Properties and predicted functions of large genes and proteins of apicomplexan parasites.

Evolutionary constraints greatly favor compact genomes that efficiently encode proteins. However, several eukaryotic organisms, including apicomplexan parasites such as Toxoplasma gondii, Plasmodium falciparum and Babesia duncani, the causative agents of toxoplasmosis, malaria and babesiosis, respectively, encode very large proteins, exceeding 20 times their average protein size. Although these large proteins represent <1% of the total protein pool and are generally expressed at low levels, their persistence throughout evolution raises important questions about their functions and possible evolutionary pressures to maintain them. In this study, we examined the trends in gene and protein size, function and expression patterns within seven apicomplexan pathogens. Our analysis revealed that certain large proteins in apicomplexan parasites harbor domains potentially important for functions such as antigenic variation, erythrocyte invasion and immune evasion. However, these domains are not limited to or strictly conserved within large proteins. While some of these proteins are predicted to engage in conventional metabolic pathways within these parasites, others fulfill specialized functions for pathogen-host interactions, nutrient acquisition and overall survival.

Predicting survival of Iranian COVID-19 patients infected by various variants including omicron from CT Scan images and clinical data using deep neural networks.

Purpose: The rapid spread of the COVID-19 omicron variant virus has resulted in an overload of hospitals around the globe. As a result, many patients are deprived of hospital facilities, increasing mortality rates. Therefore, mortality rates can be reduced by efficiently assigning facilities to higher-risk patients. Therefore, it is crucial to estimate patients' survival probability based on their conditions at the time of admission so that the minimum required facilities can be provided, allowing more opportunities to be available for those who need them. Although radiologic findings in chest computerized tomography scans show various patterns, considering the individual risk factors and other underlying diseases, it is difficult to predict patient prognosis through routine clinical or statistical analysis. Method: In this study, a deep neural network model is proposed for predicting survival based on simple clinical features, blood tests, axial computerized tomography scan images of lungs, and the patients' planned treatment. The model's architecture combines a Convolutional Neural Network and a Long Short Term Memory network. The model was trained using 390 survivors and 108 deceased patients from the Rasoul Akram Hospital and evaluated 109 surviving and 36 deceased patients infected by the omicron variant. Results: The proposed model reached an accuracy of 87.5% on the test data, indicating survival prediction possibility. The accuracy was significantly higher than the accuracy achieved by classical machine learning methods without considering computerized tomography scan images (p-value <= 4E-5). The images were also replaced with hand-crafted features related to the ratio of infected lung lobes used in classical machine-learning models. The highest-performing model reached an accuracy of 84.5%, which was considerably higher than the models trained on mere clinical information (p-value <= 0.006). However, the performance was still significantly less than the deep model (p-value <= 0.016). Conclusion: The proposed deep model achieved a higher accuracy than classical machine learning methods trained on features other than computerized tomography scan images. This proves the images contain extra information. Meanwhile, Artificial Intelligence methods with multimodal inputs can be more reliable and accurate than computerized tomography severity scores.

The beneficial effects of bare and CMC-supported α-FeOOH, Fe3O4, and α-Fe2O3 nanoparticles on growth, nutrient content, and essential oil of summer savory (Satureja hortensis L.) under Cd, Pb and Zn stresses.

This research studies the impacts of iron oxide nanoparticles (FeONPs) on alleviating the toxic effects of cadmium (Cd), lead (Pb), and zinc (Zn) on summer savory (Satureja hortensis L.). Different types of soil additives, including bare and carboxymethylcellulose (CMC)-supported hematite (α-Fe2O3), goethite (α-FeOOH), and magnetite (Fe3O4), were applied at three rates (0, 0.25, and 0.5% w/w) to a Cd, Pb, and Zn-contaminated soil sample. The experimental results showed that the application of FeONPs increased plant height, dry weights of shoot and root, and yield and content of essential oil. Bare and CMC-supported FeONPs increased the content of K, P, and Fe in the aerial parts of summer savory. However, these soil additives reduced the contents of Cd, Pb, and Zn in plant tissues. CMC-supported FeONPs proved to be more efficient additives in diminishing the toxic effects of Cd, Pb, and Zn in summer savory compared to their bare forms. Bare and CMC-supported goethite NPs were able to restrict the uptake of Cd, Pb, and Zn by summer savory roots in the metal-contaminated soil. The application of CMC-supported goethite at an application dose of 0.5% (w/w) increased shoot dry weight, shoot concentrations of K, P, and Fe, and yield of essential oil by about 62.6, 76.6, 77.1, 210, and 230%, respectively. Conversely, they reduced shoot concentrations of Cd, Pb, and Zn by about 64.6, 68.7, and 40.6%, respectively, compared to the control. These are significant results and indicate that CMC-supported goethite is likely to be the most effective soil additive in diminishing the toxicity of Cd, Pb, and Zn to metal-stressed summer savory.

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.

A Cross-sectional Survey to Assess Reasons for Therapeutic Inertia in People With Type 2 Diabetes Mellitus and Preferred Strategies to Overcome It From the Perspectives of Persons With Diabetes and General/Family Practitioners: Results From the MOTION Study.

OBJECTIVES: Although multiple causes of therapeutic inertia in type 2 diabetes mellitus (T2DM) have been identified, few studies have addressed the behavioural aspects of treatment-intensification decisions among persons with type 2 diabetes (PwT2DM) and general practitioners/family practitioners (GPFPs). METHODS: A quantitative online survey was developed to capture from 300 PwT2DM and 100 GPFPs the following information: 1) perspectives on shared decision-making (SDM) related to treatment intensification, using the 9-item Shared Decision Making Questionnaire and the Shared Decision Making Questionnaire---physician version; 2) intentions to intensify treatments, using the Theory of Planned Behaviour (TPB); and 3) preferred strategies to overcome causes of therapeutic inertia in T2DM. Regression methods were applied post hoc to examine correlations with SDM scores, behavioural intentions and behaviours. RESULTS: SDM scores showed a significantly lower level of perceived involvement in decision-making related to treatment intensification among PwT2DM compared with GPFPs. The TPB identified that, for PwT2DM, attitudes, perceived behavioural control and age were associated with variation in intention to intensify treatment and, for GPFPs, perceived behavioural control and not being in a shared/group practice were associated with intentions to intensify treatment. PwT2DM behaviour, measured as hesitancy to intensify treatment, was associated with age. PwT2DM want more information to become more comfortable with the treatment decision-making process, whereas GPFPs desired support from other health professionals, and more time to address issues among PwT2DM. CONCLUSIONS: Strategies directed at providing GPFPs with tools/approaches to increase PwT2DM involvement in the decision-making process, such as behavioural coaching, decision aids and goal setting, may increase acceptance of treatment intensification, leading to a reduction in therapeutic inertia in T2DM.

Persons With Diabetes and General/Family Practitioner Perspectives Related to Therapeutic Inertia in Type 2 Diabetes Mellitus Using Qualitative Focus Groups and the Theoretical Domains Framework: Results From the MOTION Study.

OBJECTIVES: Therapeutic inertia in type 2 diabetes (T2DM) is the failure to receive timely treatment intensification as indicated according to T2DM treatment guidelines. Multifactorial causes of therapeutic inertia in T2DM have been documented at the level of persons with diabetes (PwD), health-care providers and health-care systems. METHODS: We developed a 3-part mixed-methods research program, called the Moving to Overcome Therapeutic Inertia Obstacles Now in T2DM (MOTION) study, to inform the development of strategies to address therapeutic inertia in T2DM. We present the results from focus groups with the following objectives: 1) understanding PwD and general practitioner/family practitioner (GPFP) determinants of behaviour related to treatment intensification using the Theoretical Domains Framework (TDF); and 2) identifying the sources of behaviours contributing to therapeutic inertia in T2DM, as proposed by the Behaviour Change Wheel (BCW). Two focus groups with PwD and 4 with GPFPs were conducted. Transcripts from the focus groups were coded independently by 2 investigators to identify themes, then mapped to TDF domains and linked using the BCW. RESULTS: For PwD, the most commonly coded TDF domains were intentions, goals, knowledge, beliefs about consequences and social influences. For GPFPs, the most common domains were intentions, environmental context and resources and social/professional role and identity. The BCW identified that PwD interventions should include reflective motivation, psychological capability and social opportunity; GPFP interventions should include physical opportunity, social opportunity and reflective motivation. CONCLUSIONS: Comprehensive strategies that target both PwD and GPFP barriers would encourage a more collaborative approach toward treatment intensification decisions and reducing therapeutic inertia.

Determination of Poultry Manure and Plant Residues Effects on Zn Bioavailable Fraction in Contaminated Soil via DGT Technique.

A greenhouse experiment aimed to assess the effects of poultry manure, sorghum, and clover residues (0 and 15 g kg-1) on the zinc (Zn) bioavailable fraction in contaminated calcareous soil using two chemical assays, including diffusion gradient in thin-films (DGT) and diethylene triamine pentaacetic acid-triethanolamine (DTPA-TEA), and a bioassay with corn (Zea mase L.). The results showed that poultry manure, clover, and sorghum residues application increased dissolved organic carbon (DOC) by 53.6 and 36.1, and 9.2%, respectively, and decreased soil pH by 0.42, 0.26, and 0.06 units, respectively compared to unamended soil. These changes resulted in increases of Zn effective concentration (CE) and DTPA-Zn, and plant Zn concentration as observed by the increase in exchangeable form of Zn. In the sorghum residues-amended soils, CE-Zn decreased by 29.5% compared to other treatments. The best correlations between corn metal concentrations and soil metal bioavailability were obtained for CE-Zn using the DGT technique, which also provided the best Zn bioavailability estimate. It is concluded that sorghum residues could be used to reduce the phytotoxicity risk of Zn in calcareous contaminated soil, and the DTPA method is the less robust indicator of Zn bioavailability than the DGT technique.

Whole-Genome Analysis of De Novo Somatic Point Mutations Reveals Novel Mutational Biomarkers in Pancreatic Cancer.

It is now known that at least 10% of samples with pancreatic cancers (PC) contain a causative mutation in the known susceptibility genes, suggesting the importance of identifying cancer-associated genes that carry the causative mutations in high-risk individuals for early detection of PC. In this study, we develop a statistical pipeline using a new concept, called gene-motif, that utilizes both mutated genes and mutational processes to identify 4211 3-nucleotide PC-associated gene-motifs within 203 significantly mutated genes in PC. Using these gene-motifs as distinguishable features for pancreatic cancer subtyping results in identifying five PC subtypes with distinguishable phenotypes and genotypes. Our comprehensive biological characterization reveals that these PC subtypes are associated with different molecular mechanisms including unique cancer related signaling pathways, in which for most of the subtypes targeted treatment options are currently available. Some of the pathways we identified in all five PC subtypes, including cell cycle and the Axon guidance pathway are frequently seen and mutated in cancer. We also identified Protein kinase C, EGFR (epidermal growth factor receptor) signaling pathway and P53 signaling pathways as potential targets for treatment of the PC subtypes. Altogether, our results uncover the importance of considering both the mutation type and mutated genes in the identification of cancer subtypes and biomarkers.

Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease.

Severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) infection, the causative agent of COVID-19, now represents the sixth Public Health Emergency of International Concern (PHEIC)-as declared by the World Health Organization (WHO) since 2009. Considering that SARS-CoV-2 is mainly transmitted via the mucosal route, a therapy administered by this same route may represent a desirable approach to fight SARS-CoV-2 infection. It is now widely accepted that genetically modified microorganisms, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Previous studies have shown that the mucosal administration of therapeutic molecules is able to induce an immune response mediated by specific serum IgG and mucosal IgA antibodies along with mucosal cell-mediated immune responses, which effectively concur to neutralize and eradicate infections. Therefore, advances in the modulation of mucosal immune responses, and in particular the use of probiotics as live delivery vectors, may encourage prospective studies to assess the effectiveness of genetically modified probiotics for SARS-CoV-2 infection. Emerging trends in the ever-progressing field of vaccine development re-emphasize the contribution of adjuvants, along with optimization of codon usage (when designing a synthetic gene), expression level, and inoculation dose to elicit specific and potent protective immune responses. In this review, we will highlight the existing pre-clinical and clinical information on the use of genetically modified microorganisms in control strategies against respiratory and non-respiratory viruses. In addition, we will discuss some controversial aspects of the use of genetically modified probiotics in modulating the cross-talk between mucosal delivery of therapeutics and immune system modulation.

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.

Association of Helicobacter bilis Infection with the Development of Colorectal Cancer.

The role of Helicobacter_bilis (H.bilis) in the development of inflammatory bowel disease (IBD) and colitis-associated carcinogenesis (CAC) has seldom been investigated. We examined the abundance of H.bilis in 58 colorectal cancers (CRCs), 20 IBDs, 40 cases of normal colorectal mucosa (NCs), and 20 adenomas (ADs) by 16S rRNA fluorescence in situ hybridization (FISH). Number of CD4+CD45RB+T cell and expression of IFN-γ and TNF-α in these tissues was determined by immunofluorescence. The abundance of H.bilis was significantly higher in CRCs than that in IBDs (P = 0.006), ADs (P < 0.001) and NCs (P < 0.0001). The abundance of H.bilis in IBDs was significantly higher than that in ADs (P = 0.013). Moreover, the average number of CD4+CD45RB+T cell was significantly higher in CRCs than that in IBDs (P = 0.017) and NCs (P = 0.009). In addition, there was a positive correlation between the H.bilis abundance and density of CD4+CD45RB+T cells in 30 colorectal tissues (P < 0.0001). The frequency of co-staining for CD4+CD45RB+T cells and IFN-γ was significantly higher in H.bilis positive group than that in H.bilis negative group (P = 0.002). H.bilis may play a role in the initiation of IBD and CAC, possibly through promoting the transformation of T cells into CD4+CD45RB+T cells and increasing the expression of proinflammatory cytokines IFN-γ.

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.

Body fluids may contribute to human-to-human transmission of severe acute respiratory syndrome coronavirus 2: evidence and practical experience.

BACKGROUND: In December 2019, an unbelievable outbreak of pneumonia associated with coronavirus was reported in the city of Wuhan, Hubei Province. This virus was called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although much effort has been spent on clarifying the transmission route of SARS-CoV-2, but, very little evidence is available regarding the relationship between human body fluids and transmission of SARS-CoV-2 virus. Considerable evidence from hospital in Wuhan indicates that strict rules to avoid occupational exposure to patients' body fluids in healthcare settings, particularly among every medical staff, limited person-to-person transmission of nosocomial infections by direct or indirect contact. CONCLUSION: We tried to provide important information for understanding the possible transmission routes of SARS-CoV-2 via body fluids including bronchoalveolar-lavage, saliva, blood, urine, feces, sputum, tears, and semen in order to control coronavirus disease 2019 (COVID-19) occurrences.

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.

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.