Stable Expression Of Tuberculosis Vaccine Antigen In Lettuce Chloroplasts

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon the developmental stage and time of leaf harvest with highestlevel of accumulation in mature leaves harvested at 6PM. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Lyophilization increased CTB-ESAT6 protein content per gram of leaf material by 22 fold. Western blot analysis of lyophilized lettuce leaves showed that the CTB-ESAT6 fusion protein was stable and can be stored for prolonged period at RT. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of ESAT-6 antigen. GM-1 binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to interact with GM1 ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens fused to CTB in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB vaccine with potential for long term storage at room temperatur

The autoimmune disease-associated SNP rs917997 of IL18RAP controls IFNγ production by PBMC

AbstractType 1 Diabetes (T1D) is an autoimmune disorder characterized by aberrant T cell responses. Innate immune activation defects may facilitate a T helper 1 (Th1) phenotype. The cytokine IL-18 synergizes with IL-12 to induce IFNγ production and Th1 differentiation. The IL-18R subunit (IL18RAP) SNP rs917997 has been linked to decreased IL18RAP gene expression. Prior reports link rs917997 allele A with protection from T1D, and conversely with susceptibility to Celiac disease. However, few studies have investigated the IL-18 pathway in T1D. In this study, we analyzed responsiveness to IL-18 in T1D, and the effect of rs917997 genotype on IL18RAP gene expression post-activation. Upon IL-12 and IL-18 treatment, peripheral blood mononuclear cells from subjects carrying susceptibility alleles at rs917997 produced higher levels of IFNγ than those with protective genotypes. Additionally, the SNP modified IL18RAP surface protein expression by NK cells and gene expression in activated T cells. Taken together, these data suggest that the disease-associated rs917997 allele G permits hyperresponsiveness to IL-18, providing a novel target for therapeutic intervention in T1D

Interplay between IL-10, IFN-γ, IL-17A and PD-1 Expressing EBNA1-Specific CD4+ and CD8+ T Cell Responses in the Etiologic Pathway to Endemic Burkitt Lymphoma

Children diagnosed with endemic Burkitt lymphoma (eBL) are deficient in interferon-γ (IFN-γ) responses to Epstein–Barr Nuclear Antigen1 (EBNA1), the viral protein that defines the latency I pattern in this B cell tumor. However, the contributions of immune-regulatory cytokines and phenotypes of the EBNA1-specific T cells have not been characterized for eBL. Using a bespoke flow cytometry assay we measured intracellular IFN-γ, IL-10, IL-17A expression and phenotyped CD4+ and CD8+ T cell effector memory subsets specific to EBNA1 for eBL patients compared to two groups of healthy children with divergent malaria exposures. In response to EBNA1 and a malaria antigen (PfSEA-1A), the three study groups exhibited strikingly different cytokine expression and T cell memory profiles. EBNA1-specific IFN-γ-producing CD4+ T cell response rates were lowest in eBL (40%) compared to children with high malaria (84%) and low malaria (66%) exposures (p < 0.0001 and p = 0.0004, respectively). However, eBL patients did not differ in CD8+ T cell response rates or the magnitude of IFN-γ expression. In contrast, eBL children were more likely to have EBNA1-specific CD4+ T cells expressing IL-10, and less likely to have polyfunctional IFN-γ+IL-10+ CD4+ T cells (p = 0.02). They were also more likely to have IFN-γ+IL-17A+, IFN-γ+ and IL-17A+ CD8+ T cell subsets compared to healthy children. Cytokine-producing T cell subsets were predominantly CD45RA+CCR7+ TNAIVE-LIKE cells, yet PD-1, a marker of persistent activation/exhaustion, was more highly expressed by the central memory (TCM) and effector memory (TEM) T cell subsets. In summary, our study suggests that IL-10 mediated immune regulation and depletion of IFN-γ+ EBNA1-specific CD4+ T cells are complementary mechanisms that contribute to impaired T cell cytotoxicity in eBL pathogenesis

Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts

Mapping the Immune Landscape in Endemic Burkitt Lymphoma Tumors and Developing a Humanized Mouse Model for Exploring Inter-Patient Tumor Variation

Endemic Burkitt lymphoma (eBL) is the leading pediatric cancer in sub-Saharan Africa and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Current treatment options in Africa are combination chemotherapy with a survival rate hovering around 50%. Relapsed or refractory eBL patients have failed to receive any targeted treatments in the clinic. Our focus was to delineate immune responses in eBL, interrogate the tumor variation in responses to targeted treatments and develop mouse models that can be used to target essential mediators of tumor pathogenesis. Immune-based treatments including immune checkpoint inhibition have recently become an effective therapeutic modality in oncology. However, some B cell lymphomas such as Hodgkin Lymphoma (HL), are more receptive to checkpoint inhibition than others suggesting a need to understand the efficacy of checkpoint inhibition on different lymphoma subtypes. Checkpoint inhibitors act by blocking inhibitory receptors on T cells and improving anti-tumor responses. One of the goals of this thesis was to characterize checkpoint inhibitors on Tumor-infiltrating lymphocytes (TILs) in eBL tumors and to identify T cell subsets that exhibit increased expression of inhibitory receptors, poor cytokine production, poor proliferation and express transcription factors associated with exhaustion. Using scRNA seq, we identified T cell clusters that co-expressed inhibitory receptors, poor proliferative markers but also sustained costimulatory signals, as well as cytokine expression suggesting a pre dysfunctional state and not terminally exhausted state. Furthermore, we quantified the dominant co-inhibitory receptors PD1 and TIGIT that are upregulated in the tumor microenvironment via immunohistochemistry (IHC) and in peripheral blood of eBL patients via flow cytometry. We compared eBL patients with healthy pediatric cohorts with a history of persistent malaria exposure to those who had little to no malaria infections, to understand uniquely T cell mediated responses in BL children. Tumors had high co-expression of PD1 and TIGIT but fewer PD1 only populations, suggesting that both ligands may play a role in restraining immune activation via IHC. Next, we investigated if PD1 ligands or TIGIT ligands were overexpressed in eBL tumors. Nectin-2, TIGIT ligand was highly expressed in eBL tumors but was not highly correlated with TIGIT expression. These studies provide insights for PD1/ TIGIT blockade in Burkitt lymphoma patients. Additionally, we established new patient-derived cell lines from eBL tumors to study tumor variation and to study targeted treatments. We established five new patient-derived eBL lines BL717, BL 719, BL720, BL725, and BL740 that were interrogated for their inter-patient variation by studying their gene expression profiles. Further, we developed a patient cell-line derived xenograft (CDX) mouse model by injecting newly patient-derived BL cell lines in immunodeficient mice (NSG BL) and studying BL tumorigenesis. Having successfully established NSG BL tumors, we observed differences in tumor growth sensitivity and survival. We tested rituximab efficacy, one of the most established treatments for B cell lymphomas in our mouse model. We also identified pathways associated with unfolded protein response (UPR) and the mammalian target of rapamycin (mTOR) signaling, as well as apoptosis in one of the cell line xenografts, BL740, in response to rituximab. BL717, BL720 cell line xenograft failed to control tumor growth and was enriched in IFN-ɑ signature genes. This mouse model will prove to be useful to study combination therapy against eBL tumors as well as mechanisms of resistance to drug targets. Collectively, these studies provide insights into intratumoral variation including subtypes during tumor progression and expression profiles of TILs in eBL tumors. This will be important in designing new therapeutic strategies as well as help pose novel therapeutic targets

Isolation and identification of Candida Species in Patients with Vulvovaginal Candidiasis

Abnormal vaginal discharge is one of the frequent complaints of women of reproductive age group. This study was carried out to determine the prevalence of vulvovaginal candidiasis(VVC) among the patients attending the tertiary care hospital with complaints of suggestive of vaginitis. This study was done in a tertiary care hospital, Chennai for a period of 1 year from January 2017 to December 2017. The study included 160 women of the age group 15 - 65 years with complaints suggestive of vaginitis. High vaginal swabs were taken and subjected to direct microscopy, cultured onto Sabouraud Dextrose Agar (SDA) and Hichrome Candida differential agar. Candida species were determined by standard microbiological methods and the results were confirmed by automated VITEK2 compact. Candida species were isolated from 56 patients which included C.albicans (25), C.tropicalis (20), C.glabrata (6), C.parapsilosis(4), C.krusei(1). Our study shows higher prevalence of non albicans Candida causing VVC. Hence, we recommend that the investigations up to species identification of Candida may be routinely followed in the microbiology laboratories

Functional evaluation of CTB-ESAT6 fusion protein in fresh and lyophilized lettuce leaves.

<p>A, Ganglioside GM1 ELISA binding assay of CTB-ESAT6 derived from total leaf protein (TLP) of transplastomic plants. 1, 2∶15 µg TLP from CTB-ESAT6 transplastomic plants; WT, 15 µg TLP from untransformed lettuce plant; CTB, purified CTB standard; BSA, 50 ng bovine serum albumin. B, Ganglioside GM1 ELISA binding assay of CTB-ESAT6 from fresh and lyophilized lettuce leaves. Fresh and lyophilized CTB-ESAT6 lettuce leaf extracts starting from 15 µg equal concentrations and with decreasing two fold dilutions were analyzed for binding assay; Wild type, untransformed leaf extract; Purified CTB, 10 ng purified CTB standard.</p

Quantitation of vaccine antigens in transplastomic lines at different developmental stages and harvesting time.

<p>A, Expression levels of tobacco CTB-ESAT6 (Nt) protein in percent total soluble protein (TSP) as a function of leaf age and harvesting time under normal growth conditions determined by ELISA. B, Expression levels of tobacco CTB-Mtb72F (Nt) protein in percent TSP in young, mature and old leaves collected from the transplastomic lines by ELISA. Expression levels of CTB-ESAT6 (Ls) protein in lettuce transplastomic lines in percent total protein (TP) by densitometry at different leaf developmental stages (C) and at various time of harvest (D). 1 to 3, transplastomic lines. Expression levels of CTB-ESAT6 (Nt) and CTB-Mtb72F (Nt) are reported as percentage of the total soluble protein whereas CTB-ESAT6 (Ls) is in percentage of the total protein. Initially, ELISA was performed to quantify CTB-ESAT6 (Nt) in the total soluble protein but subsequent analysis of pellet, supernatant and homogenate fractions of leaf extracts detected fusion protein in the insoluble fraction. Therefore, CTB-ESAT6 (Ls) fusion protein was estimated based on percentage of total leaf protein. The CTB-Mtb72F fusion protein was quantitated based on percentage of the total soluble protein as the fusion protein existed only in the soluble fraction. Data presented is mean ± SD of triplicate experiments. Protein was extracted from three independent plants and amount of CTB-fusion protein was quantified either by ELISA or by densitometry on western blots. Three independent measurements were made to quantify expression levels of fusion protein and a mean ± SD value was plotted on the graph.</p

Western blot analysis to detect expression of recombinant proteins in transplastomic plants.

<p>Immunoblots with anti CTB antibody showing presence of expected size fusion protein band in CTB-ESAT6 tobacco (A) and CTB-ESAT6 lettuce (B) transplastomic lines. Immunoblots showing pentameric CTB-ESAT6 fusion protein detection with protein extracted under non-reducing/non-denaturing conditions and loaded on SDS-PAGE without boiling (C). Immunoblots showing distribution of recombinant fusion protein in the homogenate, supernatant and pellet fractions of leaf extract from CTB-ESAT6 tobacco (D), CTB-ESAT6 lettuce (E) and CTB-Mtb72F tobacco (F) transplastomic plants. (N, Untransformed; H, homogenate; S, S1 & S2: supernatant; P, pellet; 1 to 4, transplastomic plants; C, purified bacterial CTB standard; C1, C2, C3 & C4: CTB standards 25, 50, 75, 100 ng for densitometry). Immunoblot using rat anti-LipY antibody showing presence of expected band in LipY transplastomic tobacco plants (G). Lane1, untransformed; Lanes 2 to 7, transformants. Immunoblot using rat anti-LipY antibody showing presence of expected band in LipY transplastomic lettuce plants (H). Lane 1, Supernatant fraction of leaf extract from transplastomic plant; Lane 2, Homogenate fraction of leaf extract from transplastomic plant; Lane 3, untransformed.</p