Overexpression Of Antigens Research Articles

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Self-replicating RNA viruses in vaccine development

Self-replicating RNA viruses such as alphaviruses, flaviviruses, paramyxoviruses, and rhabdoviruses have been engineered as expression vectors for vaccine development. The prominent feature of self-replicating RNA viruses is their RNA-dependent RNA polymerase activity, which generates massive self-amplification of RNA in the cytoplasm of infected host cells leading to extreme levels of transgene expression. Infectious diseases have been targeted by overexpression of surface proteins of pathogens as antigens for vaccine development. Moreover, overexpression of tumor-associated antigens and immunostimulatory genes has been the basis for cancer vaccines. Proof-of-concept of robust antigen-specific immune responses and protection against challenges with lethal doses of infectious agents have been demonstrated. Likewise, vaccine development against various cancers has elicited strong immune responses and resulted in tumor regression and eradication, cure, and prolonged survival in animal tumor models. Good safety and immune responses have been achieved in clinical trials. The ERVEBO® vaccine, based on the vesicular stomatitis virus, has been approved for immunization against the Ebola virus disease.

The complexity of cancer immunotherapy illustrated through skin tumors.

Skin tumours are among the cancer types most sensitive to immunotherapy, due to their unique immunogenic features including skin-associated lymphoid tissue, high mutational load, overexpression of tumour antigens, and high frequency of viral antigens. Despite this high immunotherapy response rate, however, ultimately most skin tumours develop similar treatment resistance to most other malignant tumours, which highlights the need for in-depth study of mechanisms of response and resistance to immunotherapy. A bibliographic review of the most recent publications regarding currently in use and emerging biomarkers on skin tumors has been done. Predictive biomarkers of treatment response, biomarkers that warn of possible resistance, and emerging markers, the majority of a systemic nature, are described. Including factors affecting not only genomics, but also the immune system, nervous system, microbiota, tumour microenvironment, metabolism and stress. For accurate diagnosis of tumour type, knowledge of its functional mechanisms and selection of a comprehensive therapeutic protocol, this inclusive view of biology, health and disease is fundamental. This field of study could also become a valuable source of practical information applicable to other areas of oncology and immunotherapy.

Mesothelin: An Immunotherapeutic Target beyond Solid Tumors.

Simple SummaryThis review summarizes the current knowledge on mesothelin’s function, its role in cancer, and opportunities for immunotherapeutic targeting of mesothelin. Immunotherapies including monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T and NK-cells, targeted alpha therapies, and bispecific T cell engaging molecules are reviewed. We show future directions for mesothelin targeting in hematological malignancies, including acute myeloid leukemia.Modern targeted cancer therapies rely on the overexpression of tumor associated antigens with very little to no expression in normal cell types. Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein that has been identified in many different tumor types, including lung adenocarcinomas, ovarian carcinomas, and most recently in hematological malignancies, including acute myeloid leukemia (AML). Although the function of mesothelin is widely unknown, interactions with MUC16/CA125 indicate that mesothelin plays a role in the regulation of proliferation, growth, and adhesion signaling. Most research on mesothelin currently focuses on utilizing mesothelin to design targeted cancer therapies such as monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T and NK cells, bispecific T cell engaging molecules, and targeted alpha therapies, amongst others. Both in vitro and in vivo studies using different immunotherapeutic modalities in mesothelin-positive AML models highlight the potential impact of this approach as a unique opportunity to treat hard-to-cure AML.

DUSP22 Rearrangements Identify a Molecularly Distinct Type of Anaplastic Large Cell Lymphoma Characterized By DNA Hypomethylation and Lack of Activated STAT3

Background: Anaplastic large cell lymphomas (ALCLs) comprise a group of T-cell non-Hodgkin lymphomas that share common pathologic features such as CD30 expression, but demonstrate marked heterogeneity in clinical presentation, genetic features, and outcomes. Current classification of ALCLs is based on site of presentation (systemic or localized) and ALK status (positive or negative). Rearrangement of the DUSP22 locus is seen in ~30% of ALK-negative ALCLs and is considered a prognostically favorable biomarker, but the molecular features of these cases are incompletely understood. Specifically, their relationship to convergent mechanisms that activate STAT3 in both ALK-positive and ALK-negative ALCL has not been studied.Methods: Gene expression profiling (GEP) data from 31 frozen ALCL samples were analyzed using hierarchical clustering and gene-set enrichment analysis (GSEA). Immunohistochemistry for phosphorylated (p) STAT3 (Tyr705) was performed on whole-tissue paraffin sections and scored in a blinded fashion based on percentage of tumor cell nuclei staining. Reduced representation bisulfite sequencing (RRBS) was performed on DNA extracted from the 31 frozen ALCLs and differentially methylated regions (DMRs) were identified among genetic subtypes using methylKit software. RRBS and RNA sequencing also was performed on 6 T-cell lymphoma cell lines treated with the demethylating agent decitabine or vehicle alone, and gene expression and methylation profiles were evaluated.Results: Unsupervised clustering of GEP data identified two main groups of ALCLs, Cluster 1 (n=10) and Cluster 2 (n=21). All DUSP22- rearranged ALCLs (n=7) were in Cluster 1 whereas all ALK-positive ALCLs (n=8) were in Cluster 2. Both clusters contained ALK-negative ALCLs lacking DUSP22 rearrangements. GSEA identified significant depletion of IL6-JAK-STAT3 signaling pathway genes in Cluster 1 (NES=-2.05; p<0.001). Immunohistochemistry in 100 ALCLs confirmed low or absent pSTAT3 in DUSP22- rearranged cases (n=23; 9.2 ± 21.5 % versus 55.7 ± 35.2 % for ALCLs without DUSP22 rearrangements; p<0.0001). However, absent pSTAT3 alone could not explain the favorable prognosis in DUSP22- rearranged ALCLs, and the 3 ALK-negative ALCLs in Cluster 1 that lacked DUSP22- rearrangements showed aggressive clinical behavior. We previously found that DUSP22- rearranged ALCLs showed upregulation of cancer-testis antigen genes. Because these genes are regulated largely by methylation, we performed RRBS and found marked hypomethylation of DUSP22- rearranged ALCLs across all categories of gene-associated methylation sites (p≤0.0001). Furthermore, this hypomethylation could be recapitulated in vitro using decitabine, which led to upregulation of a DUSP22- specific gene expression signature in T-cell lymphoma cell lines.Conclusions: DUSP22- rearranged ALCLs are a molecularly distinct type of ALCL. These cases belong to a group of ALCLs that show down-regulation of STAT3-associated genes and lack activated STAT3 protein. Furthermore, DUSP22- rearranged ALCLs show global hypomethylation that contributes to their unique GEP signature, including up-regulation of cancer-testis antigens. These data suggest DUSP22- rearranged ALCLs may be less sensitive to therapies targeting JAK-STAT3 that have been proposed in ALCL but might benefit from immunotherapy approaches, which have efficacy in other cancers demonstrating hypomethylation and overexpression of cancer-testis antigens. DisclosuresAnsell:Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering .

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.

Aberrant activation of Wnt/catenin signaling and overexpression of ABCG2 contributes to apoptosis down regulation and tumor progression of high grade ovarian cancer.

Side Population (SP) cells are the small pool of CSC like progenitor cells, which are drug resistant and recapitulate tumor generation. The occurrence of SP cells is the major inference for attaining a better treatment and improved patient survival. In this work, we have isolated 6% SP cells from a high grade ovarian carcinoma. Our functional characterization of SP cells revealed that elevated ABCG2 and anti-apoptotic factors contribute to chemoresistance and increased life span of SP cells. Further, the overexpression of surface antigens, such as CD133 and CD117 in SP cells, are the key driving forces for high clonogenic and invasion properties of SP cells. More importantly, we found by RT-PCR aberrant activation and upregulation of Wnt/ β-catenin and its downstream targeting genes, such as DKK1 and AXIN2 in SP cells. These findings suggest that development of new anticancer drugs which target Wnt/β-catenin signaling might effectively exterminate the SP cells and aid in disease free survival.

Mechanism of EBV inducing anti-tumour immunity and its therapeutic use.

SummaryTumour-associated antigens (TAAs) comprise a large collection of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as immunotherapy targets has been explored for over two decades1, yet the genesis of TAA-specific T cells remains elusive. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses including Epstein-Barr virus (EBV) and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here, we show that expression of the EBV signaling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signaling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on MHC-I and -II (mainly through the endogenous pathway), and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a novel mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in patient tumour B cells and thereby empowering them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a broad array of endogenous tumour antigens, such as TAAs and neoantigens, for treating B-cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches for cancers.

Overexpression of Schistosome-specific Antigens for Selection of scFv Antibody from Phage Display Libraries of Water Buffalo for Potential Use in Schistosomiasis Diagnosis

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes of the genus Schistosoma. The Kato-Katz method is the standard for the diagnosis of intestinal schistosomiasis because of its specificity, simplicity, and low cost. However, Kato-Katz is less useful in light infections and other available detection methods are neither practical nor sensitive for routine or large-scale screening. Thus, improved tests are needed for the assessment of infection. The water buffalo is a natural host and reservoir of Schistosoma but is schistosomiasis-resistant, suggesting a robust immune repertoire, which can be tapped as a source of antibodies for diagnosis of schistosomiasis. Two schistosome antigens had been previously shown to be recognized by a phage display single-chain fragment of variation (scFv) library derived from a schistosome-infected water buffalo. However, specific antibodies have not been isolated using these antigens due to a lack of sufficient amounts and purified forms of the antigens. In this study, the schistosome antigens were cloned and overexpressed in vector with solubility tag, purified by immobilized metal affinity chromatography (IMAC), and used to interrogate the immune scFv library to isolate and identify specific schistosome antibody. An scFv specific to schistosome SAP domain-containing hypothetical protein was isolated but was only 105-bp long although certain regions align with the IgG VL, tetraglycylserine (Gly4Ser) linker, and IgG VH sequences of synthetic scFv constructs. Despite its length, scFv A63 can be potentially designed as a ligand in direct ELISA (enzyme-linked immunosorbent assay) by fusion with enzyme conjugates such as alkaline phosphatase to detect S. japonicum schistosomule and adult worm. The results in this study also suggest the need to construct a bigger and more diverse scFv library and to test a wider range of schistosome antigens, specifically those that will be present on the surface of the schistosome at all stages of S. japonicum life cycle.

MicroRNA (miR) dysregulation during Helicobacter pylori-induced gastric inflammation and cancer development: critical importance of miR-155.

Dysregulation of noncoding microRNA molecules has been associated with immune cell activation in the context of Helicobacter pylori induced gastric inflammation as well as carcinogenesis, but also with downregulation of mismatch repair genes, and may interfere with immune checkpoint proteins that lead to the overexpression of antigens on gastric tumor cells. Numerous miR-molecules have been described as important tools and markers in gastric inflammation and cancer development —including miR-21, miR-143, miR-145, miR-201, and miR-335— all of which are downregulated in gastric tumors, and involved in cell cycle growth or tumor invasion. Among the many microRNAs involved in gastric inflammation, adenocarcinoma development and immune checkpoint regulation, miR-155 is notable in that its upregulation is considered a key marker of chronic gastric inflammation that predisposes a patient to gastric carcinogenesis. Among various other miRs, miR-155 is highly expressed in activated B and T cells and in monocytes/macrophages present in chronic gastric inflammation. Notably, miR-155 was shown to downregulate the expression of certain MMR genes, such as MLH1, MSH2, and MSH6. In tumor-infiltrating miR-155-deficient CD8+ T cells, antibodies against immune checkpoint proteins restored the expression of several derepressed miR-155 targets, suggesting that miR-155 may regulate overlapping pathways to promote antitumor immunity. It may thus be of high clinical impact that gastric pathologies mediated by miR-155 result from its overexpression. This suggests that it may be possible to therapeutically attenuate miR-155 levels for gastric cancer treatment and/or to prevent the progression of chronic gastric inflammation into cancer.

Abstract B70: Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults. The current standard-of-care treatment, which includes surgical resection, radiation, and chemotherapy, extends the survival of patients to merely 14.6 months. GBM is virtually incurable due to its heterogeneity, which is represented by tumor cells with different patterns of gene expression (proneural, neural, classical, and mesenchymal GBM), the presence of GBM cancer stem (like) cells (CSC), and immune evasion. Thus, new therapeutic strategies directed at eliminating GBM CSC in particular are urgently needed. Utilizing fresh patient material from surgical resections, GBM CSC were successfully enriched and cultivated in serum-free medium and subsequently differentiated by switching to medium containing FBS. The gene expression pattern of both, GBM CSC and differentiated cells, was characterized. Furthermore, a screening platform was tailored for flow-cytometric characterization of patient-specific antigen expression of all newly established glioblastoma cell lines. In this work we present a promising immunotherapeutic approach towards eradication of GBM CSC by combining the universal targeting and controllability of our recently developed adapter chimeric antigen receptor (AdCAR) system with the “off-the-shelf” properties of the continuously expandable and well-characterized NK cell line NK-92. The AdCAR system is based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin, which can be linked to monoclonal antibodies (mAb). Utilizing these biotinylated mAb as adapter molecules, the system allows precise quantitative (on-/off switch) as well as qualitative (change and combination of target antigen) regulation of immune cell function. AdCAR-transduced NK-92 cells demonstrated significant target cell lysis, which, importantly, is largely independent from activation of endogenously expressed NK receptors and the presence of their ligands on target cells but mainly mediated by AdCAR activation through binding to the respective tumor antigen. In the presence of the respective antigen, first and foremost biotinylated antibodies directed at CD9, CD146, CD276, and EGFR were capable of inducing significant AdCAR NK-92-mediated lysis of GBM cells after 2 hours. Interestingly, due to overexpression of various target antigens on GBM cancer stem cells, CSC lysis was noticeably higher as compared to AdCAR-mediated lysis of differentiated glioblastoma cells. In conclusion, we have successfully generated a CAR-modified NK cell line, AdCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens, allowing universal and tunable targeting of glioblastoma cells. Most importantly, AdCAR NK-92 cells provide a promising strategy to eradicate GBM cancer stem (like) cells. Citation Format: Stefan Grote, Chun-Ho Chan, Caroline Baden, Stephan M. Huber, Franziska Eckert, Joerg Mittelstaet, Andrew Kaiser, Christian Seitz, Patrick Schlegel, Rupert Handgretinger, Sabine Schleicher. Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B70.

Deep Learning to Estimate Human Epidermal Growth Factor Receptor 2 Status from Hematoxylin and Eosin-Stained Breast Tissue Images

Context: Several therapeutically important mutations in cancers are economically detected using immunohistochemistry (IHC), which highlights the overexpression of specific antigens associated with the mutation. However, IHC panels can be imprecise and relatively expensive in low-income settings. On the other hand, although hematoxylin and eosin (H&E) staining used to visualize the general tissue morphology is a routine and low cost, it does not highlight any specific antigen or mutation. Aims: Using the human epidermal growth factor receptor 2 (HER2) mutation in breast cancer as an example, we strengthen the case for cost-effective detection and screening of overexpression of HER2 protein in H&E-stained tissue. Settings and Design: We use computational methods that reliably detect subtle morphological changes associated with the over-expression of mutation-specific proteins directly from H&E images. Subjects and Methods: We trained a classification pipeline to determine HER2 overexpression status of H&E stained whole slide images. Our training dataset was derived from a single hospital containing 26 (11 HER2+ and 15 HER2–) cases. We tested the classification pipeline on 26 (8 HER2+ and 18 HER2–) held-out cases from the same hospital and 45 independent cases (23 HER2+ and 22 HER2–) from the TCGA-BRCA cohort. The pipeline was composed of a stain separation module and three deep neural network modules in tandem for robustness and interpretability. Statistical Analysis Used: We evaluate our trained model through area under the curve (AUC)-receiver operating characteristic. Results: Our pipeline achieved an AUC of 0.82 (confidence interval [CI]: 0.65–0.98) on held-out cases and an AUC of 0.76 (CI: 0.61–0.89) on the independent dataset from TCGA. We also demonstrate the region-level correspondence of HER2 overexpression between a patient’s IHC and H&E serial sections. Conclusions: Our work strengthens the case for automatically quantifying the overexpression of mutation-specific proteins in H&E-stained digital pathology, and it highlights the importance of multi-stage machine learning pipelines for added robustness and interpretability.

Tumor-associated antigens identified early in mouse mammary tumor development can be effective vaccine targets

Breast cancer vaccines composed of antigens identified by serological analysis of cDNA expression libraries (SEREX) induce antigen specific immune responses in patients but have had disappointing clinical benefits. While many attempts to modify the adjuvants and vaccine method have been tried, one issue not addressed was whether the SEREX tumor-associated antigens identified from late stages of disease were ideal targets. We questioned in the transgenic TgMMTV-neu mouse model whether the antigen repertoire is distinct between early and late stage breast cancer and whether the antigens identified via SEREX from transgenic mice with early or late stage tumors would elicit differential anti-tumor effects to address this question.Three early stage antigens, Pdhx, Stk39, and Otud6B, were identified from a SEREX screen of mice prior to development of palpable lesions. Formulated into a vaccine, each early antigen inhibited tumor growth (p < 0.0001). The antigens identified from mice with late stage tumors (Swap70, Gsn, and Arhgef2) were unable to inhibit tumor growth when used as vaccines (for example Gsn p = 0.26). Each of the three early stage antigens were essential for tumor survival in syngeneic mouse tumor cells and in human breast cancer cell lines across breast cancer subtypes. Silencing protein expression of the early antigens increased apoptosis (p < 0.0001 for all antigens in mouse and p < 0.05 for all antigens in human triple negative breast cancer) and decreased survival (p < 0.0001 for all antigens in mouse and human triple negative and HER2 positive breast cancer). Overexpression of the early stage antigens in women with breast cancer predicted worse prognosis (p = 0.03) while overexpression of late stage antigens did not impact prognosis (p = 0.09). These data suggest that antigens expressed earlier in breast tumor development and functionally relevant to breast tumor growth may be more effective targets for therapeutic breast cancer vaccines than antigens identified in later disease.

Development of an auxotrophic, live-attenuated Brucella suis vaccine strain capable of expressing multimeric GnRH

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Targeting HPV-16 antigens to the endoplasmic reticulum induces an endoplasmic reticulum stress response.

Very promising results have been observed with a deoxyribonucleic acid (DNA) vaccine based on human papillomavirus type-16 (HPV-16) antigen retention and delivery system in the endoplasmic reticulum (ER). However, the mechanism by which these antigens are processed once they reach this organelle is still unknown. Therefore, we evaluated whether this system awakens a stress response in the ER. Different DNA constructs based on E6 and E7 mutant antigens fused to an ER signal peptide (SP), a signal for retention in the ER (KDEL), or both signals (SPK), were transfected into HEK-293 cells. Overexpression of E6 and E7 antigens targeted to the ER (SP, and SPK constructs) induced ER stress, which was indicated by an increase of the ER-stress markers GRP78/BiP and CHOP. Additionally, the ER stress response was mediated by the ATF4 transcription factor, which was translocated into the nucleus. Besides, the overexpressed antigens were degraded by the proteasome. Through a cycloheximide-chase assay, we demonstrated that when both protein synthesis and proteasome were inhibited, the overexpressed antigens were degraded. Interestingly, when proteasome was blocked autophagy was increased and the ER stress response decreased. Taken together, these results indicate that the antigens are initially degraded by the ERAD pathway, and autophagy degradation pathway can be induced to compensate the proteasome inhibition. Therefore, we provided a new insight into the mechanism by which E6 and E7 mutant antigens are processed once they reach the ER, which will help to improve the development of more effective vaccines against cancer.

ClearColi BL21(DE3)-based expression of Zika virus antigens illustrates a rapid method of antibody production against emerging pathogens

Available rapid, simple and accurate methods for detection and diagnosis of emerging viral diseases are required. Recently, there was an urgent need for specific antibodies against mosquito-borne Zika virus (ZIKV), which is an emerging zoonotic disease of medical concern in different regions of the world. Here, we showed that overexpression of ZIKV antigens in ClearColi BL21(DE3), a bacteria strain expressing a non-endotoxic form of LPS, is suitable for the production of specific ZIKV antisera. Two major ZIKV antigenic domains, the domain III from envelope E glycoprotein, which brings the virus-specific epitopes, and the N-terminal region of nonstructural NS1 glycoprotein, which is responsible for pathophysiological conditions, were overexpressed in ClearColi BL21(DE3). Immunization of adult rat with insoluble recombinant ZIKV antigens in inclusion bodies resulted in the production of specific antibodies in a few weeks. Anti-E and anti-NS1 antibodies are efficient as biological tools for ZIKV detection by indirect ELISA and immunoblot assay. This method could successfully be applied to any emerging viruses.

Paired Expression Analysis of Tumor Cell Surface Antigens.

Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or with one another provided the most promising paired-hits. We propose that targeting these markers together would increase the specificity and thereby the safety of CAR-based therapy for neuroblastoma.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin.

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker.OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo.METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression.FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector.MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Detecting of p16 Autoantibody as a Potential Early Diagnostic Serum Biomarker in Patients with Cervical Cancer.

Over-expression of tumor-associated antigens (TAAs) may trigger secretion of their auto-antibodies. The present work was designed to test whether circulating antibody to P16 protein-derived antigens was altered in cervical cancer. 141 cases of cervical cancer patients, 133 cases of cervical benign tumor patients, and 153 healthy volunteers matched in age were recruited. The level of circulating P16 auto-antibody was tested using an ELISA developed in-house with linear peptide antigens derived from the P16 protein. The P16 auto-antibody in the malignant tumor group had a significantly higher level than the healthy control group and the benign tumor group (t = 4.016, p < 0.001; t = 3.879, p < 0.001). Patients with stage I cervical cancer have the highest level of P16 autoantibody and the sensitivity against > 90% specificity was 20.3%. The circulating auto-antibody to P16 may be one of a series of potential biomarkers with early prognostic values for cervical cancer.

Outer membrane vesicles as platform vaccine technology.

Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.