Exploring the cancer-testis antigen BORIS to design a novel multi-epitope vaccine against breast cancer based on immunoinformatics approaches.

Recently, cancer immunotherapy has gained lots of attention to replace the current chemoradiation approaches and multi-epitope cancer vaccines are manifesting as the next generation of cancer immunotherapy. Therefore, in this study, we used multiple immunoinformatics approaches along with other computational approaches to design a novel multi-epitope vaccine against breast cancer. The most immunogenic regions of the BORIS cancer-testis antigen were selected according to the binding affinity to MHC-I and II molecules as well as containing multiple cytotoxic T lymphocyte (CTL) epitopes by multiple immunoinformatics servers. The selected regions were linked together by GPGPG linker. Also, a T helper epitope (PADRE) and the TLR-4/MD-2 agonist (L7/L12 ribosomal protein from mycobacterium) were incorporated by A(EAAAK)3A linker to form the final vaccine construct. Then, its physicochemical properties, cleavage sites, TAP transport efficiency, B cell epitopes, IFN-γ inducing epitopes and population coverage were predicted. The final vaccine construct was reverse translated, codon-optimized and inserted into pcDNA3.1 to form the DNA vaccine. The final vaccine construct was a stable, immunogenic and non-allergenic protein that contained numerous CTL epitopes, IFN-γ inducing epitopes and several linear and conformational B cell epitopes. Also, the final vaccine construct formed stable and significant interactions with TLR-4/MD-2 complex according to molecular docking and dynamics simulations. Moreover, its world population coverage for HLA-I and HLA-II were about 93% and 96%, respectively. Taking together, these preliminary results can be used as an appropriate platform for further experimental investigations. Communicated by Ramaswamy H. Sarma.

Immunoprotective effect of an in silico designed multiepitope cancer vaccine with BORIS cancer-testis antigen target in a murine mammary carcinoma model.

In our previous study, immunoinformatic tools were used to design a novel multiepitope cancer vaccine based on the most immunodominant regions of BORIS cancer-testis antigen. The final vaccine construct was an immunogenic, non-allergenic, and stable protein consisted of multiple cytotoxic T lymphocytes epitopes, IFN-γ inducing epitopes, and B cell epitopes according to bioinformatic analyzes. Herein, the DNA sequence of the final vaccine construct was placed into the pcDNA3.1 vector as a DNA vaccine (pcDNA3.1-VAC). Also, the recombinant multiepitope peptide vaccine (MPV) was produced by a transfected BL21 E. coli strain using a recombinant pET-28a vector and then, purified and screened by Fast protein liquid chromatography technique (FPLC) and Western blot, respectively. The anti-tumor effects of prophylactic co-immunization with these DNA and protein cancer vaccines were evaluated in the metastatic non-immunogenic 4T1 mammary carcinoma in BALB/c mice. Co-immunization with the pcDNA3.1-VAC and MPV significantly (P < 0.001) increased the serum levels of the MPV-specific IgG total, IgG2a, and IgG1. The splenocytes of co-immunized mice exhibited a significantly higher efficacy to produce interleukin-4 and interferon-γ and proliferation in response to MPV in comparison with the control. The prophylactic co-immunization regime caused significant breast tumors' growth inhibition, tumors' weight decrease, inhibition of metastasis formation, and enlarging tumor-bearing mice survival time, without any considerable side effects. Taking together, this cancer vaccine can evoke strong immune response against breast tumor and inhibits its growth and metastasis.

Efficacy of co-immunization with the DNA and peptide vaccines containing SYCP1 and ACRBP epitopes in a murine triple-negative breast cancer model.

Multiepitope cancer vaccines have gained lots of attention for prophylactic and therapeutic purposes in cancer patients. In our previous study, multiepitope DNA and peptide cancer vaccines consisted of the most immunodominant epitopes of ACRBP and SYCP1 antigens were designed by bioinformatic tools. In this study, the effect of prophylactic co-immunization with these DNA and peptide cancer vaccines in the 4T1 breast cancer animal model was assessed. Serum levels of the peptide-specific IgG total, IgG2a and IgG1 were measured by enzyme-linked immunosorbent assay (ELISA). Also, the efficacy of the immunized mice splenocytes' for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) was evaluated. The co-immunization caused a significant (P < .05) increase in the serum levels of IgG1 and IgG2a. The co-immunized mice splenocytes exhibited significantly enhanced IL-4 (6.6-fold) and IFN-γ (19-fold) production. Also, their lymphocytes exhibited higher proliferation rate (3-fold) and granzyme B production (6.5-fold) in comparison with the control. The prophylactic co-immunization significantly decreased the breast tumors' volume (78%) and increased the tumor-bearing mice survival time (37.5%) in comparison with the control. Taking together, prophylactic co-immunization with these multiepitope DNA and peptide cancer vaccines can activate the immune system against breast cancer. However, further experiments are needed to evaluate their efficacy from different angles.

Exploring the out of sight antigens of SARS-CoV-2 to design a candidate multi-epitope vaccine by utilizing immunoinformatics approaches.

SARS-CoV-2 causes a severe respiratory disease called COVID-19. Currently, global health is facing its devastating outbreak. However, there is no vaccine available against this virus up to now. In this study, a novel multi-epitope vaccine against SARS-CoV-2 was designed to provoke both innate and adaptive immune responses. The immunodominant regions of six non-structural proteins (nsp7, nsp8, nsp9, nsp10, nsp12 and nsp14) of SARS-CoV-2 were selected by multiple immunoinformatic tools to provoke T cell immune response. Also, immunodominant fragment of the functional region of SARS-CoV-2 spike (400-510 residues) protein was selected for inducing neutralizing antibodies production. The selected regions' sequences were connected to each other by furin-sensitive linker (RVRR). Moreover, the functional region of ß-defensin as a well-known agonist for the TLR-4/MD complex was added at the N-terminus of the vaccine using (EAAAK)3 linker. Also, a CD4 + T-helper epitope, PADRE, was used at the C-terminal of the vaccine by GPGPG and A(EAAAK)2A linkers to form the final vaccine construct. The physicochemical properties, allergenicity, antigenicity, functionality and population coverage of the final vaccine construct were analyzed. The final vaccine construct was an immunogenic, non-allergen and unfunctional protein which contained multiple CD8 + and CD4 + overlapping epitopes, IFN-γ inducing epitopes, linear and conformational B cell epitopes. It could form stable and significant interactions with TLR-4/MD according to molecular docking and dynamics simulations. Global population coverage of the vaccine for HLA-I and II were estimated 96.2% and 97.1%, respectively. At last, the final vaccine construct was reverse translated to design the DNA vaccine. Although the designed vaccine exhibited high efficacy in silico, further experimental validation is necessary.

Spirulina extract enriched for Braun-type lipoprotein (Immulina®) for inhibition of 4T1 breast tumors' growth and metastasis.

Spirulina platensis extracts have exhibited considerable anti-cancer effects. To investigate the efficacy of the Spirulina extract enriched for Braun-type lipoprotein (Immulina®) for breast cancer treatment, 4T1 breast tumor-bearing mice were treated with 40 mg/kg Immulina® daily and the tumors' growth and metastasis were assessed. Also, CD4, CD8, and CD56 staining were performed to investigate the Immulina® effect on the immune cells' recruitment to the tumors by immunohistochemistry. Immulina® could significantly (P < 0.001) inhibit 4T1 breast tumors' growth. Immulina®-treated group exhibited a 63% decrease in the tumors' volume in comparison with control (P < 0.001). Also, Immulina® could significantly (P < 0.001) decrease metastatic burden at the vital organs as 68% and 61% decrease in the liver and lungs metastatic colonies were observed, respectively. Also, Immulina® could increase mean survival time of the tumor-bearing mice for 29 days. The Spirulina-treated mice tumors contained significantly more infiltrated NK, CD4+, and CD8+ T lymphocytes in comparison with control. Taking together, Immulina® can be a safe anti-cancer supplement with the ability to cause direct apoptosis to the cancer cells and activate the immune system against tumor. This supplement with natural origin seems to have bright future to help breast cancer patients.

C-phycocyanin: a natural product with radiosensitizing property for enhancement of colon cancer radiation therapy efficacy through inhibition of COX-2 expression.

Different chemical and nanomaterial agents have been introduced for radiosensitizing purposes. However, many researchers believe these agents are far away from clinical application due to side effects and limited knowledge about their behavior in the human body. In this study, C-phycocyanin (C-PC) was used as a natural radiosensitizer for enhancement of radiation therapy (RT) efficacy. C-PC treatment's effect on the COX-2 expression of cancer cells was investigated by flow cytometry, western blot, qRT-PCR analyses in vitro and in vivo. Subsequently, the radiosensitizing effect of C-PC treatment was investigated by MTT and clonogenic cell survival assays for CT-26, DLD-1, HT-29 colon cancer cell lines and the CRL-1831 as normal colonic cells. In addition, the C-PC treatment effect on the radiation therapy efficacy was evaluated according to CT-26 tumor's growth progression and immunohistochemistry analyses of Ki-67 labeling index. C-PC treatment (200 µg/mL) could significantly enhance the radiation therapy efficacy in vitro and in vivo. Synergistic interaction was detected at C-PC and radiation beams co-treatment based on Chou and Talalay formula (combination index <1), especially at 200 µg/mL C-PC and 6 Gy radiation dosages. The acquired DEF of C-PC treatment was 1.39, 1.4, 1.63, and 1.05 for CT-26, DLD-1, HT-29, and CRL-1831 cells, respectively. Also, C-PC + RT treated mice exhibited 35.2% lower mean tumors' volume and about 6 days more survival time in comparison with the RT group (P < 0.05). In addition, C-PC + RT group exhibited 54% lower Ki-67 index in comparison with the RT group. Therefore, C-PC can exhibit high radiosensitizing effects. However, the potential cardiovascular risks of C-PC as a COX-2 inhibitor should be evaluated with extensive preclinical testing before developing this agent for clinical trials.

Identifying the Most Effective Hydatid Cyst Fluid Fraction for Anticancer Vaccination of 4T1 Breast Tumor-Bearing Mice.

BACKGROUND: The hydatid cyst fluid antigens have high homology with cancer cell antigens and also exhibit considerable immunogenicity. Therefore, their utilization for cancer immunization can cause an effective antitumor immune response. However, the main challenge is identifying the most effective antigens for this purpose. METHODS: Hydatid cyst fluid fractions including the glycolipid fraction, glycoprotein fraction, 78 kDa fraction, and antigen B fraction were prepared. Then, the BALB/c mice were immunized against different antigens and, subsequently, 4T1 cells were subcutaneously implanted. The tumors' growth, metastasis, and tumor-bearing mice survival were assessed in different immunized groups. In addition, IL-2, IL-4, IFN-γ, and TNF-α serum levels were estimated to evaluate the immune system response. RESULTS: BALB/c mice immunization against the complete hydatid cyst fluid antigens exhibited more significant inhibition of the tumors' growth and metastasis and increase of tumor-bearing mice survival in comparison with its derived fractions. However, the 78 kDa fraction exhibited the best results according to the same factors in comparison with all the prepared fractions. CONCLUSIONS: The 78 kDa fraction of the hydatid cyst fluid was the most effective fraction of hydatid cyst fluid for immunization against 4T1 breast tumors.

Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine.

Melanoma cells are significantly resistance to the current treatments. Therefore, the best option for high-risk populations is prevention. Recently, many preventive cancer vaccines have been developed. In our previous study, several bioinformatic tools were employed for selection of the most immunodominant epitopes of acrosin binding protein (ACRBP) and synaptonemal complex protein 1 (SYCP1) antigens to design multiepitope DNA and peptide cancer vaccines. In the current study, the final construct of the multiepitope DNA vaccine was placed into a pcDNA3.1 vector and then, subcloned into a pET-28a (+) expression vector for transfecting BL21 E. coli strain. The recombinant multiepitope peptide vaccine, weighing 6.35 kDa, was purified by Fast protein liquid chromatography technique (FPLC) and detected by western blotting. Subsequently, C57BL/6 mice were immunized by a mixture of the peptide vaccine and incomplete Freund's adjuvant (IFA) (four vaccinations with one-week intervals). Two weeks after the last vaccination, the serum levels of the peptide-specific IgG total, IgG2a, and IgG1 were measured by enzyme-linked immunosorbent assays (ELISA). Also, the immunized mice splenocytes efficacy for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) after stimulation with the peptide vaccine was evaluated. At last, the prophylactic effect of the peptide vaccine immunization was evaluated in B16-F10 murine melanoma model. The peptide vaccine immunization caused a significant increase in the serum levels of IgG1, IgG2a, and IgG2a. Also, the immunized mice splenocytes exhibited significantly higher ability to produce IL-4 (10-fold) and IFN-γ (16-fold) after stimulation with the peptide vaccine, in comparison with the PBS and IFA groups. The peptide immunized mice exhibited 50.2% and 43% decrease in the mean tumors' volume in comparison with PBS and IFA groups. Also, the mean survival time for the peptide immunized mice was 33 ±â€¯1.3 days which was 5 and 6 days more than the PBS and IFA groups, respectively. The obtained results exhibit high efficacy of the designed multiepitope peptide vaccine for the immune system activation and anti-tumor prophylactic effects in the murine melanoma model.

Immunization using male germ cells and gametes as rich sources of cancer/testis antigens for inhibition of 4T1 breast tumors' growth and metastasis in BALB/c mice.

Recently cancer/testis antigens (CTA) have gained lots of attention as targets of immune therapy. However, the therapeutic efficacy of the CTAs single-antigen vaccines is not satisfying due to tumor heterogenicity. Therefore, many studies have focused on the enhancement of their efficacy by utilizing rich sources of tumor-associated antigens for anti-cancer vaccination. In the present study, the testicular germ cells and sperm cells as well-known sources of cancer/testis antigens were investigated for anti-4T1 breast cancer vaccination in BALB/c mice. The testicular germ cells (TGCs) and sperm cells were isolated from male BALB/c mice. The definite number of cells were homogenized and mixed with Bacillus Calmette-Guerin (BCG) for vaccination of female BALB/c mice. The treatment groups underwent 3 times of immunizations with one-week intervals and one week after the last injection, all groups were injected with 4T1 cancer cells. The TGCs + BCG (259.7 ±â€¯39 mm3) and Sperm + BCG (426 ±â€¯52 mm3) groups exhibited a significant decrease in the tumors' volume in comparison with BCG (641.3 ±â€¯102 mm3) and no-treatment (788.1 ±â€¯117 mm3) groups. Therefore, the TGCs + BCG immunized mice had the smallest tumors in comparison with all groups (P < 0.05). Also, the vital organs of TGCs + BCG (lungs: 6.8 ±â€¯2, liver: 10.1 ±â€¯2) immunized mice exhibited lowest metastatic burden in comparison with the Sperm + BCG (lungs: 13.5 ±â€¯3, liver: 21.1 ±â€¯4), BCG (lungs: 24.3 ±â€¯4, liver: 33 ±â€¯4), and no-treatment (lungs: 26.5 ±â€¯6, liver: 37.3 ±â€¯3) groups. These observations were inconsistent with the tumor-bearing mice survival evaluations as the TGCs + BCG group had longer mean survival time (79.6 ±â€¯12 days) in comparison with other groups (no-treatment: 49.8 ±â€¯8, BCG: 50.5 ±â€¯10, BCG + Sperm: 64.6 ±â€¯7 days). Therefore, TGCs can be a potential source of antigens for the anti-breast cancer immunization and more investigations are necessary.

In silico analysis of transmembrane protein 31 (TMEM31) antigen to design novel multiepitope peptide and DNA cancer vaccines against melanoma.

Multiepitope cancer vaccines are announcing themselves as the future of melanoma treatment. Herein, high immunogenic regions of transmembrane protein 31 (TMEM31) antigen were selected according to cytotoxic T lymphocytes' (CTL) epitopes and major histocompatibility complex (MHC) binding affinity through in silico analyses. The 32-62, 77-105, and 125-165 residues of the TMEM31 were selected as the immunodominant fragments. They were linked together by RVRR and HEYGAEALERAG motifs to improve epitopes separation and presentation. In addition, to activate helper T lymphocytes (HTL), Pan HLA DR-binding epitope (PADRE) peptide sequence and tetanus toxin fragment C (TTFrC) were incorporated into the final construct. Also, the Beta-defensin conserved domain was utilized in the final construct as a novel adjuvant for Toll-like receptor 4/myeloid differentiation factor (TLR4-MD) activation. The CTL epitopes, cleavage sites, post-translational modifications, TAP transport efficiency, and B cells epitopes were predicted for the peptide vaccine. The final construct contained multiple CTL and B cell epitopes. In addition, it showed 93.55% and 99.13% population coverage in the world for HLA I and HLA II, respectively. According to these preliminary results, the multiepitope cancer vaccine can be an appropriate choice for further experimental investigations.