Potential links between the microbiota and T cell immunity determine the tumor cell fate.

The central role of the microbiota as a pivotal factor regulating anti-tumor immune responses has recently been appreciated. Increasing evidence has put a spotlight on the connection of microbiota to T cells, by showing impaired effector and/or memory responses in germ-free (GF) mice or in the presence of dysbiotic communities, and association with tumor growth and overall survival (OS). These observations also have significant implications for anti-tumor therapy and vaccination, suggesting that the communication between T cells and the microbiota involves soluble mediators (microbiota-derived metabolites) that influence various functions of T cells. In addition, there is growing appreciation of the role of bacterial translocation into the peritumoral milieu from the intestinal tract, as well as of locally developed tumor microbial communities, spatially separated from the gut microbiota, in shaping the tumor microbiome. Collectively, these findings have added new support to the idea that tonic inputs mirroring the existence of tumor microbiome could regulate the function of tumor-infiltrating T cells and tissue-resident memory T (TRM) cells. In this review, we focus on recent advances and aspects of these active areas of investigation and provide a comprehensive overview of the unique mechanisms that play a pivotal role in the regulation of anti-tumor immunity by the microbiota, some of which could be of particular relevance for addressing problems caused by tumor heterogeneity. It is our hope that this review will provide a theoretical foundation for future investigations in this area.

Role of Gut Microbiota and Probiotics in Colorectal Cancer: Onset and Progression.

The gut microbiota plays an important role in maintaining homeostasis in the human body, and the disruption of these communities can lead to compromised host health and the onset of disease. Current research on probiotics is quite promising and, in particular, these microorganisms have demonstrated their potential for use as adjuvants for the treatment of colorectal cancer. This review addresses the possible applications of probiotics, postbiotics, synbiotics, and next-generation probiotics in colorectal cancer research.

Modulation of the PI3K/Akt/mTOR signaling pathway by probiotics as a fruitful target for orchestrating the immune response.

The mammalian target of rapamycin (mTOR) and the phosphatidylinositol-3-kinase (PI3K)/protein kinase B or Akt (PKB/Akt) signaling pathways are considered as two but somewhat interconnected significant immune pathways which play complex roles in a variety of physiological processes as well as pathological conditions. Aberrant activation of PI3K/Akt/mTOR signaling pathways has been reported to be associated in a wide variety of human diseases. Over the past few years, growing evidence in in vitro and in vivo models suggest that this sophisticated and subtle cascade mediates the orchestration of the immune response in health and disease through exposure to probiotics. An expanding body of literature has highlighted the contribution of probiotics and PI3K/Akt/mTOR signaling pathways in gastrointestinal disorders, metabolic syndrome, skin diseases, allergy, salmonella infection, and aging. However, longitudinal human studies are possibly required to verify more conclusively whether the investigational tools used to understand the regulation of these pathways might provide effective approaches in the prevention and treatment of various disorders. In this Review, we summarize the experimental evidence from recent peer-reviewed studies and provide a brief overview of the causal relationship between the effects of probiotics and their metabolites on the components of PI3K/Akt/mTOR signaling pathways and human disease.

Twenty years of research on HPV vaccines based on genetically modified lactic acid bacteria: an overview on the gut-vagina axis.

Most cervical cancer (CxCa) are related to persistent infection with high-risk human papillomavirus (HR-HPV) in the cervical mucosa, suggesting that an induction of mucosal cell-mediated immunity against HR-HPV oncoproteins can be a promising strategy to fight HPV-associated CxCa. From this perspective, many pre-clinical and clinical trials have proved the potential of lactic acid bacteria (LAB) genetically modified to deliver recombinant antigens to induce mucosal, humoral and cellular immunity in the host. Altogether, the outcomes of these studies suggest that there are several key factors to consider that may offer guidance on improvement protein yield and improving immune response. Overall, these findings showed that oral LAB-based mucosal HPV vaccines expressing inducible surface-anchored antigens display a higher potential to induce particularly specific systemic and mucosal cytotoxic cellular immune responses. In this review, we describe all LAB-based HPV vaccine investigations by reviewing databases from international studies between 2000 and 2020. Our aim is to promote the therapeutic HPV vaccines knowledge and to complete the gaps in this field to empower scientists worldwide to make proper decisions regarding the best strategies for the development of therapeutic HPV vaccines.

Intervention on gut microbiota may change the strategy for management of colorectal cancer.

Dysbiosis in the gut microbiota composition due to environmental or genetic variations can disrupt the immune system and may promote several diseases such as colorectal cancer (CRC). Gut microbiota can alter the toxicity and efficiency of an extensive range of CRC treatment methods, especially surgery, chemotherapy, radiotherapy, and immunotherapy. The recent scientific evidence suggested that gut microbiota modulation exhibits an essential positive influence on inhibition and treatment of CRC. The literature survey revealed that modulating the gut microbiota composition by probiotics, prebiotics, and diets protects CRC patients from treatment-associated adverse effects. This review summarizes the recent advancements in the association between interventions on gut microbiota and CRC to provide innovative strategies for enhancing the safety and efficiency of CRC therapy.

Gut microbiota-derived metabolites and colorectal cancer: New insights and updates.

Abundant evidence from in-vitro as well as in-vivo studies supports the gut microbiota-derived metabolites as crucial executors of diet effect on the host physiology. As such, a number of microbiota-derived metabolites produced from diet have been connected to complex forms of human diseases such as colorectal cancer (CRC). Despite current unresolved questions concerning molecular mechanisms between metabolites, host signaling pathways, and CRC, some new progresses promise continued advancement of the field. Therefore, clarification of the molecular events underlying which metabolites may regulate proliferation of colonocytes will hopefully open up new avenues for seeking the possibilities affecting host health and exploitation of these capabilities for therapeutic purpose. In this Review, we will discuss recent insights into contributions of the gut microbiota-derived metabolites to CRC and argue that the cumulative effects of metabolites should be considered with the intention of better predict and prevent cancer progression. We will also discuss the signaling pathways induced by specific metabolites toward down-regulation and/or up-regulation of immune system that eventually trigger progression and/or inhibition of CRC.

The First Clinical Use of a Recombinant Lactococcus lactis Expressing Human Papillomavirus Type 16 E7 Oncogene Oral Vaccine: A Phase I Safety and Immunogenicity Trial in Healthy Women Volunteers.

A dose-escalation, randomized, double-blind, placebo-controlled phase I clinical trial was performed in healthy Iranian volunteer women to assess the safety, tolerability, and immunogenicity of NZ8123-HPV16-optiE7 vaccine involving recombinant Lactococcus lactis expressing the codon-optimized human papillomavirus (HPV)-16 E7 oncogene. Fifty-five eligible subjects were divided into 6 cohorts based on the dosages (1 × 109, 5 × 109, and 1 × 1010 CFU/mL) of either vaccine or placebo, which were administrated orally a total of 4 times at weeks 1, 2, 4, and 8. Then, adverse events, specific serum IgG and vaginal IgA, and E7-specific IFNγ-secreting CD8+ CTL responses were evaluated. The vaccination was well tolerated by 40 subjects who completed the immunization schedule, and no serious adverse effects were reported. The IgG and IgA levels peaked at day 60, and the levels for the 5 × 109 CFU/mL and 1 × 1010 CFU/mL dose groups were higher than those for the 1 × 109 CFU/mL dose group. Time-to-peak stimulation in E7-specific IFNγ-secreting CD8+ CTL responses was seen in cervical lymphocytes 1 month after the last vaccination. Again, no significant increase was seen in the peripheral blood mononuclear cells (PBMC) of the same volunteers. CTL responses in cervical lymphocytes and PBMCs at day 90 were markedly higher in the 5 × 109 and 1 × 1010 CFU/mL groups than in the 1 × 109 CFU/mL group, demonstrating the dose dependency of NZ8123-HPV16-optiE7 vaccine following oral administration. The 6-month follow-up revealed that antibody levels decreased up to day 240; nevertheless, long-term E7-specific IFNγ-secreting CD8+ CTL responses were recorded during follow-up. Overall, the safety and immunogenicity profile achieved in this study encourages further phase II trials with the 5 × 109 CFU/mL dose vaccine.

Phase 1 Safety and Immunogenicity Trial of Recombinant Lactococcus lactis Expressing Human Papillomavirus Type 16 E6 Oncoprotein Vaccine.

The present study purposed to investigate the safety, tolerability, and immunogenicity of the therapeutic NZ8123-HPV16-optiE6 vaccine, following oral vaccination. The safety and tolerability were evaluated. Specific serum immunoglobulin G (IgG) and vaginal IgA antibodies were calculated by ELISA, and E6-specific IFN-γ-secreting T cells were counted by enzyme-linked immune absorbent spot (ELISpot) assay in cervical lymphocytes and PBMC samples. The vaccine was well tolerated, and no serious adverse effects were observed in vaccine recipients. Statistical analysis showed that all vaccine groups had significant increases in antibody levels at day 60 after baseline. The time to peak activation in E6-specific IFN-γ-secreting CD8+ CTL responses was seen at month 1 after last vaccination. According to the results, the humoral immune and cell-mediated responses for the vaccine groups that received 5 × 109 and 1 × 1010 CFU/mL of vaccine were similar and were higher than those of the 1 × 109 CFU/mL group, indicating the dose-dependency of the NZ8123-HPV16-optiE6 vaccine following oral administration. Low antibody levels compared with the placebo groups were recorded at month 6 after the last vaccination. Interestingly, long-term E6-specific CTL responses were observed during follow-up. It was concluded that oral immunization with the NZ8123-HPV-16-optiE6 vaccine is safe, induces persistent immunity, and is reasonably well tolerated.

Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis.

BACKGROUND: We aimed at constructing Lactococcus lactis strains expressing HPV-16 recombinant E7 (rE7) oncoprotein and examining its overproduction ability followed by optimizing batch and fed-batch fermentations. Thereafter, in order to assess the immunogenicity of recombinant L. lactis cells, C57BL/6 mice were immunized by oral gavage. RESULTS: The results suggested that recombinant strains harboring optiE7 and E7 genes produced a maximum of 4.84 and 1.91 µg/mL of rE7 in static flask experiments, while the corresponding strains gave a maximum yield of 35.49 and 14.24 µg/mL in batch experiments, respectively. Fed-batch study indicated that the concentration of rE7 protein significantly increased after feeding yeast extract plus GM17 medium. The rE7 production of the best performing strains was 2.09- and 1.48-fold higher than that of the strains during the batch fermentation. Furthermore, biomass levels were 1.98- and 1.92-fold higher than those in batch cultivation. Oral immunization of C57BL/6 mice with recombinant L. lactis produced significant specific IgG and IgA antibody responses in serum and vaginal fluids, respectively. Our outcomes suggest that vaccination with L. lactis expressing rE7 can generate significant protective effects against E7-expressing cell line. Also, our study provides evidence that the presence of large amounts of E7-specific CD4+ T helper and CD8+ T cell precursors was stimulated. Significantly higher frequencies of HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells were detected in antigen-stimulated splenocytes and intestinal mucosal lymphocytes, when compared to the control groups. CONCLUSIONS: We conclude that optimization of culture conditions along with recombinant protein expression can highly stimulate both specific humoral and cell-mediated immune responses in mice after oral immunization. These promising results represent a step towards fast-tracking a vaccine against HPV-16-associated cervical cancer.

Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein.

Recombinant strains of Lactococcus lactis NZ9000 that express native and codon-optimized E6 protein (fused to the SPusp45 secretion signal) were successfully constructed by using the nisin-controlled gene expression (NICE) system. Expression of the recombinant strains was evaluated by Western blot analysis. Female mice of strain C57BL/6 were immunized orally with recombinant lactococci expressing inducible E6 oncoprotein and the antigen-specific antibody production (IgA and IgG) and cytokines were measured by ELISA and ELISPOT assay, respectively. Our outcomes indicate that the HPV-16 E6 specific IL-2- and IFN-γ-secreting lymphocytes in the antigen-stimulated intestinal mucosal lymphocytes, splenocytes and vaginal lymphocytes were significantly higher than the control groups. We showed that L. lactis having codon-optimized E6 oncogene had better inhibitory effect on tumor growth, better treatment effects on progression of tumor size, and better survival rate in comparison with L. lactis having native E6 oncogene, (P < 0.0001). In conclusion, the rE6 protein displayed by L. lactis can induce humoral and cellular immunity. Taken together, these preclinical results represent a promising step towards the development of recombinant L. lactis as a live oral vector vaccine to treat the HPV-16 associated with cervical cancer.

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice.

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.

Molecular Screening and Single Nucleotide Polymorphism Typing of Molluscum Contagiosum Virus (MCV) from Genital Specimens, between 2012 and 2015

Background: The present study is the first comprehensive report of the Molluscum contagiosum virus (MCV) in Iran based on the molecular technique for differentiation and typing of the MCV1 and MCV2. Methods: Patients were diagnosed as having tumor-like genital warts less than 5 mm in diameter, and HIV seronegative samples were chosen for this cross-sectional study. TaqMan real-time PCR was used to identify MCV following clinical examination. Typing of the MCV-positive specimens was performed in the SNP A27451G region of MC021L gene. Results: Of 1470 samples, 114 (7.75%) samples were positive for the MCV. From MCV-positive samples, 71.05% sequences were found to be related to the MCV1 and 28.95% to the MCV2. Conclusion: This assay constitutes a reliable method for identification and typing of the MCV genomic variants that could be valuable for reviewing the pathogenesis, molecular epidemiology, and the natural history of MCV-related situations.

Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H Strain

Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins. GS115 and KM71H are two kinds of Pichia pastoris strains whilst production of recombinant proteins in these strains is not predictable. The aim of the study was evaluation of t-PA expression in KM71H strains. Methods: In this study, the cDNA of the t-PA gene was amplified by PCR, sequenced and cloned into Pichia pastoris KM71H host strain using pPICZalphaA expression vector that allows methanol-induced expression and secretion of the protein. Results: Dot blotting results confirmed the presence oft-PA in the cell supernatant. Western blotting test revealed the approximate size of 70 KDa for recombinant t-PA. Quantitative ELISA experiment showed 810 µg/L of t-PA in the supernatant samples. Zymography analysis confirmed the proteolytic activity and biological function of the expressed recombinant t-PA. Conclusions: Correspondingly, Pichia pastoris KM71H is an appropriate strain for production of active recombinant protein.

Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system.

The present work was aimed at investigating the expression and optimization of a human papillomavirus (HPV) type 16 gene encoding oncoprotein E7 in Lactococcus lactis. We genetically engineered Lactococcus lactis using nisin-controlled gene expression (NICE) system pNZ8148 to express the native and codon optimized E7 oncogenes isolated from Iranian HPV-16. The results of optimizing fermentation showed, the concentration of produced protein was expressively improved by 10 ng/mL nisin after 3.5, and 4 h induction for NZ9000 harboring the codon-optimized, and native E7 respectively. Furthermore the recombinant NZ9000 strains expressed rE7 by maximum value of 4.7 (Codon-optimized), and 1.82 µg/mL (Native) in static flask experiments at initial glucose concentrations of 50 and 75 g/L respectively. The rE7 yield was further enriched in batch fermenter experiments using controlled pH. Thus, the overall production of rE7 under optimized conditions accumulated in the cytoplasm to nearly 33.25 µg/mL by L. lactis NZ9000 containing codon-optimized E7, which was over ∼2.7-fold higher compared to the NZ9000 having native E7 strain (12.01 µg/mL). Accordingly, the maximum biomass production was calculated 4.87, and 1.51 g/L respectively.

Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363

HPV 16 intratypic sequence variations has been recognized in association with oncogenic potential diverge and geographic distribution. This study aimed to investigate nucleotide modifications and optimization of HPV 16 E7 regions from Iranian infected women. Cervical biopsies from 79/163 HPV 16 positive cancer patients detected in our study were analyzed by PCR in a couple of cloning of a complete ORF of the E7 gene, and sequencing. The most frequently observed variant was C196T in E7 which led to an amino acid change of R66W. In addition, only one common variant T234G was identified from all specimens, but it did not lead to any amino acid change. We also detected nucleotide variations A86G, and C188T in samples. Among 99 codons in E7 gene, 56 codons were improved for Lactococcus lactis subsp. cremoris MG1363 resulting in a reduced G+C content from 43.1% to 34.0%. Also, the AT%, ENC, and CAI values were 66, 20±1.1, and 1.000 instead of 56.90, 60 ±1.1, and 0.406 respectively. Finally we constructed expression vector pNZ8148 encoding optimized E7 oncoprotein of HPV 16. This study declared for the first time, the genetic variations of HPV 16 E7 in IRAN. We conclude that plasmid pNZ8148-HPV 16-opti E7 can be potential vaccine candidates in the future.