The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform

Chuan Wang - One of the best experts on this subject based on the ideXlab platform.

  • Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses.
    Scientific reports, 2020
    Co-Authors: Ming-juan Jiang, Xiang Zhang, Si-jing Liu, Tian Tang, Chuan Wang
    Abstract:

    We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.

  • Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii
    Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition, 2020
    Co-Authors: Ting Liu, Si-jing Liu, Yu-zhen Zhou, Yun-wen Zhang, Chuan Wang
    Abstract:

    Objective To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. Methods T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. Results A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P

  • construction and evaluation of a tuberculosis multistage vaccine candidate based on recombinant Listeria ivanovii
    Journal of Sichuan University. Medical science edition, 2020
    Co-Authors: Ting Liu, Si-jing Liu, Yu-zhen Zhou, Yun-wen Zhang, Chuan Wang
    Abstract:

    Objective To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. Methods T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. Results A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P<0.005). Additionally, the frequency of specific TNF-α + CD4 + T cell in LI -msv immunized group was significantly higher than that of vector control ( P<0.01) and NS control group ( P<0.005), and TNF-α + CD8 + T cell frequency obviously increased than NS control group ( P<0.005). Conclusions A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.

  • The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation.
    Journal of molecular microbiology and biotechnology, 2017
    Co-Authors: Si-jing Liu, Xiang Zhang, Ming-juan Jiang, Chenyan Ren, Tian Tang, Li Yongyu, Chuan Wang
    Abstract:

    Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

  • Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein
    Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition, 2017
    Co-Authors: Xiang Zhang, Si-jing Liu, Ming-juan Jiang, Huan Huang, Chuan Wang
    Abstract:

    OBJECTIVE Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSION The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

Si-jing Liu - One of the best experts on this subject based on the ideXlab platform.

  • Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses.
    Scientific reports, 2020
    Co-Authors: Ming-juan Jiang, Xiang Zhang, Si-jing Liu, Tian Tang, Chuan Wang
    Abstract:

    We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.

  • Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii
    Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition, 2020
    Co-Authors: Ting Liu, Si-jing Liu, Yu-zhen Zhou, Yun-wen Zhang, Chuan Wang
    Abstract:

    Objective To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. Methods T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. Results A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P

  • construction and evaluation of a tuberculosis multistage vaccine candidate based on recombinant Listeria ivanovii
    Journal of Sichuan University. Medical science edition, 2020
    Co-Authors: Ting Liu, Si-jing Liu, Yu-zhen Zhou, Yun-wen Zhang, Chuan Wang
    Abstract:

    Objective To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. Methods T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. Results A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P<0.005). Additionally, the frequency of specific TNF-α + CD4 + T cell in LI -msv immunized group was significantly higher than that of vector control ( P<0.01) and NS control group ( P<0.005), and TNF-α + CD8 + T cell frequency obviously increased than NS control group ( P<0.005). Conclusions A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.

  • The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation.
    Journal of molecular microbiology and biotechnology, 2017
    Co-Authors: Si-jing Liu, Xiang Zhang, Ming-juan Jiang, Chenyan Ren, Tian Tang, Li Yongyu, Chuan Wang
    Abstract:

    Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

  • Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein
    Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition, 2017
    Co-Authors: Xiang Zhang, Si-jing Liu, Ming-juan Jiang, Huan Huang, Chuan Wang
    Abstract:

    OBJECTIVE Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSION The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

Jingli Liu - One of the best experts on this subject based on the ideXlab platform.

  • Prevalence and Characteristics of Listeria ivanovii Strains in Wild Rodents in China
    Vector borne and zoonotic diseases (Larchmont N.Y.), 2018
    Co-Authors: Xiaolong Cao, Lu Zhang, Yan Wang, Yi Wang, Lijuan Luo, Pengfei Wang, Jingli Liu
    Abstract:

    Listeria is ubiquitous in natural environments and can be isolated from animal hosts, including rodents. Listeria ivanovii and Listeria monocytogenes are the main pathogenic species that can cause serious listeriosis in animals and human. In this study, we investigated the prevalence of Listeria in wild rodents from six regions in China, and analyzed the molecular characteristics and antibiotics resistance of the L. ivanovii isolates. A total of 702 fecal samples of 25 different species of wild rodents were examined, and 75 were Listeria positive, including 26 L. ivanovii strains, 2 L. monocytogenes strains, and 47 Listeria innocus strains. The 26 L. ivanovii isolates (including 2 subspecies) were divided into 5 different sequence types by multilocus sequence typing with ST6 being the dominant type, and 5 different pulsotypes by pulsed-field gel electrophoresis. The results of antimicrobial susceptibility revealed that all L. ivanovii isolates were sensitive to rifampin, chloramphenicol, gentamicin, erythromycin, tetracycline, vancomycin, penicillin G, imipenem, trimethoprim-sulfamethoxazole, but some isolates (including ST1, ST7, and ST8) were resistant to clindamycin. The results suggest that the prevalence of L. ivanovii (3.7%, 26/702) in wild rodents was higher in some regions, and the genetic diversity of L. ivanovii isolates in the wild rodents is relatively low and most belong to one lineage. These wild rodents may act as the natural host for L. ivanovii and possibly transmit the pathogen between wild animals and humans.

Hao Shen - One of the best experts on this subject based on the ideXlab platform.

  • Listeria ivanovii Infection in Mice: Restricted to the Liver and Lung with Limited Replication in the Spleen
    Frontiers in microbiology, 2016
    Co-Authors: Mengying Zhou, Si-jing Liu, Ming-juan Jiang, Hao Shen, Chenyan Ren, Howard Goldfine, Chuan Wang
    Abstract:

    Listeria monocytogenes (LM) vectors have shown much promise in delivery of viral and tumor antigens for the development of vaccines. Listeria ivanovii (LI) is a closely related bacterium with a similar intracellular life cycle that may offer advantages over LM because it is not a human pathogen, but can infect other animal species. Recent studies show that recombinant LI expressing M. tuberculosis antigens is effective in inducing protective immunity in mouse models, demonstrating the potential of LI as a live vaccine vector. However, a key barrier in the development of LI into a live vaccine vector is that its pathogenic and immunogenic characteristics have yet to be fully understood. Therefore, in this research, C57BL/6J mice were inoculated with LM or LI intravenously or intranasally, and bacterial loads, histopathologic changes, and cytokine production were determined at indicated days post inoculation. Results showed that after intravenous infection with LM or LI, bacteria were found proliferating in the liver, spleen and lung. However, LI could only reach a heavy burden in the liver and its ability to multiply and to resist host immunity seemed limited in the spleen and lung. After intranasal inoculation with LI, bacteria were mainly localized in the lung and failed to infect liver or spleen, while LM could. In organs with heavy LI burden, lesions were isolated, localized and densely packed, compared to lesions caused by LM, which were invasive. In the liver of intravenously inoculated mice and lung of intranasally inoculate mice, LI was able to elicit comparable cytokine production with LM and cause less severe histopathologic damages, and thus could be considered as a vector for treating or preventing hepatic or pulmonary diseases.

  • Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.
    Journal of biotechnology, 2015
    Co-Authors: Qingqing Lin, Mengying Zhou, Asma Khanniche, Hao Shen, Chuan Wang
    Abstract:

    Bacillus Calmette-Guerin (BCG) has failed in complete control of tuberculosis (TB), thus, novel tuberculosis vaccines are urgently needed. We have constructed several TB vaccine candidates, which are characterized by the use of Listeria ivanovii (LI) strain as an antigen delivery vector. Two L. ivanovii attenuated recombinant strains L. ivanovii△actAplcB-Rv0129c and L. ivanovii△actAplcB-Rv3875 were successfully screened. Results from genome PCR and sequencing showed that the Mycobacterium tuberculosis antigen gene cassette coding for Ag85C or ESAT-6 protein respectively had been integrated into LI genome downstream of mpl gene. Western blot confirmed the secretion of Ag85C or ESAT-6 protein from the recombinant LI strains. These two recombinant strains showed similar growth curves as wide type strain in vitro. In vivo, they transiently propagated in mice spleen and liver, and induced specific CD8(+) IFN-γ secretion. Therefore, in this paper, two novel LI attenuated strains expressing specific TB antigens were successfully constructed. The promising growth characteristics in mice immune system and the capability of induction of IFN-γ secretion make them of potential interest for development of TB vaccines.

  • Expression of porcine respiratory and reproductive syndrome virus membrane-associated proteins in Listeria ivanovii via a genome site-specific integration and expression system.
    Journal of molecular microbiology and biotechnology, 2014
    Co-Authors: Chuan Wang, Asma Khanniche, Fuqin Zhang, Jianglong Yang, Hao Shen
    Abstract:

    Listeria ivanovii (LI) possesses a facultative intracellular life cycle in macrophages and non-professional phagocytes thus making it a potential novel vaccine vector. We have developed a genome site-specific integration system as a universal tool for constructing the live LI-based vaccines. Using this system, the porcine reproductive and respiratory syndrome virus (PRRSV) orf5 and orf6 genes, coding for PRRSV membrane-associated protein GP5 and M, respectively, were integrated into the LI genome. Genome PCR and sequencing results showed that the orf5 or orf6 gene had been integrated into the LI genome downstream of orfXYZ or mpl gene. Western blot analysis of the non-secreted and secreted bacterial protein samples showed the expression and secretion of GP5 or M protein from the recombinant LI strains. The secretion of the heterologous proteins is a key step for bacterial vaccine vectors to deliver the antigens to the immune system to stimulate antigen-specific immune responses. Therefore, this integration system may be a potential tool for constructing novel vaccines using live LI as vector.

Ming-juan Jiang - One of the best experts on this subject based on the ideXlab platform.

  • Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses.
    Scientific reports, 2020
    Co-Authors: Ming-juan Jiang, Xiang Zhang, Si-jing Liu, Tian Tang, Chuan Wang
    Abstract:

    We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.

  • The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation.
    Journal of molecular microbiology and biotechnology, 2017
    Co-Authors: Si-jing Liu, Xiang Zhang, Ming-juan Jiang, Chenyan Ren, Tian Tang, Li Yongyu, Chuan Wang
    Abstract:

    Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

  • Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein
    Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition, 2017
    Co-Authors: Xiang Zhang, Si-jing Liu, Ming-juan Jiang, Huan Huang, Chuan Wang
    Abstract:

    OBJECTIVE Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSION The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

  • Listeria ivanovii Infection in Mice: Restricted to the Liver and Lung with Limited Replication in the Spleen
    Frontiers in microbiology, 2016
    Co-Authors: Mengying Zhou, Si-jing Liu, Ming-juan Jiang, Hao Shen, Chenyan Ren, Howard Goldfine, Chuan Wang
    Abstract:

    Listeria monocytogenes (LM) vectors have shown much promise in delivery of viral and tumor antigens for the development of vaccines. Listeria ivanovii (LI) is a closely related bacterium with a similar intracellular life cycle that may offer advantages over LM because it is not a human pathogen, but can infect other animal species. Recent studies show that recombinant LI expressing M. tuberculosis antigens is effective in inducing protective immunity in mouse models, demonstrating the potential of LI as a live vaccine vector. However, a key barrier in the development of LI into a live vaccine vector is that its pathogenic and immunogenic characteristics have yet to be fully understood. Therefore, in this research, C57BL/6J mice were inoculated with LM or LI intravenously or intranasally, and bacterial loads, histopathologic changes, and cytokine production were determined at indicated days post inoculation. Results showed that after intravenous infection with LM or LI, bacteria were found proliferating in the liver, spleen and lung. However, LI could only reach a heavy burden in the liver and its ability to multiply and to resist host immunity seemed limited in the spleen and lung. After intranasal inoculation with LI, bacteria were mainly localized in the lung and failed to infect liver or spleen, while LM could. In organs with heavy LI burden, lesions were isolated, localized and densely packed, compared to lesions caused by LM, which were invasive. In the liver of intravenously inoculated mice and lung of intranasally inoculate mice, LI was able to elicit comparable cytokine production with LM and cause less severe histopathologic damages, and thus could be considered as a vector for treating or preventing hepatic or pulmonary diseases.