Leptospira borgpetersenii Leucine-Rich Repeat Proteins Provide Strong Protective Efficacy as Novel Leptospiral Vaccine Candidates.

Leucine-rich repeat (LRR) proteins are advocated for being assessed in vaccine development. Leptospiral LRR proteins were identified recently in silico from the genome of Leptospira borgpetersenii serogroup Sejroe, the seroprevalence of leptospiral infections of cattle in Thailand. Two LRR recombinant proteins, rKU_Sej_LRR_2012M (2012) and rhKU_Sej_LRR_2271 (2271), containing predicted immunogenic epitopes, were investigated for their cross-protective efficacies in an acute leptospirosis model with heterologous Leptospira serovar Pomona, though, strains from serogroup Sejroe are host-adapted to bovine, leading to chronic disease. Since serovar Pomona is frequently reported as seropositive in cattle, buffaloes, pigs, and dogs in Thailand and causes acute and severe leptospirosis in cattle by incidental infection, the serogroup Sejroe LRR proteins were evaluated for their cross-protective immunity. The protective efficacies were 37.5%, 50.0%, and 75.0% based on the survival rate for the control, 2012, and 2271 groups, respectively. Sera from 2012-immunized hamsters showed weak bactericidal action compared to sera from 2271-immunized hamsters (p < 0.05). Therefore, bacterial tissue clearances, inflammatory responses, and humoral and cell-mediated immune (HMI and CMI) responses were evaluated only in 2271-immunized hamsters challenged with virulent L. interrogans serovar Pomona. The 2271 protein induced prompt humoral immune responses (p < 0.05) and leptospiral tissue clearance, reducing tissue inflammation in immunized hamsters. In addition, protein 2271 and its immunogenic peptides stimulated splenocyte lymphoproliferation and stimulated both HMI and CMI responses by activating Th1 and Th2 cytokine gene expression in vaccinated hamsters. Our data suggest that the immunogenic potential renders rhKU_Sej_LRR_2271 protein a promising candidate for the development of a novel cross-protective vaccine against animal leptospirosis.

Expression of a leptospiral leucine-rich repeat protein using a food-grade vector in Lactobacillus plantarum, as a strategy for vaccine delivery.

In this study, a first food-grade mucosal vaccine against leptospirosis was developed without the use of antibiotic resistance gene. This expression system is based on a food-grade host/vector system of Lactobacillus plantarum and a new vaccine candidate antigen, a leucine-rich repeat (LRR) protein of Leptospira borgpetersenii. The LRR of interest from serovar Sejroe is encoded by two overlapping genes and these genes were fused together by site-directed mutagenesis. The mutant gene thus obtained could be successfully expressed in this system as was shown by western blot analysis and liquid chromatography-mass spectrometry (LC-MS/MS) analysis. In addition, this analysis showed that the mutant LRR protein fused to a homologous signal peptide of L. plantarum could be exported to the cell surface as a result of the native LPXAG motif of the heterologous LRR protein, which presumably is responsible for anchoring the protein to the cell wall of L. plantarum. This new strategy could be an essential tool for further studies of leptospirosis mucosal vaccine delivery.

Dimorphism in the T-cell receptor constant region affects T-cell function, phenotype and HIV outcome.

OBJECTIVES: CD8 T cells recognize human leukocyte antigen-peptide complex through the T-cell receptor. Although amino acid variation in T-cell receptor variable chains often affects antigen specificity, dimorphism in the beta chain constant region (TRBC1 and TRBC2) is not thought to affect T-cell function. A recent study suggested that adoptive transfer of TRBC1-specific chimeric antigen-receptor-T cells provided an option for T-cell leukemia therapy that preserved T-cell immunity in the TRBC2 subset. This raises an important question as to whether TRBC1T cells are qualitatively different from TRBC2T cells. DESIGN: Cross-sectional study. METHODS: Sixty-six antiretroviral therapy-naive HIV-infected individuals, including 19 viraemic controllers and 47 noncontrollers, were enrolled. Peripheral blood mononuclear cells were isolated for T-cell functional assays, tetramer analyses, TRBC1 staining and immunophenotyping. RESULTS: Viraemic controllers had a higher proportion of circulating TRBC1T cells than noncontrollers, raising the possibility that TRBC1T cells might be associated with HIV control. TRBC1T cells also showed more functional T-cell responses against both HIV and cytomegalovirus (P < 0.01). The immunophenotypes of TRBC1-bearing T cells were skewed towards naive and central memory phenotypes, whereas the majority of TRBC2-expressing T cells were terminally differentiated. Inverse correlations were observed between %TRBC1T cells and HIV plasma viral load, which was most pronounced for CD8 T cells (r = -0.7096, P = 0.00002357). CONCLUSION: These data suggest that TRBC1T-cell responses are of better quality than their TRBC2 counterparts, which should be considered in immunotherapeutic strategies for HIV infection. Conversely, depletion of TRBC1T cells as part of the treatment of TRBC1 T-cell malignancies may lead to compromised T-cell response quality.

Clinical outcome of HIV viraemic controllers and noncontrollers with normal CD4 counts is exclusively determined by antigen-specific CD8+ T-cell-mediated HIV suppression.

In this cross-sectional study we evaluated T-cell responses using several assays to determine immune correlates of HIV control that distinguish untreated viraemic controllers (VC) from noncontrollers (NC) with similar CD4 counts. Samples were taken from 65 ART-naïve chronically HIV-infected VC and NC from Thailand with matching CD4 counts in the normal range (>450 cells/µl). We determined HIVp24-specific T-cell responses using standard Interferon-gamma (IFNγ) ELISpot assays, and compared the functional quality of HIVp24-specific CD8+ T-cell responses using polychromatic flow cytometry. Finally, in vitro HIV suppression assays were performed to evaluate directly the activity of CD8+ T cells in HIV control. Autologous CD4+ T cells were infected with primary patient-derived HIV isolates and the HIV suppressive activity of CD8+ T cells was determined after co-culture, measuring production of HIVp24 Ag by ELISA. The HIVp24-specific T-cell responses of VC and NC could not completely be differentiated through measurement of IFNγ-producing cells using ELISpot assays, nor by the absolute cell numbers of polyfunctional HIVp24-specific CD8+ T cells. However, in vitro HIV suppression assays showed clear differences between VC and NC. HIV suppressive activity, mediated by either ex vivo unstimulated CD8+ T cells or HIVp24-specific T-cell lines, was significantly greater using cells from VC than NC cells. Additionally, we were able to demonstrate a significant correlation between the level of HIV suppressive activity mediated by ex vivo unstimulated CD8+ T cells and plasma viral load (pVL) (Spearman r = -0.7345, p = 0.0003). This study provides evidence that in vitro HIV suppression assays are the most informative in the functional evaluation of CD8+ T-cell responses and can distinguish between VC and NC.

Poor HIV control in HLA-B*27 and B*57/58 noncontrollers is associated with limited number of polyfunctional Gag p24-specific CD8+ T cells.

OBJECTIVES: Analysis of immune response in HIV controllers, a unique group of infected individuals who are able to control HIV naturally, has provided us a chance to investigate the roles of host immune responses in HIV control. DESIGN: In this study, the functional quality of HIV Gag p24-specific CD8 T-cell responses was assessed in two groups of clinically distinct, HLA-B*27, HLA-B*57/58-matched individuals, viremic controllers [plasma HIV load (pVL) ≤ 2000 copies/ml) and noncontrollers (pVL >2000 copies/ml) to determine its impacts on natural HIV clinical outcome. METHODS: An ex-vivo interferon (IFN)-γ ELISpot assay was used to screen for each individual's HIV Gag p24-specific T-cell responses. Intracellular cytokine staining assay was used to determine their functional quality (as number of cytokine being produced). RESULTS: We found that, in contrast to previous studies, all Thai volunteers with HLA-B*5801 were uniformly noncontrollers. Viremic controllers were observed with a significantly larger number of high functional quality p24-specific CD8 T cells than noncontrollers (P < 0.05). This superior quality of responses was observed at both total p24 and epitope-specific level. Moreover, the absolute number of high functional quality Gag p24-specific CD8 T cells was significantly in a negative correlation with pVL (r = -0.6984, P = 0.0006) and also in a positive correlation with CD4 T-cell count (r = 0.5648, P = 0.0095). CONCLUSION: We concluded that an adequate number of high functional quality Gag p24-specific CD8 T cells is strongly associated with a natural HIV controller status.