RESUMO
The T cell response is an important detection index in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development. The present study was undertaken to determine the T cell epitopes in the spike (S) protein of SARS-CoV-2 that dominate the T cell responses in SARS-CoV-2-infected patients. PBMCs from rhesus macaques vaccinated with a DNA vaccine encoding the full-length S protein were isolated, and an ELISPOT assay was used to identify the recognized T cell epitopes among a total of 158 18-mer and 10-aa-overlapping peptides spanning the full-length S protein. Six multipeptide-based epitopes located in the S1 region, with four of the six located in the receptor-binding domain, were defined as the most frequently recognized epitopes in macaques. The conservation of the epitopes across species was also verified, and peptide mixtures for T cell response detection were established. Six newly defined T cell epitopes were found in the current study, which may provide a novel potential target for T cell response detection and the diagnosis and vaccine design of SARS-CoV-2 based on multipeptide subunit-based epitopes.
Assuntos
Epitopos de Linfócito T/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Macaca mulattaRESUMO
BACKGROUND: Hepatitis B virus (HBV) X protein (HBX) has been reported to be responsible for the epithelial-mesenchymal transition (EMT) in HBV-related hepatocellular carcinoma (HCC). Vimentin is an EMT-related molecular marker. However, the importance of vimentin in the pathogenesis of HCC mediated by HBX has not been well determined. METHODS: The expression of vimentin induced by HBX, and the role of LIM and SH3 domain protein 1 (LASP1) in HBX-induced vimentin expression in hepatoma cells were examined by western blot and immunohistochemistry analysis. Both the signal pathways involved in the expression of vimentin, the interaction of HBX with vimentin and LASP1, and the stability of vimentin mediated by LASP1 in HBX-positive cells were assessed by western blot, Co-immunoprecipitation, and GST-pull down assay. The role of vimentin in EMT, proliferation, and migration of HCC cells mediated by HBX and LASP1 were explored with western blot, CCK-8 assay, plate clone formation assay, transwell assay, and wound healing assay. RESULTS: Vimentin expression was increased in both HBX-positive hepatoma cells and HBV-related HCC tissues, and the expression of vimentin was correlated with HBX in HBV-related HCC tissues. Functionally, vimentin was contributed to the EMT, proliferation, and migration of hepatoma cells mediated by HBX. The mechanistic analysis suggested that HBX was able to enhance the expression of vimentin through LASP1. On the one hand, PI3-K, ERK, and STAT3 signal pathways were involved in the upregulation of vimentin mediated by LASP1 in HBX-positive hepatoma cells. On the other hand, HBX could directly interact with vimentin and LASP1, and dependent on LASP1, HBX was capable of promoting the stability of vimentin via protecting it from ubiquitination mediated protein degradation. Besides these, vimentin was involved in the growth and migration of hepatoma cells mediated by LASP1 in HBX-positive hepatoma cells. CONCLUSION: Taken together, these findings demonstrate that, dependent on LASP1, vimentin is crucial for HBX-mediated EMT and hepatocarcinogenesis, and may serve as a potential target for HBV-related HCC treatment. Video abstract.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , Proteínas do Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Proteínas com Domínio LIM/metabolismo , Neoplasias Hepáticas/patologia , Transativadores/metabolismo , Vimentina/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Ligação Proteica , Estabilidade Proteica , Transdução de Sinais , Regulação para CimaRESUMO
Hepatitis B virus (HBV) is a human hepatotropic virus. However, HBV infection also occurs at extrahepatic sites, but the relevant host factors required for HBV infection in non-hepatic cells are only partially understood. In this article, a non-hepatic cell culture model is constructed by exogenous expression of four host genes (NTCP, HNF4α, RXRα and PPARα) in human non-hepatic 293T cells. This cell culture model supports HBV entry, transcription and replication, as evidenced by the detection of HBV pgRNA, HBV cccDNA, HBsAg, HBeAg, HBcAg and HBVDNA. Our results suggest that the above cellular factors may play a key role in HBV infection of non-hepatic cells. This model will facilitate the identification of host genes that support extrahepatic HBV infection.
Assuntos
Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Hepatócitos/virologia , Linhagem Celular , Linhagem Celular Tumoral , DNA Viral/genética , Células HEK293 , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Replicação Viral/genéticaRESUMO
Neuraminidase 1 (NEU1) has been reported to be associated with hepatocellular carcinoma (HCC). However, the function and associated molecular mechanisms of NEU1 in hepatitis B virus (HBV)-related HCC have not been well investigated. In the present study, the expression of NEU1 mediated by HBV and HBV core protein (HBc) was measured in hepatoma cells. The expression of NEU1 protein was detected via immunohistochemical analysis in HBV-associated HCC tissues. The role of NEU1 in the activation of signaling pathways and epithelial-mesenchymal transition (EMT) and the proliferation and migration of hepatoma cells mediated by HBc was assessed. We found that NEU1 was upregulated in HBV-positive hepatoma cells and HBV-related HCC tissues. HBV promoted NEU1 expression at the mRNA and protein level via HBc in hepatoma cells. Mechanistically, HBc was able to enhance the activity of the NEU1 promoter through NF-κB binding sites. In addition, through the increase in NEU1 expression, HBc contributed to activation of downstream signaling pathways and EMT in hepatoma cells. Moreover, NEU1 facilitated the proliferation and migration of hepatoma cells mediated by HBc. Taken together, our findings provide novel insight into the molecular mechanism underlying the oncogenesis mediated by HBc and demonstrate that NEU1 plays a vital role in HBc-mediated functional abnormality in HCC. Thus, NEU1 may serve as a potential therapeutic target in HBV-associated HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Hepatite B/metabolismo , Neoplasias Hepáticas/metabolismo , Neuraminidase/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuraminidase/genética , Proteínas do Core Viral/genéticaRESUMO
Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45â% after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.
Assuntos
Técnicas de Introdução de Genes , Genoma Viral , Herpesvirus Humano 1/genética , Mutagênese Insercional , Animais , Sistemas CRISPR-Cas , Chlorocebus aethiops , Reparo do DNA por Junção de Extremidades , Células HEK293 , Humanos , Plasmídeos , Células VeroRESUMO
Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans in late 2019, it has rapidly spread worldwide. To identify the biological characteristics of SARS-CoV-2 in a normal laboratory environment (biosafety level 2 [BSL-2]), a lentiviral-based nucleocapsid was used to carry the spike protein of SARS-CoV-2 onto the surface of pseudoviral particles as a surrogate model to evaluate the infective characterization of SARS-CoV-2. This study indicated that SARS-CoV-2 has extensive tissue tropism for humans and may infect monkeys and tree shrews but not rodents. More importantly, the use of pseudoviral particles in this study allows rapid assessment of neutralizing antibodies in serum in a BSL-2 laboratory. This study will provide a quick and easy tool for evaluating neutralizing antibodies in the serum of recovering patients and assessing the potency of candidate vaccines.
Assuntos
COVID-19/virologia , Engenharia Genética , Recombinação Genética , SARS-CoV-2/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Linhagem Celular , Células Cultivadas , Genes Reporter , Humanos , Testes de Neutralização , Especificidade de Órgãos , SARS-CoV-2/fisiologia , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Transdução Genética , Vacinas Virais , Vírion/genéticaRESUMO
Interactions between hepatitis C virus (HCV) and lipoproteins in humans play an important role in the efficient establishment of chronic infection. Apolipoprotein E (ApoE) on the HCV envelope mediates virus attachment to host cells as well as immune evasion. This interaction is thought to occur in hepatocytes, as ApoE plays dual functions in HCV assembly and maturation as well as cell attachment. In the present study, we found that secreted ApoE (sApoE) can also bind to viral particles via its C-terminal domain after HCV is released from the cell. Furthermore, the binding affinity of interactions between the sApoE N terminus and cell surface receptors affected HCV infectivity in a dose-dependent manner. The extracellular binding of sApoE to HCV is dependent on HCV envelope proteins, and recombinant HCV envelope proteins are also able to bind to sApoE. These results suggest that extracellular interactions between HCV and sApoE may potentially complicate vaccine development and studies of viral pathogenesis.IMPORTANCE End-stage liver disease caused by chronic HCV infection remains a clinical challenge, and there is an urgent need for a prophylactic method of controlling HCV infection. Because host immunity against HCV is poorly understood, additional investigations of host-virus interactions in the context of HCV are important. HCV is primarily transmitted through blood, which is rich in lipoproteins. Therefore, it is of interest to further determine how HCV interacts with lipoproteins in human blood. In this study, we found that secreted ApoE (sApoE), an exchangeable component found in lipoproteins, participates in extracellular interactions with HCV virions. More significantly, different variants of sApoE differentially affect HCV infection efficiency in a dose-dependent manner. These findings provide greater insight into HCV infection and host immunity and could help propel the development of new strategies for preventing HCV infection.
Assuntos
Apolipoproteínas E/metabolismo , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Humanos , Ligação ProteicaRESUMO
A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.
Assuntos
Engenharia Genética/métodos , Genoma Viral , Edição de RNA/genética , RNA Guia de Cinetoplastídeos/genética , Ribonucleases/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Dados de Sequência Molecular , Ribonucleases/genética , Células Vero , Vacinas Virais/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Neutrophils, underestimated in multiple sclerosis (MS), are gaining increased attention for their significant functions in patients with MS and the experimental autoimmune encephalomyelitis (EAE) animal model. However, the precise role of neutrophils in cervical lymph nodes (CLNs), the primary CNS-draining lymph nodes where the autoimmune response is initiated during the progression of EAE, remains poorly understood. METHODS: Applying single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive immune cell atlas of CLNs during development of EAE. Through this atlas, we concentrated on and uncovered the transcriptional landscape, phenotypic and functional heterogeneity of neutrophils, and their crosstalk with immune cells within CLNs in the neuroinflammatory processes in EAE. RESULTS: Notably, we observed a substantial increase in the neutrophil population in EAE mice, with a particular emphasis on the significant rise within the CLNs. Neutrophils in CLNs were categorized into 3 subtypes, and we explored the specific roles and developmental trajectories of each distinct neutrophil subtype. Neutrophils were found to engage in extensive interactions with other immune cells, playing crucial roles in T-cell activation. Moreover, our findings highlighted the strong migratory ability of neutrophils to CLNs, partly regulated by triggering the receptor expressed on myeloid cells 1 (TREM-1). Inhibiting TREM1 with LR12 prevents neutrophil migration both in vivo and in vitro. In addition, in patients with MS, we confirmed an increase in peripheral neutrophils with an upregulation of TREM-1. DISCUSSION: Our research provides a comprehensive and precise single-cell atlas of CLNs in EAE, highlighting the role of neutrophils in regulating the periphery immune response. In addition, TREM-1 emerged as an essential regulator of neutrophil migration to CLNs, holding promise as a potential therapeutic target in MS.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Neutrófilos , Análise de Célula Única , Receptor Gatilho 1 Expresso em Células Mieloides , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/genética , Neutrófilos/metabolismo , Neutrófilos/imunologia , Animais , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Camundongos , Feminino , Análise de Sequência de RNA , Linfonodos/metabolismoRESUMO
The current study aims to develop a safe and highly immunogenic COVID-19 vaccine. The novel combination of a DNA vaccine encoding the full-length Spike (S) protein of SARS-CoV-2 and a recombinant S1 protein vaccine induced high level neutralizing antibody and T cell immune responses in both small and large animal models. More significantly, the co-delivery of DNA and protein components at the same time elicited full protection against intratracheal challenge of SARS-CoV-2 viruses in immunized rhesus macaques. As both DNA and protein vaccines have been proven safe in previous human studies, and DNA vaccines are capable of eliciting germinal center B cell development, which is critical for high-affinity memory B cell responses, the DNA and protein co-delivery vaccine approach has great potential to serve as a safe and effective approach to develop COVID-19 vaccines that provide long-term protection.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , DNA/imunologia , Células HEK293 , Humanos , Contagem de Linfócitos , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Linfócitos T/imunologiaRESUMO
Preclinical studies require an immune response similar to that of humans in a small animal model that is convenient to operate. Based on genome alignment, tree shrews are small animals considered to be more similar to primates than are rodents, and many human disease models have been established with tree shrews. However, the characteristics of the humoral immune response of tree shrews remain to be elucidated. In this study, the genetic sequence of the heavy chain constant region of tree shrew immunoglobulin (Ig) was complemented, and the results of immunoglobulin domain homology and transcriptome analysis showed that the tree shrew genome encodes only four classes of antibodies and does not encode IgD. The oldest IgM antibody has the highest homology with primates. After the complete sequence of each type of antibody was obtained, the tree shrew antibody protein was further expressed and purified by in vitro recombination, and an IgG quantitative evaluation system was established. The highly effective immuno protective effect induced by HSV-1 infection and the significant bactericidal effect induced by Neisseria meningitidis group C polysaccharide immunization showed that tree shrews exhibited immune responses more similar to humans than to mice. This may provide better predictive value for vaccine preclinical research.
Assuntos
Sistema Imunitário/imunologia , Imunidade Humoral/imunologia , Tupaiidae/imunologia , Sequência de Aminoácidos , Animais , Células CHO , Sequência Conservada , Cricetinae , Cricetulus , DNA Complementar/genética , Feminino , Loci Gênicos , Genoma , Imunoglobulina G/química , Imunoglobulina G/genética , Masculino , Camundongos Endogâmicos BALB C , Filogenia , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Tupaiidae/genéticaRESUMO
OBJECTIVES: Interleukin-34 (IL-34) is associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, the role and associated mechanisms of IL-34 in HBV-related HCC remain unclear. In this study, the expression, biological function and associated mechanisms of IL-34 in HBV-related HCC cells were investigated. METHODS: IL-34 expression induced by HBV and HBV X (HBX) gene was measured in hepatoma cells. The role of CCAAT/enhancer-binding protein α (CEBP/α) in HBX-induced IL-34 expression was examined. The signal pathways involved in the expression of CEBP/α and IL-34 induced by HBX were assessed. The role of IL-34 in the proliferation and migration of HCC cells, and related mechanisms were explored. RESULTS: Dependent on HBX, HBV increased IL-34 expression in hepatoma cells, and HBX upregulated and interacted with CEBP/α to enhance the activity of IL-34 promoters. CEBP/α mediated by HBX was associated with the activation of PI3-K and NF-κB pathways to promote IL-34 expression. Via CSF1-R and CD138, IL-34 promoted the proliferation and migration of hepatoma cells, and contributed to the activation of ERK and STAT3 pathways and the upregulation of Bcl-xl and c-Myc mediated by HBX. CONCLUSION: We demonstrate that IL-34 contributes to HBX-mediated functional abnormality of HCC cells and provides a novel insight into the molecular mechanism of carcinogenesis mediated by HBX.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Interleucinas/metabolismo , Transativadores/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Hepatite B , Humanos , Neoplasias Hepáticas/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2-/- mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12â¯weeks, Gcn2-/- mice were less obese than wild-type (WT) mice, and Gcn2-/- significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/genética , Proteínas Serina-Treonina Quinases/deficiência , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Resistência à Insulina , Lipogênese , Fígado/metabolismo , Fígado/fisiopatologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Obesidade/induzido quimicamente , Obesidade/metabolismo , Obesidade/fisiopatologia , Estresse OxidativoRESUMO
The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes/métodos , Genoma Viral , Herpesvirus Humano 1/genética , RNA Guia de Cinetoplastídeos/genética , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endonucleases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Herpesvirus Humano 1/metabolismo , Recombinação Homóloga , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Seleção Genética , Células Vero , Replicação ViralRESUMO
AIM: To express Helicobacter pylori (Hp) alpA gene in a live delivery vehicle of lactococcus lactis (L. lactis) and assay the efficacy of the L. lactis-alpA oral vaccine in recipient mice. METHODS: The alpA gene of Hp was amplified by PCR and cloned into the prokaryotic expression vector pNICE: sec. The recombinant vector pNICE: sec-alpA was transformed into Lactococcus lactis strain NZ9000. Then the engineered strain was induced to express recombinant alpA as shown by SDS-PAGE and Western blot. Female ICR mice (CV Grade) were randomly divided into 4 groups and administrated orally with PBS, L. lactis pNICE: sec, L. lactis pNICE: sec-alpA, and the inactivated Hp, respectively. After immunized seven times, the mice were detected for their alpA-specific IgG and IgA. RESULTS: The alpA gene was obtained and successfully cloned into the vector pNICE: sec. The recombinant alpA protein (56,000) was accumulated in L. lactis after the induction of the nisin, accounting for 9.6% of the total bacterial protein. Western bolt confirmed that the alpA protein could be recognized specifically by the anti-Hp serum. The titer of anti-alpA IgG in the pNICE: sec-alpA group, comparable to that in the inactivated Hp group, was higher than that in the pNICE: sec group. The titer of anti-alpA IgA in the pNICE: sec-alpA group was higher than all other groups (P<0.05). CONCLUSION: The oral administration of the engineered alpA-expressing L. lactis induced protective immunity against Hp. Our study provides a certain experimental basis for the use of L. lactis as an antigen-delivering vehicle for the development of Hp oral vaccines.