raiva/fisiologia , Análise de Sobrevida , Carga Viral
18.
Virol Sin ; 26(2): 81-94, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21468931

RESUMO

Severe Acute Respiratory Syndrome (SARS) is a deadly infectious disease caused by SARS Coronavirus (SARS-CoV). Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV. However, safe and potent adjuvants, especially with more efficient and economical needle-free vaccination are always needed more urgently in a pandemic. The development of a safe and effective mucosal adjuvant and vaccine for prevention of emergent infectious diseases such as SARS will be an important advancement. PIKA, a stabilized derivative of Poly (I:C), was previously reported to be safe and potent as adjuvant in mouse models. In the present study, we demonstrated that the intraperitoneal and intranasal co-administration of inactivated SARS-CoV vaccine together with this improved Poly (I:C) derivative induced strong anti-SARS-CoV mucosal and systemic humoral immune responses with neutralizing activity against pseudotyped virus. Although intraperitoneal immunization of inactivated SARS-CoV vaccine alone could induce a certain level of neutralizing activity in serum as well as in mucosal sites, co-administration of inactivated SARS-CoV vaccine with PIKA as adjuvant could induce a much higher neutralizing activity. When intranasal immunization was used, PIKA was obligatorily for inducing neutralizing activity in serum as well as in mucosal sites and was correlated with both mucosal IgA and mucosal IgG response. Overall, PIKA could be a good mucosal adjuvant candidate for inactivated SARS-CoV vaccine for use in possible future pandemic.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Poli I-C/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos BALB C , Poli I-C/administração & dosagem , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
19.
Biochem Biophys Res Commun ; 392(4): 582-7, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20102702

RESUMO

Flagellin contains conserved N/C domains for TLR5 binding to activate innate immunity and a middle hypervariable domain harboring the major antigenic epitopes. However, conflict results existed in the previous studies as to whether the hypervariable domain was involved in the cytokine production and adjuvancy of flagellin. Here we constructed three flagellin variants (designated as FliCDelta190-278, FliCDelta220-320, and FliCDelta180-400) with deletions in the hypervariable domain. Our data demonstrated that all deletion variants lost substantial antigenicity but not mucosal adjuvancy. Surprisingly, the variant with deletion of amino acids 220-320 (FliCDelta220-320) induced higher production of IL-8, MCP-1, and TNF-alpha, and showed higher mucosal adjuvancy than full-length FliC flagellin. Our data supported the notion that the hypervariable domain was involved in the cytokine production by flagellin and more importantly demonstrated that the hypervariable domain was important for the mucosal adjuvancy of flagellin.


Assuntos
Adjuvantes Imunológicos/farmacologia , Flagelina/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Proteínas Recombinantes/imunologia , Salmonella enterica/imunologia , Animais , Variação Antigênica/genética , Citocinas/biossíntese , Flagelina/genética , Flagelina/farmacologia , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Deleção de Sequência
20.
Clin Vaccine Immunol ; 16(11): 1595-600, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19776200

RESUMO

Viral antigens complexed to heat shock proteins (HSPs) can enhance antiviral immunity. The present study evaluated the immunogenicity of a novel human immunodeficiency virus type 1B' (HIV-1B')-specific, human leukocyte antigen A2 (HLA-A2)-restricted peptide (FLQSRPEPTA, Gag(448-457)) and the cellular immune adjuvant effect of HSP gp96 using the HLA-A2 transgenic mouse model. It was found that gp96 could augment cytotoxic-T-lymphocyte responses specific for the 10-mer peptide of HIV-1B'. This study also evaluated the humoral immune adjuvant effect of HSP gp96 and its N-terminal fragment (N336) and found that immunization of BALB/c mice with a mixture of gp96 or its N-terminal fragment and HIV-1 p24 antigen or with an p24-N336 fusion protein resulted in a significant increase in anti-HIV p24 antibody titer. These results demonstrate the possibility of using gp96 and its N fragment as adjuvants to augment cellular and humoral immune responses against HIV-1 infection.


Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Neoplasias/farmacologia , Anticorpos Anti-HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Feminino , HIV-1/imunologia , Humanos , Camundongos , Proteínas Recombinantes de Fusão/imunologia
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