Immunized Mice Research Articles

The Role of Dendritic Cells in Adaptive Immune Response Induced by OVA/PDDA Nanoparticles

Background/Objective: Cationic polymers were shown to assemble with negatively charged proteins yielding nanoparticles (NPs). Poly-diallyl-dimethyl-ammonium chloride (PDDA) combined with ovalbumin (OVA) yielded a stable colloidal dispersion (OVA/PDDA-NPs) eliciting significant anti-OVA immune response. Dendritic cells (DCs), as sentinels of foreign antigens, exert a crucial role in the antigen-specific immune response. Here, we aimed to evaluate the involvement of DCs in the immune response induced by OVA/PDDA. Methods: In vivo experiments were used to assess the ability of OVA/PDDA-NPs to induce anti-OVA antibodies by ELISA, as well as plasma cells and memory B cells using flow cytometry. Additionally, DC migration to draining lymph nodes following OVA/PDDA-NP immunization was evaluated by flow cytometry. In vitro experiments using bone marrow-derived DCs (BM-DCs) were used to analyze the binding and uptake of OVA/PDDA-NPs, DC maturation status, and their antigen-presenting capacity. Results: Our data confirmed the potent effect of OVA/PDDA-NPs inducing anti-OVA IgG1 and IgG2a antibodies with increased CD19+CD138+ plasma cells and CD19+CD38+CD27+ memory cells in immunized mice. OVA/PDDA-NPs induced DC maturation and migration to draining lymph nodes. The in vitro results showed higher binding and the uptake of OVA/PDDA-NPs by BM-DCs. In addition, the NPs were able to induce the upregulation of costimulatory and MHC-II molecules on DCs, as well as TNF-α and IL-12 production. Higher OVA-specific T cell proliferation was promoted by BM-DCs incubated with OVA/PDDA-NPs. Conclusions: The data showed the central role of DCs in the induction of antigen-specific immune response by OVA-PDDA-NPs, thus proving that these NPs are a potent adjuvant for subunit vaccine design.

Inactivation of Zika Virus with Hydroxypropyl-Beta-Cyclodextrin

Background/Objectives: Zika virus (ZIKV) infection is associated with life-threatening diseases in humans. To date, there are no available FDA-approved therapies or vaccines for the specific treatment or prevention of ZIKV infection. Variation in the ZIKV envelope protein (Env), along with its complex quaternary structure, presents challenges to synthetic approaches for developing an effective vaccine and broadly neutralizing antibodies (bnAbs). We hypothesized that beta-cyclodextrin (BCD) could be used to uniquely inactivate infectious ZIKV without disruption of Env. Methods: ZIKV was propagated in Vero cells and admixed with BCD. The BCD-treated ZIKV was evaluated for infectivity using immunofluorescence and quantitative RT-PCR (qRT-PCR) assays, for immunoreactivity in Western blots, structural integrity by electron microscopy, and immunogenicity in mice. Results: Here, we show that 200 mM BCD-treated ZIKV is non-infectious in cell culture, remains immunoreactive with an Env-specific antibody, retains its virion shape and size, and elicits the production of immunogen-specific antibodies in immunized mice. Conclusions: These results indicate that BCD can be used to safely inactivate ZIKV, and they provide insights for vaccine and antibody development.

Macrophage-specific in vivo RNA editing promotes phagocytosis and antitumor immunity in mice.

Macrophages play a central role in antitumor immunity, making them an attractive target for gene therapy strategies. However, macrophages are difficult to transfect because of nucleic acid sensors that can trigger the degradation of foreign plasmid DNA. Here, we developed a macrophage-specific editing (MAGE) system by which compact plasmid DNA encoding a CasRx editor can be delivered to macrophages by a poly(β-amino ester) (PBAE) carrier to bypass the DNA sensor and enable RNA editing in vitro and in vivo. We identified a four-arm branched PBAE with 1-(2-aminoethyl)-4-methylpiperazine end-capping (PBAE29) that enables highly efficient macrophage transfection. PBAE29-mediated transfection of cultured macrophages stimulated less inflammatory cytokine production and inflammasome activation compared with traditional lipofectamine or electroporation-mediated plasmid delivery. Transfection efficiency was further improved by delivering CasRx by minicircle plasmid. The MAGE system incorporated a layer of carboxylated-mannan coating to target macrophage mannose receptors and a macrophage-specific promoter for enhanced selectivity. The delivery of CasRx with guide RNA targeting the transcripts for sialic acid-binding immunoglobulin similar to lectin 10 and signal regulatory protein alpha expression resulted in effective protein knockdown, improving macrophage phagocytosis. The MAGE system also showed efficacy in targeting macrophages in vivo, stimulating antitumor immune responses and reducing tumor volume in murine tumor models, including patient-derived pancreatic adenocarcinoma xenografts in humanized mice. In sum, the MAGE system presents a promising platform for in vivo macrophage-specific delivery of RNA editing tools that can be applied as a cancer therapy.

Evaluation of Synthetic Peptides from Schistosoma mansoni ATP Diphosphohydrolase 1: In Silico Approaches for Characterization and Prospective Application in Diagnosis of Schistosomiasis.

Schistosomiasis is the infection caused by Schistosoma mansoni and constitutes a worldwide public health problem. The parasitological recommended method and serological methods can be used for the detection of eggs and antibodies, respectively. However, both have limitations, especially in low endemicity areas. Thus, new approaches for the diagnosis of schistosomiasis are essential. In this study, a six-amino acid peptide and derived sequences from SmATPDase1 were synthesized for the evaluation of immunogenicity. SmATPDase1 is included in a protein group in S. mansoni tegument; therefore, its peptides could be potential candidates for diagnostic antigens. In the hypothetical SmATPDase1 three-dimensional structure, peptides are located in a region exposed and accessible to antibody binding. In addition, peptide amino acid sequences are conserved in the most relevant Schistosoma species and have low identity with human NTPDases isoforms. Swiss mice immunization resulted in significant anti-peptide polyclonal antibodies production, which recognized a 63 kDa protein in tegument and adult worm preparations. By immunofluorescence microscopy, polyclonal antibodies also identified this enzyme in cercariae. Sera of infected animals presented high seropositivity in ELISA-peptides, with an area under curve (AUC) greater than 0.96 for all peptides. In mice with low parasite burden, we observed a seropositivity AUC > 0.9. Reactivity in the prepatent period exhibited AUC values greater than 0.94 for all peptides. Anti-P1425 monoclonal antibodies were successfully produced, and mAbs recognized the integral protein in ELISA and Western blots. The data indicate that peptides from SmATPDase1 are potential biomarkers for schistosomiasis, and anti-peptide antibodies are interesting tools for the detection of the infection.

Altering the intracellular trafficking of Necator americanus GST-1 antigen yields novel hookworm mRNA vaccine candidates.

The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1's thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.

Substructure-Specific Antibodies Against Fentanyl Derivatives.

Structural variants of the synthetic opioid fentanyl are a major threat to public health. Following an investigation showing that many derivatives are poorly detected by commercial lateral flow and related assays, we created hapten conjugate vaccines using an immunogenic virus-like particle carrier and eight synthetic fentanyl derivatives designed to mimic the structural features of several of the more dangerous analogues. Immunization of mice elicited strong antihapten humoral responses, allowing the screening of hundreds of hapten-specific hybridomas for binding strength and specificity. A panel of 13 monoclonal IgG antibodies were selected, each showing a different pattern of recognition of fentanyl structural variations, and all proving to be highly efficient at capturing parent fentanyl compounds in competition ELISA experiments. These results provide antibody reagents for assay development as well as a demonstration of the power of the immune system to create binding agents capable of both broad and specific recognition of small-molecule targets.

Low-avidity T cells drive endogenous tumor immunity in mice and humans.

T cells recognize neoepitope peptide-major histocompatibility complex class I on cancer cells. The strength (or avidity) of the T cell receptor-peptide-major histocompatibility complex class I interaction is a critical variable in immune control of cancers. Here, we analyze neoepitope-specific CD8 cells of distinct avidities and show that low-avidity T cells are the sole mediators of cancer control in mice and are solely responsive to checkpoint blockade in mice and humans. High-avidity T cells are ineffective and immune-suppressive. The mechanistic basis of these differences lies in the higher exhaustion status of high-avidity cells. High-avidity T cells have a distinct transcriptomic profile that is used here to calculate an 'avidity score', which we then use for in silico identification of low-avidity and high-avidity T cells in mice and humans. Surprisingly, CD8+ T cells with identical T cell receptors exhibit wide variation in avidities, suggesting an additional level of regulation of T cell activity. Aside from providing a better understanding of endogenous T cell responses to cancer, these findings might instruct future immunotherapy strategies.

Enhanced RNAi does not provide efficient innate antiviral immunity in mice.

In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.

Evaluating the Immunoprotective and Diagnostic Potential of Schistosoma mansoni Epitopes from Sm050890 and Sm141290 Proteins Identified Through Reverse Vaccinology.

Schistosomiasis remains a parasitic disease affecting millions of people worldwide, requiring interventions like vaccination. In previous work, our group used reverse vaccinology to identify two epitopes from the Schistosoma mansoni proteins, Sm050890 (44-58) and Sm141290 (225-239). This study evaluated the immune response profile and protection induced by peptides, as a mixture of immunogens, in murine vaccination trials. Additionally, the diagnostic potential of these peptides was assessed on immunoassays. Mice were immunized with a formulation containing the mixture of the peptides, subsequently infected, and perfused for worm burden recovery and quantification. Liver and blood samples from animals were used to evaluate the effect of immunization on the formation of granulomas and specific anti-peptide antibodies (IgG). Additionally, cytokine measurement was performed in splenocyte cultures from immunized mice, and peripheral blood serum from individuals infected with S. mansoni was used to assess the recognition of the peptides by IgG antibodies. The vaccine stimulated an increase in the production of IgG and IgG2c antibodies, associated with a significant reduction of 44 - 29% in worm burden. Although the vaccine did not reduce liver pathology, it enhanced the production of IFN-γ while decreasing IL-10 production by splenocytes. Furthermore, the peptides Sm050890 (44-58) and Sm141290 (225-239) were not recognized by IgG antibodies in the serum from infected individuals. Overall, our data suggest that the peptides Sm050890 (44-58) and Sm141290 (225-239) are promising vaccine candidates against schistosomiasis and can be used to compose a multiepitope/chimeric vaccine in future studies.

Human DBR1 deficiency impairs stress granule-dependent PKR antiviral immunity.

The molecular mechanism by which inborn errors of the human RNA lariat-debranching enzyme 1 (DBR1) underlie brainstem viral encephalitis is unknown. We show here that the accumulation of RNA lariats in human DBR1-deficient cells interferes with stress granule (SG) assembly, promoting the proteasome degradation of at least G3BP1 and G3BP2, two key components of SGs. In turn, impaired assembly of SGs, which normally recruit PKR, impairs PKR activation and activity against viruses, including HSV-1. Remarkably, the genetic ablation of PKR abolishes the corresponding antiviral effect of DBR1 in vitro. We also show that Dbr1Y17H/Y17H mice are susceptible to similar viral infections in vivo. Moreover, cells and brain samples from Dbr1Y17H/Y17H mice exhibit decreased G3BP1/2 expression and PKR phosphorylation. Thus, the debranching of RNA lariats by DBR1 permits G3BP1/2- and SG assembly-mediated PKR activation and cell-intrinsic antiviral immunity in mice and humans. DBR1-deficient patients are prone to viral disease because of intracellular lariat accumulation, which impairs G3BP1/2- and SG assembly-dependent PKR activation.

Loss of Mist1 alters the characteristics of Paneth cells and impacts the function of intestinal stem cells in physiological conditions and after radiation injury.

Intestinal stem cells (ISCs) and Paneth cells (PCs) reside at the bottom of the crypts of Lieberkühn in the small intestine. Recent studies have shown that the transcription factor Mist1, also named BHLHA15, plays an important role in the maturation of PCs. Since there is an intimate interaction between PCs and ISCs, we speculated that the loss of Mist1 could impact these two neighboring cell types. Here, we report that mice lacking Mist1 had fewer but larger PCs with shrunken secretory granules, accompanied by an increase in goblet cells and tuft cells. Mist1 loss significantly decreased the number of proliferative crypt cells, especially columnar basal cells (CBCs). In addition, Mist1-deficient enteroids needed supplemental Wnt3a to support their growth. Results from RNA sequencing (RNA-seq) demonstrated an apparent deficiency of innate immunity in Mist1-knockout mice. Intriguingly, Mist1 loss increased the survival rate of mice subjected to whole abdominal irradiation (WAI). Moreover, radiation injury was ameliorated in Mist1-knockout mice compared with their wild-type littermates based on histological analysis and enteroid culture, which might be a consequence of increased contents of the endoplasmic reticulum (ER) and the increased activity of mTORC1 in Paneth cells. In summary, our data uncover that Mist1 plays an important functional role in PCs and regulates the maintenance of ISCs and their response to radiation injury. © 2025 The Pathological Society of Great Britain and Ireland.

Relieving Effect of Artemisia ordosica Krasch Extract on DSS-Induced Colitis by Regulating Immunity, Antioxidant Function, Gut Microbiota, and Bile Acid Metabolism in Mice

Artemisia ordosica Krasch, a traditional Chinese herbal medicine, possesses antibacterial, antiviral, and anti-inflammatory properties. The aim of this experiment was to investigate the therapeutic effect of Artemisia ordosica Krasch extraction (AOE) in treating colitis induced by dextran sulfate sodium (DSS) in mice. The in vitro antioxidant activity of AOE was evaluated by assessing its iron reduction capacity and scavenging capacity towards 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals (·OH). The protective effect of AOE on colitis in mice was determined by monitoring key indicators such as body weight, colon length, and survival rate in mice, as well as by assessing the expression of colon-related genes and cytokine levels. We evaluated the impact of AOE on intestinal microbiota by measuring the 16s sequencing of cecal contents and bile acid metabolism. The results showed that the iron reduction capacity of AOE was positively correlated with its concentration. The half-maximal inhibitory concentrations (IC50) for scavenging DPPH and hydroxyl radicals were 3.126 mg/mL and 6.139 mg/mL, with a 95% confidence interval of 95%. In vivo studies demonstrated that AOE reduced DSS-induced colitis in mice by increasing the colon length, enhancing antioxidant enzyme activity, inhibiting inflammatory cell infiltration, suppressing the formation of TNF-α and IL-6, and reducing malondialdehyde (MDA) levels. qPCR analysis revealed that AOE reversed the down-regulation of Claudin mRNA expression, and altered the composition of cecal microbiota, thus mitigating DSS-induced colitis. AOE plays a crucial role in alleviating colitis in mice and effectively improves DSS-induced colitis, highlighting its potential as a therapeutic agent for inflammatory bowel diseases.

Membrane palmitoylated protein MPP1 inhibits immune escape by regulating the USP12/ CCL5 axis in urothelial carcinoma.

The response rate to immunotherapy in patients with urothelial carcinoma remains limited. Studies have shown that membrane palmitoylated proteins (MPPs) play key roles in tumor progression. However, the mechanisms by which MPP1 regulates immune escape in urothelial carcinoma are not well understood. The TCGA and BEST databases were used to analyze the associations between the expression of members of the MPP family and the prognosis or immunotherapy sensitivity of urothelial carcinoma patients. MPP1 was identified due to its significant association with survival and immunotherapy sensitivity. MPP1 expression in urothelial carcinoma tissues and cell lines was examined. An MPP1 overexpression vector was used to transfect urothelial carcinoma cells. The functional assays included proliferation, migration, urothelial carcinoma cell-CD8+ T-cell coculture, CD8+ T-cell chemotaxis, and tumorigenesis in human immune reconstitution NOG mice (HuNOG). Bioinformatics, coimmunoprecipitation (CO-IP), mass spectrometry, quantitative real-time polymerase chain reaction (RT-qPCR), and western blotting were used to validate the activity of the MPP1/USP12/CCL5 cascade. Analysis of the BEST data revealed that, compared with other MPP family genes, MPP1 was more strongly associated with urothelial carcinoma prognosis and immunotherapy response. Low MPP1 expression was observed in urothelial carcinoma patients and was positively associated with better survival. MPP1 inhibited the proliferation and migration of urothelial carcinoma cells. Bioinformatics, in vitro coculture assays, and in vivo tumorigenesis experiments demonstrated that MPP1 promotes CCL5 production and CD8+ T-cell chemotaxis in the urothelial carcinoma tumor microenvironment (TME). Mechanistically, bioinformatics, mass spectrometry, co-IP, RT-qPCR, and western blot analyses indicated that MPP1 increases CCL5 expression by binding to and promoting USP12. MPP1 significantly inhibits urothelial carcinoma cell proliferation and immune escape via the MPP1/USP12/CCL5 cascade. MPP1 has the potential to serve as a biomarker for guiding immunotherapy in patients with urothelial carcinoma.

Oral Immunisation With Non-GMO Surface Displayed SARS-CoV-2 Spike Epitopes on Bacteria-Like Particles Provokes Robust Humoral and Cellular Immune Responses, and Modulated the Gut Microbiome in Mice.

The coronavirus disease 2019 (COVID-19) is a fatal disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To date, several vaccines have been developed to combat the spread of this virus. Mucosal vaccines using food-grade bacteria, such as Lactobacillus spp., are promising strategies for developing safe and effective vaccines against SARS-CoV-2. In this study, we designed a non-GMO surface-displayed SARS-CoV-2 spike S1 epitope on Limosilactobacillus fermentum-derived bacteria-like particles (BLPs). After that, we evaluated its efficacy to induce immune responses in immunocompetent mice. Moreover, we examined the influence of oral immunisation on the gut microbiome and microbiota metabolites. Twenty-eight 6-week-old male C57BL/6 mice were orally immunised with the following: PBS (control), Lm. fermentum-derived BLPs only, BLPs displaying SARS-CoV-2 spike S1-2, or BLPs displaying SARS-CoV-2 spike S1-3 epitopes. Our results showed that mucosal immunisation of mice with surface-displayed SARS-CoV-2 spike epitopes provoked high-level secretory IgA and systemic IgG production. Moreover, the immunisation exhibited a Th1-like immune response, characterised by an elevated IgG2a-to-IgG1 ratio and high antiviral IFN-γ production. In addition, we observed gut microbiome modulation and increased butyrate production in immunised mice. Overall, the use of Lm. fermentum-derived BLPs and the anchor CshA to display SARS-CoV-2 spike S1epitopes is a promising novel strategy in developing a cost-effective, non-GMO mucosal vaccine alternative against SARS-CoV-2.

Protection induced by recombinant vaccinia virus targeting the ROP4 of Toxoplasma gondii in mice.

Toxoplasmosis is a parasitic disease affecting a significant portion of the global population, whose etiology can be attributed to the protozoan organism Toxoplasma gondii. Despite its public health importance, an efficacious vaccine to prevent human toxoplasmosis remains unavailable. To this end, we designed an experimental toxoplasmosis vaccine using recombinant vaccinia virus vectors (rVacv) expressing the T. gondii rhoptry protein 4 (ROP4) antigen and evaluated its efficacy in a murine model. Intranasal vaccination with ROP4-rVacvs induced parasite-specific serum antibody responses as early as 3 weeks post-immunization, with subsequent immunizations elevating antibody responses to a greater extent. When challenged with T. gondii ME49 strain, significantly enhanced levels of mucosal IgA antibodies were detected in the intestines of immunized mice. Additionally, ROP4-rVacv immunization ensured that T cells and germinal center B cell populations were retained at high levels. These immune responses mitigated the severity of neuroinflammation, reduced tissue cyst formation, and ensured the survival of immunized mice. Our findings indicate that ROP4-rVacv could be a promising toxoplasmosis vaccine candidate.

Protection induced by Streptococcus pneumoniae extracellular vesicles against nasal colonization and invasive infection in mice and the role of PspA

Diseases caused by Streptococcus pneumoniae (pneumococcus) produce a great impact on public health, killing about one million people annually despite available vaccines. Recent research has revealed that the pneumococcus produces extracellular vesicles (pEVs), which display selective cargo and hold potential for vaccine development. Here, we evaluated the immunogenicity and protective potential of pEVs derived from a non-encapsulated pneumococcal strain (R6) using murine models of pneumococcal colonization and invasive pneumonia. Characterization of the immune response revealed that while pEVs contain multiple virulence determinants, immunization with these nanoparticles only induces antibodies against a subset of them. Specifically, subcutaneous immunization elicits a high antibody response against PspA, a modest response against PrsA, and limited response against Ply, MalX and PsaA. The antibody response was further supported by agglutination studies, showing that sera from pEV immunized mice agglutinate pneumococci and that PspA contributes to this response in a strain-dependent manner. Subcutaneous immunization with pEVs provides protection in the invasive pneumonia model whereas nasal immunization results in one log reduction in pneumococcal colonization of the upper respiratory tract. Finally, PspA is a strong contributor to protection in the invasive model and provides a degree of protection even across heterologous families of PspA. We conclude that pEVs demonstrate potential for vaccine development, protecting across capsular types and providing some degree of protection across heterologous PspA variants.

Selection signatures associated with adaptation in South African Drakensberger, Nguni, and Tuli beef breeds

In the present study 1,709 cattle, including 1,118 Drakensberger (DRB), 377 Nguni (NGI), and 214 Tuli (TUL), were genotyped using the GeneSeek® Genomic Profiler™ 150 K bovine SNP panel. A genomic data set of 122,632 quality-filtered single nucleotide polymorphisms (SNPs) were used to identify selection signatures within breeds based on conserved runs of homozygosity (ROH) and heterozygosity (ROHet) estimated with the detectRUNS R package. The mean number of ROH per animal varied across breeds ranging from 36.09 ± 12.82 (NGI) to 51.82 ± 21.01 (DRB), and the mean ROH length per breed ranged between 2.31 Mb (NGI) and 3.90 Mb (DRB). The smallest length categories i.e., ROH < 4 Mb were most frequent, indicating historic inbreeding effects for all breeds. The ROH based inbreeding coefficients (FROH) ranged between 0.033 ± 0.024 (NGI) and 0.081 ± 0.046 (DRB). Genes mapped to candidate regions were associated with immunity (ADAMTS12, LY96, WDPCP) and adaptation (FKBP4, CBFA2T3, TUBB3) in cattle and genes previously only reported for immunity in mice and human (EXOC3L1, MYO1G). The present study contributes to the understanding of the genetic mechanisms of adaptation, providing information for potential molecular application in genetic evaluation and selection programs.

Vaccination with a DNA vaccine cocktail encoding TgROP2, TgROP5, TgROP9, TgROP16, TgROP17, and TgROP18 confers limited protection against Toxoplasma gondii in BALB/c mice.

Toxoplasmosis is a foodborne zoonotic parasitic disease caused by Toxoplasma gondii, which seriously threatens to human health and causes economic losses. At present, there is no effective vaccine strategy for the prevention and control of toxoplasmosis. T. gondii rhoptry proteins (ROPs) are important proteins secreted by the parasite during the early stage of invasion into host cells. In this study, we constructed six individual plasmids (pVAX1-ROP2, pVAX1-ROP5, pVAX1-ROP9, pVAX1-ROP16, pVAX1-ROP17, and pVAX1-ROP18) encoding T. gondii rhoptry proteins and then used an equimolar amount of each as a vaccine cocktail. Following booster immunization, serum antibody levels, splenic lymphocyte proliferation, cytokine production, and survival time after infection with T. gondii RH strain were measured in immunized mice. The results showed that the mice immunized with the DNA vaccine cocktail developed a higher level of the specific anti-T. gondii IgG in serum and the cytokines such as IFN-γ, IL-2, IL-12, and IL-4 (P < 0.01). The stimulation index (SI) of spleen lymphocytes (P < 0.01), the frequencies of CD4+ T lymphocytes (P < 0.01), and the ratio of CD4+/CD8+ T lymphocytes (P < 0.05 or P < 0.01) in the vaccine-immunized mice were significantly increased compared to the control group. After challenge with the virulent T. gondii RH strain tachyzoites, the survival time of mice in the DNA vaccine cocktail group (18.1 ± 1.81 d) was significantly longer (P < 0.01) than that in the control group (8.4 ± 1.02 or 7.9 ± 0.83 d). The results indicated that the DNA vaccine cocktail could elicit strong humoral and cellular immune responses in mice and could also improve the resistance of mice to acute T. gondii infection.

Modification of Fc-fusion protein structures to enhance efficacy of cancer vaccine in plant expression system.

Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM-like structures as anti-cancer vaccines in Nicotiana tabacum. High-mannose glycan structures were generated by adding a C-terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM-Fc and EpCAM-FcK proteins in transgenic plants via Agrobacterium-mediated transformation. Cross-fertilization of EpCAM-Fc (FcK) transgenic plants with Joining chain (J-chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM-IgA Fc (EpCAM-A) and IgM-like (EpCAM-M) proteins. Immunoblotting, SDS-PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti-EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti-EpCAM IgGs. Flow cytometry indicated that the EpCAM-Fc fusion proteins significantly activated CD8+ cytotoxic Tcells, CD4+ helper Tcells and B cells, particularly with EpCAM-FcKP and EpCAM-FcP (FcKP) × JP (JKP). The induced anti-EpCAM IgGs captured human prostate cancer PC-3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down-regulated proliferation markers (PCNA, Ki-67) and epithelial-mesenchymal transition markers (Vimentin); and up-regulated E-cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti-cancer immune responses.

Development of an Enzyme-Linked Immunosorbent Assay Based on a Monoclonal Antibody for the Rapid Detection of Citrinin in Wine.

The ingestion of food contaminated with citrinin (CIT) poses a variety of health risks to humans and animals. The immunogens (CIT-COOH-BSA, CIT-H-BSA) and detection antigen (CIT-COOH-OVA, CIT-H-OVA) were synthesised using the active ester method (-COOH) and formaldehyde addition method (-H). A hybridoma cell line (3G5) that secretes anti-CIT monoclonal antibodies (mAbs) was screened via CIT-H-BSA immunisation of mice, cell fusion, and ELISA screening technology. The cell line was injected intraperitoneally to prepare ascites. The reaction conditions for the indirect competitive ELISA (ic-ELISA) were optimised, and an ic-ELISA method for detecting CIT was preliminarily established. The results revealed that the IC50 of CIT from optimised ic-ELISA was 37 pg/mL, the linear detection range was 5.9~230 pg/mL, and the cross-reaction (CR) rate with other analogues was less than 0.01%. The intra-assay and interassay sample recovery rates of CIT were 84.7~92.0% and 83.6~91.6%, and the coefficients of variation (CVs) were less than 10%. The ic-ELISA of CIT established in this study was not significantly different from the HPLC results and is rapid, highly sensitive and strongly specific, providing technical support for the detection of CIT.