Cutaneous Leishmaniasis (CL), a neglected vector-borne disease caused by protozoan parasite Leishmania major (L. major), is a major public health concern, and the development of new strategies to reduce the disease incidence has become a top priority. Advances in immunoinformatics and in-silico epitope prediction could be a promising approach to designing a finest vaccine candidate. In this study, we aimed to design a peptide-based vaccine against CL using computational tools and identified ten B-cell-derived T-cell epitopes from the glycoprotein gp63 of L. major. All of the potential immunodominant epitopes were used to design a vaccine construct along with a linker and an adjuvant at the N-terminal for enhancing its immunogenicity. Additionally, many characteristics of the proposed vaccine were examined, and it was confirmed to be non-allergenic, non-toxic, and thermally stable. To assess the vaccine interaction with the innate immune toll-like receptor-4 (TLR-4), a 3D structure of the vaccine construct was developed. Molecular docking and molecular dynamic simulation were used to confirm the binding and to assess the stability of the vaccine-TLR4 complex and interactions, respectively. In conclusion, our multi-epitope vaccine will provide a gateway to analyze the protein function of a potential vaccine candidate against CL.

Introduction: Toxoplasma gondii is an opportunistic protozoan and can be grown using several human cell lines. Breast cancer is the second leading cause of cancer death in women. Her2/Neu-expressing mammary cancer cell lines called TUBO can be grown in vitro. In recent years, protozoan parasites have become popular means of use in cancer therapy research. In this study, we analyzed whether T. gondii tachyzoites can destroy TUBO cells using a novel continuous feed medium approach. Methodology: Two sets of flasks (each containing four groups) containing TUBO cells were inoculated with T. gondii Ankara strain tachyzoites. First set containing 5×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 1:2, 1:1, 2:1, and 4:1 and second set containing 1×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 10:1, 100:1, 1000:1, and 2000:1. Thereafter, culture supernatants were harvested at various days until TUBO cells were destroyed and tachyzoites were counted. Results: In the first and second sets of flasks, TUBO cells were destroyed between days 8 to 12 and 12 to 25, respectively. In addition, the amount of tachyzoites increased 7- 43 and 595 to 112500 times in the first and second set of flasks, respectively. Conclusions: These results show that T. gondii tachyzoites successfully destroy Her2/Neu-expressing mammary cancer cells using a continuous feed medium approach. Although this idea may be too premature for the moment, the approach defined herein may support future researchers investigating the relationship between cancer and parasites which can make important progress toward saving cancer patient lives.

The heterogeneity of cancer strongly suggests the need to explore additional pathways to target. As cancer cells have increased proteotoxic stress, targeting proteotoxic stress-related pathways such as endoplasmic reticulum stress is attracting attention as a new anticancer treatment. One of the downstream responses to endoplasmic reticulum stress is endoplasmic reticulum-associated degradation (ERAD), a major degradation pathway that facilitates proteasome-dependent degradation of unfolded or misfolded proteins. Recently, SVIP (small VCP/97-interacting protein), an endogenous ERAD inhibitor, has been implicated in cancer progression, especially in glioma, prostate, and head and neck cancers. Here, the data of several RNA-sequencing (RNA-seq) and gene array studies were combined to evaluate the SVIP gene expression analysis on a variety of cancers, with a particular focus on breast cancer. The mRNA level of SVIP was found to be significantly higher in primary breast tumors and correlated well with its promoter methylation status and genetic alterations. Strikingly, the SVIP protein level was found to be low despite increased mRNA levels in breast tumors compared to normal tissues. On the other hand, the immunoblotting analysis showed that the expression of SVIP protein was significantly higher in breast cancer cell lines compared to non-tumorigenic epithelial cell lines, while most of the key proteins of gp78-mediated ERAD did not exhibit such an expression pattern, except for Hrd1. Silencing of SVIP enhanced the proliferation of p53 wt MCF-7 and ZR-75-1 cells but not p53 mutant T47D and SK-BR-3 cells; however, it increased the migration ability of both types of cell lines. Importantly, our data suggest that SVIP may increase p53 protein levels in MCF7 cells by inhibiting Hrd1-mediated p53 degradation. Overall, our data reveal the differential expression and function of SVIP on breast cancer cell lines together with in silico data analysis.

The heterogeneity of cancer strongly suggests the need to explore additional pathways to target. As cancer cells have increased proteotoxic stress, targeting proteotoxic stress-related pathways such as endoplasmic reticulum stress is attracting attention as new anticancer treatments. One of the downstream responses to endoplasmic reticulum stress is endoplasmic reticulum-associated degradation (ERAD), a major degradation pathway that facilitates proteasome-dependent degradation of unfolded or misfolded proteins. Recently, SVIP, an endogenous ERAD inhibitor, has been implicated in cancer progression, especially in glioma, prostate, and head and neck cancers. Here, the data of several RNA-seq and gene array studies were combined to evaluate the SVIP gene expression analysis on a variety of cancers, with a particular focus on breast cancer. SVIP was found to be overexpressed in primary breast tumors compared to normal tissues correlated well with its promoter methylation status and genetic alterations. Similarly, immunoblotting analysis showed that SVIP was expressed significantly higher in breast cancer cell lines compared to non-tumorigenic epithelial cell line. On the other hand, the expression of the key proteins of gp78-mediated ERAD did not exhibit such a pattern. Interestingly, silencing of SVIP enhanced the proliferation of p53 wt MCF7 cells but not p53 mutant T47D cells, however increased migration ability of both cell lines. Interestingly, SVIP expression is high in primary breast tumors but low in breast metastatic tumors. This correlates well with a lower probability of survival of breast cancer patients with lower SVIP expression compared to the patients with overexpressed SVIP. Overall, our data revealing the differential expression and function of SVIP on breast cancer cell lines together with in silico data analysis suggest that SVIP may have complex functions in breast cancer progression and has the potential to be a therapeutic target for breast cancer.