Umthong, Supawadee
(Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University)
,
Buaklin, Arun
(Department of Microbiology, Faculty of Medicine, Chulalongkorn University)
,
Jacquet, Alain
(Chulalongkorn University Vaccine Research Center, Chulalongkorn University)
,
Sangjun, Noppadol
(Armed Forces Research Institute of Medical Sciences)
,
Kerdkaew, Ruthairat
(Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University)
,
Patarakul, Kanitha
(Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University)
,
Palaga, Tanapat
(Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University)
Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigate...
Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.
Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.
To monitor the encapsulation efficacy, naked pMax-GFP plasmid (Amaxa, USA), a model plasmid, and CS/pMax-GFP nanoparticles prepared at N/P ratios of 1:1, 2:1, 4;1, 8:1, 10:1, and 20:1 were loaded onto 1% agarose gels in 1× TAE buffer. Naked plasmid pMax-GFP was used as a control to compare the efficacy of chitosan in encapsulating DNA.
To compare the formulation of the DNA vaccine containing two ORFs within a single plasmid with the co-administration of lipL32 and loa22 in different plasmids, mice were immunized three times by intramuscular (i.m.) injection with 10 pmol of CS/pVITRO-lipL32-loa22 or co-immunized with CS/pVITRO-lipL32+CS/pVITRO-loa22 at 10 pmol each, on days 1, 30, and 44 (Fig. 3).
대상 데이터
Female BALB/c mice (8 weeks old) were purchased from the National Laboratory Animal Centre, Mahidol University, Thailand. They were housed at the Department of Pathology, Faculty of Medicine, Chulalongkorn University, Thailand.
성능/효과
1B) indicated that LipL32 and Loa22 were secreted extracellularly rather than released as a result of cell necrosis. Comparison of the expression levels of LipL32 and Loa22 in pVITRO-lipL32-loa22-transfected cells with pVITRO-lipL32 and pVITRO-loa22 co-transfected cells revealed that pVITRO-lipL32-loa22, which carries both inserts in the same plasmid, resulted in a slightly higher expression of both proteins than the co-transfection method, especially for the level of Loa22 (Fig. 1B).
CFSE was used to monitor cell proliferation after restimulation with recombinant antigens, and gated CD4+ T cells were analyzed for cell proliferation by flow cytometry. Mice immunized with the combination of LipL32 and Loa22 antigens or vaccinated with a single LipL32 or Loa22 antigen showed no significant differences in CD4+ T cell proliferation upon restimulation with 20 µg/ml of LipL32 or Loa22 (Fig. 4A).
Therefore, in this study, a combination of LipL32 and Loa22 was investigated as a candidate vaccine by using biopolymer chitosan as a delivery system for lipL32 and loa22 DNA vaccines. Furthermore, since our unpublished data indicated that DNA vaccines of either lipL32 and loa22 alone induced antibody response poorly, the heterologous prime-boost immunization regimen was carried out using DNA vaccine for priming and recombinant protein vaccine as a booster.
Therefore, in this study, a combination of LipL32 and Loa22 was investigated as a candidate vaccine by using biopolymer chitosan as a delivery system for lipL32 and loa22 DNA vaccines. Furthermore, since our unpublished data indicated that DNA vaccines of either lipL32 and loa22 alone induced antibody response poorly, the heterologous prime-boost immunization regimen was carried out using DNA vaccine for priming and recombinant protein vaccine as a booster.
The first formulation was CS/pVITRO-lipL32-loa22 (two ORFs in one plasmid) and the other consisted of a CS/pVITRO-lipL32+CS/pIVTRO-loa22 co-administration (two ORFs in two plasmids). There was no significant difference in LipL32 protein expression levels in vitro after transfection with the two formulations, in contrast to Loa22 expression levels, in which the transfection of CS/pVITRO-lipL32-loa22 (two ORFs in one plasmid) resulted in higher levels in both cell lysates and culture supernatants than the cotransfection method. This result was consistent with the in vivo humoral responses observed after vaccination with equimolar concentrations of the DNA vaccines.
This result was consistent with the in vivo humoral responses observed after vaccination with equimolar concentrations of the DNA vaccines. The levels of total Loa22-specific IgG were higher in mice immunized with CS/pVITRO-lipL32-loa22 than those receiving co-immunization by the two plasmids. In addition to differences in protein antigen expression, other mechanisms may also contribute to the observed diminished effect in the two-plasmid formulation.
Cytokine measurements of in vitro restimulated splenocytes revealed that all vaccinated groups showed significantly increased levels of Th1 (IFN-γ and IL-2) but not Th2 (IL-4 and IL-10) cytokines compared with the control groups. More importantly, splenocytes from mice receiving two antigens produced significantly more IFN-γ than those from mice receiving a single antigen.
Using intracellular cytokine staining, however, we could not detect any differences in the percentage of IFN-γ CD4+ T cells or in fluorescent intensities for these cytokines in CD4+ T cells between these two groups (data not shown). Therefore, it is possible that other cell subsets in the spleen may be responsible for the greater production of cytokines observed.
Combining LipL32 and Loa22 antigens did not provide a synergistic effect in stimulating humoral immune responses against LipL32 or Loa22, and the combination of both antigens did not act antagonistically against each other. We observed only a synergistic effect in an in vitro restimulation assay for Th1 cytokines.
후속연구
Therefore, the use of LipL32 antigen alone may be insufficient to achieve complete protection. The objective of this study was to therefore test whether a combination of LipL32 with another antigen could promote better humoral and cell-mediated immune responses than when using LipL32 alone.
Although the synergistic effect on the humoral immune response between LipL32 and Loa22 may not have been observed, a combination of the two antigens may promote better protective efficacy than using a LipL32 or Loa22 vaccine alone. The protective efficacy of LipL32 and Loa22 in combination needs to be studied further in pathogen-challenged animal models. To determine the precise synergistic protective effects of LipL32-Loa22 vaccine candidates and their protective efficiency, hamster or guinea pig models should be tested in the future.
The protective efficacy of LipL32 and Loa22 in combination needs to be studied further in pathogen-challenged animal models. To determine the precise synergistic protective effects of LipL32-Loa22 vaccine candidates and their protective efficiency, hamster or guinea pig models should be tested in the future.
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