The Effect of Different Immunization Cycles of a Recombinant Mucin1-Maltose-Binding Protein Vaccine on T Cell Responses to B16-MUC1 Melanoma in Mice.

We explored the effect of a recombinant mucin1-maltose-binding protein vaccine, including immunization cycles of recombinant mucin1-maltose-binding protein (MUC1-MBP) and CpG 2006 on T cell responses to human MUC1-overexpressing mouse melanoma B16 cells (B16-MUC1) melanoma in mice. We found that the vaccine had a significant antitumor effect, with the most obvious tumor-suppressive effect being observed in mice immunized five times. After more than five immunizations, the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations). To study the possible mechanism, Mucin-1(MUC1)-specific antibodies, IFN-γ secretion by lymphocytes, and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and a real-time cell analyzer (RTCA). T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. These results showed that five immunizations activated MUC1-specific Th1 and CTL and reduced the ratio of myeloid-derived suppressor cells (MDSCs) and Th17 in mice more significantly than eight immunizations, indicating that excessive frequency of the immune cycle leads to the increased numbers of immunosuppressive cells and decreased numbers of immunostimulatory cells, thereby inhibiting antitumor immune activity. This data provide an experimental foundation for the clinical application of a recombinant MUC1-MBP vaccine.

A peptide/maltose-binding protein fusion protein used to replace the traditional antigen for immunological detection of deoxynivalenol in food and feed.

A deoxynivalenol (DON) epitope clone (D8) was obtained by phage display technology using anti-DON monoclonal antibodies as a target molecule. Subsequently, a DON antigen mimic (D8-maltose-binding protein [MBP]) was synthesized by fusing the mimic epitope peptide with MBP. An enzyme-linked immunosorbent assay (ELISA) and urchin-like gold nanoparticle immunochromatographic assay was developed based on D8-MBP for detection of DON in maize and wheat. The half-maximal inhibitory concentration, lower detection limit, and linear range of the D8-MBP ELISA were 57.98 ±â€¯0.97, 9.83, and 11.32-286.77 ng/mL, respectively. The sensitivity of the D8-MBP ELISA was nearly 2.5 times higher than that of traditional ELISA using DON-bovine serum albumin (BSA). The detection threshold of the colloidal gold immunochromatographic assay for D8-MBP was 25 ng/mL. Thus, D8-MBP could be used to replace the traditional DON-BSA antigen for the immunological detection of DON, permitting low cost, rapid detection of DON.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation.

Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4+ T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy.

Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

Immunization against recombinant GnRH-I alters testicular structure in an experimental boar model.

The aim of this study was to evaluate and to compare testicular tissue in immunized and control boars. Eighteen male piglets, aged 12 weeks, were vaccinated twice intramuscularly with a maltose-binding protein-gonadotropin-releasing hormone I hexamer peptide (MBP-GnRH-I6). Blood samples were taken at 12, 18, 21 and 24 weeks of age. Serum concentrations of testosterone and GnRH-I antibodies were determined by radioimmunoassay. The pigs were sacrificed 6 weeks after the second immunization. Testicular weight and size were recorded and tissue samples were collected for histological examination. The results demonstrated that active immunization against MBP-GnRH-I6 increased serum GnRH-I antibody levels (P < 0.05) and reduced serum concentrations of testosterone (P < 0.05) when compared with controls. Histological studies performed on testicular tissue revealed clear signs of atrophy in the MBP-GnRH-I6 immunized pigs, and a significant reduction (P < 0.05) in paired testes weight and size were seen in the treated boars. Microscopically, the mean diameter of the seminiferous tubules was markedly reduced (P < 0.01). Spermatogonia were visible, as well as few spermatocytes, but no spermatozoa were detected in the seminiferous tubules. Ultramicroscopic analysis of testicular tissue revealed an increase in the thickness of the basement membrane and extensive damage in the cell organelles of the treated animals, including small spermatogonial size, decreased number of mitochondria and endoplasmic reticulum in the primary spermatocyte and spermatid, a shallow hollow for nuclear membranes in Sertoli cells and mitochondrial vacuolation in Leydig cells. We conclude that MBP-GnRH-I6 induces severe atrophy in the testes of immunized boars.

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.

Immunization against recombinant GnRH-I alters ultrastructure of gonadotropin cell in an experimental boar model.

BACKGROUND: Gonadotropin cell is the main responsible for the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and immunocastration reduces the concentrations of serum FSH and LH. A few studies have reported the histological structure of gonadotropin cells obtained from immunocastration animals at the light microscopy level. However, the ultrastructure of gonadotropin cells remains largely unexplored. The aim of this study was to evaluate and to compare ultrastructure of gonadotropin cell in gonadally intact boars and immunologically castrated male animals. FINDINGS: In this study, serum and adenohypophysis tissue were collected from nine gonadally intact boars and nine male pigs treated with recombinant gonadotropin releasing hormone I (GnRH-I). Anti-GnRH-I antibodies in serum and the ultrastructure of gonadotropin cell in adenohypophysis were determined by enzymelinked immunosorbent assay and electron microscopy, respectively. The results demonstrated that active immunization against recombinant GnRH-I increased serum GnRH-I antibody levels (P<0.05). Ultramicroscopic analysis of gonadotropin cell revealed a decrease (P<0.05) in the number and size of the large granules and small granules in the recombinant GnRH-I immunized animals. CONCLUSIONS: We conclude that immunization against recombinant GnRH-I induces severe atrophy of granules in gonadotropin cell of boars, possibly reflecting GnRH-I regulation of gonadotropin cell.

The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini.

Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.

Maltosylated polyethylenimine-based triple nanocomplexes of human papillomavirus 16L1 protein and DNA as a vaccine co-delivery system.

To improve vaccine delivery, we herein designed a co-delivery system using a protein antigen and its encoding plasmid linked in nanocomplexes via maltosylated PEI (mPEI). Cationic mPEI was electrostatically complexed to a plasmid encoding the human papillomavirus (HPV) type 16L1 protein (pHPV16L1), and further complexed to a maltose binding protein (MBP)-fused human papillomavirus type 16L1 fusion protein (HPV16L1-MBP). The HPV16L1-MBP/mPEI/pHPV16L1 complexes were characterized by gel-retardation properties, zeta potentials and sizes. The intracellular co-delivery of protein and plasmid DNA vaccines was significantly higher for mPEI-based triple nanocomplexes than for a simple physical mixture of the proteins and DNA. Moreover, the cellular delivery of plasmid DNA using mPEI-based triple nanocomplexes resulted in higher expression levels comparable to those obtained using dual complexes of mPEI and the plasmid DNA. In vivo, co-immunization of mice with HPV16L1-MBP/mPEI/pHPV16L1 nanocomplexes triggered the highest levels of humoral immune responses among various vaccination groups. Moreover, the mPEI-based nanocomplexes significantly enhanced the number of interferon-γ producing CD8(+) T cells compared with the use of mixed proteins and plasmid DNA. These results suggest that the effective cellular co-delivery of MBP-fused antigen proteins and plasmid DNA using maltosylated PEI-based triple nanocomplexes could enhance the immunogenicity of HPV16L1 vaccines.

Synergistic antitumor effects of Escherichia coli maltose binding protein and Bacillus Calmette-Guerin in a mouse lung carcinoma model.

Maltose binding protein (MBP) is a component of the maltose transport system in the periplasm of Escherichia coli. It is commonly believed that MBP has minimal effects on the bioactivity, thus, it is widely used in the purification of recombinant proteins. Here, we found that the combined immunization with MBP and Bacillus Calmette-Guerin (BCG) significantly inhibited tumor growth compared with MBP or BCG immunization alone in a mouse lung carcinoma model. Further studies showed that MBP nonspecifically activated T helper 1 (Th1) cells and enhanced the BCG-induced Th1 cell activation. Moreover, MBP or BCG immunization alone increased the activities of natural killer (NK) cells and macrophages, and the combined immunization with MBP and BCG induced a synergistic effect on the activities of NK cells and macrophages. These results suggest that MBP possesses potent immune enhancement activities, and that the combination of MBP and BCG-induced synergistic antitumor effects might be mediated mainly through the activation of Th1 cells, NK cells and macrophages.

Establishment of a mouse IgA nephropathy model with the MBP-20-peptide fusion protein.

Here, we aimed to determine whether immunoglobulin-A nephropathy (IgAN) could be induced in Balb/c mice by immunizing them with a fusion protein (MBP-20 peptide) comprising the maltose-binding protein (MBP) and a 20-amino-acid peptide derived from Staphylococcus aureus. A recombinant plasmid encoding the fusion protein was constructed and expressed in bacterial cells. The synthetic 20-peptide was used to prepare the monoclonal antibody. Balb/c mice were immunized with the MBP-20-peptide fusion protein over a 21-week course before renal histology was examined at the light and electron microscopic levels. Direct immunofluorescence staining with the anti-20-peptide monoclonal antibody was also performed using renal biopsy tissue from human IgAN patients as a comparison. IgA and IgG specific for the 20-peptide in human and mice serum were detected. The IgAN experimental mice developed a clinical and pathological profile that closely resembled that of human IgAN patients, including the induction of hematuria and numerous histopathological features. Levels of IgA and IgG specific for the 20-peptide were significantly increased in serum from the IgAN experimental mice and IgAN patients compared with control mice and non-IgAN patients. In IgAN model mice, the anti-20-peptide antibody labeled glomeruli, while the antibody strongly labeled glomeruli and weakly labeled tubular epithelial cells in renal tissue from human IgAN patients. In conclusion, immunization with an MBP-20-peptide fusion protein is able to induce clinical and pathological features closely resembling IgAN in Balb/c mice, indicating a potentially useful role for the model in the study of IgAN and related diseases.