Yanping Luo

Subunit vaccine candidate AMM down-regulated the regulatory T cells and enhanced the protective immunity of BCG on a suitable schedule.

Mycobacterium bovis bacillus Calmette‐Guérin (BCG) priming and subunit vaccine boosting strategies are urgently needed to improve the protective efficacy of BCG in adult population. However, the schedule of subunit vaccine boosting is not well investigated, especially the optimal immune responses and vaccine immunization schedules are still not clear. We have constructed a novel subunit vaccine candidate consisting of fusion protein Ag85B‐Mpt64 (190‐198)‐Mtb8.4 (AMM) in a complex adjuvant composed of dimo‐thylidioctyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG‐PSN). In this study, we compared the effect of different boosting schedules of the subunit vaccine in the prime‐boost strategies. C57BL/6 mice were primed with BCG first and then boosted with the AMM vaccine once at 10th week, twice at 8th, 10th week, or thrice at 6th, 8th, 10th week, respectively. The immune responses were evaluated at the 14th and 20th weeks, respectively. Twelve weeks after the last immunization, the mice were challenged with virulent Mycobacterium tuberculosis strain H37Rv, and the protective effect was evaluated. The results showed that BCG priming and the AMM vaccine boosting twice induced the strongest antigen‐specific IFN‐γ and IL‐2 production, down‐regulated CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) and had the best protective effect among all groups, while boosting thrice induced the strongest IL‐4 production and did not improve BCG‐primed protection significantly. Boosting BCG with the AMM vaccine twice instead of once or thrice induced strong Th1‐type immunity and down‐regulated Tregs significantly, which correlated with the best protection against M. tuberculosis infection in mice.

A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice.

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38log10CFU) than traditional vaccine Bacillus Calmette-Guerin (BCG) (6.01 ± 0.33log10CFU) and PBS control (6.53 ± 0.26log10CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.

Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.

A multistage mycobacterium tuberculosis subunit vaccine LT70 including latency antigen Rv2626c induces long-term protection against tuberculosis

ABSTRACT To develop an effective subunit vaccine which could target tubercle bacilli with different metabolic states and provide effective protective immunity, we fused antigens ESAT6, Ag85B, peptide 190–198 of MPT64, and Mtb8.4 mainly expressed by proliferating bacteria and latency-associated antigen Rv2626c together to construct a multistage protein ESAT6-Ag85B-MPT64(190–198)-Mtb8.4-Rv2626c (LT70 for short) with the molecular weight of 70 kDa. The human T-cell responses to LT70 and other antigens were analyzed. The immune responses of LT70 in the adjuvant of DDA and Poly I:C and its protective efficacy against Mycobacterium tuberculosis (M. tuberculosis) infection in C57BL/6 mice were evaluated. The results showed that LT70 was stably produced in Escherichia coli and could be purified by successive salting-out and chromatography. LT70 could be strongly recognized by human T cells from TB patients and persons who are supposed latently infected with M. tuberculosis. The subunit vaccine LT70 generated strong antigen-specific humoral and cell-mediated immunity, and induced higher protective efficacy (5.41±0.37 Log10 CFU in lung) than traditional vaccine Bacillus Calmette-Guerin (6.01±0.33 Log10 CFU) and PBS control (6.53±0.26 Log10 CFU) at 30 weeks post vaccination (10 weeks post-challenge) against M. tuberculosis infection (p < 0.05). These findings suggested that LT70 would be a promising subunit vaccine candidate against M. tuberculosis infection.

Effects of immunomodulators on liquefaction and ulceration in the rabbit skin model of tuberculosis

To better control tuberculosis (TB) epidemics in developing countries a real need exists to study the liquefaction and cavity formation that occur in pulmonary TB lesions. This report is the first to evaluate the effects of immunomodulators on these two processes in a rabbit skin model. The effects of recombinant human interferon-γ (rIFN-γ), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) were evaluated in TB lesions produced by intradermal injection of 5 × 10(6) viable BCG bacilli. Recombinant IL-2 and rIFN-γ accelerated the liquefaction and healing of the lesions, and reduced the bacterial load. In contrast, dexamethasone inhibited the liquefaction of the lesions, and increased the bacterial load. The effect of CTX was similar to dexamethasone but not as pronounced. Serum levels of IL-2 were higher during the liquefaction and healing phases in the rIL-2 and rIFN-γ groups. Therefore, immunomodulators affect both the development of TB lesions and the survival of the mycobacteria within them. This study suggests that the rabbit skin model can be a valuable method to select therapeutic agents that could inhibit liquefaction and cavity formation in pulmonary tuberculosis.

Comparison of student perception and performance between case-based learning and lecture-based learning in a clinical laboratory immunology course

Abstract Background: Case-based learning (CBL), an educational method of problem-based learning, provides students with a venue to relate content learned in the classroom to performance in professional practice. This study compared CBL in the teaching of a clinical laboratory immunology (CLI) course to lecture-based learning (LBL), and evaluated the effect on students regarding the CBL. Methods: Data were collected from senior students (n=85; 46% males, 54% females) at Lanzhou University in China. The students were divided into two groups, one group was offered CBL, while the other LBL as a teaching instrument. After intervention, perceptions of both the groups about their respective teaching method were evaluated using questionnaires, the resulting scores were compared to those obtained in the LBL group. Results: The CBL group showed significantly better scores in course examination (p<0.05) as compared to the LBL group. Seventy-seven (90.6%) students in the CBL group opined that CBL improved their learning and clinical problem-solving skills. CBL also provided them with better understanding (90.6%) and preparation for examinations (90.6%). CBL group improved markedly in comparison to the LBL group with regard to learning motivation (p=0.040), clinical reasoning ability (p=0.023) and clinical problem-solving ability (p=0.022). Conclusions: Our findings demonstrate that CBL is a more effective teaching strategy as compared to LBL in a CLI course. Consequently, the implementation of CBL in teaching a CLI course helps students to improve their learning motivation, problem solving abilities and mastery of knowledge.

A flavonoid compound from Chrysosplenium nudicaule inhibits growth and induces apoptosis of the human stomach cancer cell line SGC-7901

Abstract Context: Gastric cancer remains highly prevalent, but treatment options are limited. Natural products have proved to be a rich source of anticancer drugs. Chrysosplenium nudicaule Ledeb. (Saxifragaceae) is a perennial herb that grows in the highlands of China. It has been used as a traditional Chinese medicine to treat digestive diseases for hundreds of years. Recent studies revealed that this herb had anticancer activity, and the flavonoids were speculated to be the effective components. 6,7,3′-Trimethoxy-3,5,4′-trihydroxy flavone (TTF) and 5,4′-dihydroxy-3,6,3′trimethoxy-flavone-7-O-β-d-glucoside (DTFG) are flavonoid compounds isolated from Chrysosplenium nudicaule. Objective: This study examined the effect of TTF and DTFG on SGC-7901 human stomach cancer cell in vitro to determine the anticancer and induction of apoptosis properties of TTF. Materials and methods: The proliferation of cells treated with 32, 16, 8, 4, and 2 μg/mL of TTF or DTFG for 24, 48, and 72 h was assessed by the MTT assay. After being treated with TTF, the apoptosis of SGC-7901 cells was assessed by acridine orange staining, ultrastructure, electrophoresis of DNA fragmentation, and flow cytometry. Results: Results indicated that TTF inhibited the growth of cancer cells with an IC50 value of 8.33 μg/mL after 72 h incubation. However, DTFG showed no inhibitory effect on the growth of the cancer cell. Further studies on TTF also confirmed that it was able to induce apoptosis of SGC-7901 cells at a concentration as low as 4 μg/mL. Discussion and conclusion: The apoptotic effect of TTF makes it a promising candidate for future chemotherapeutic application in treating stomach cancer.

Prolonged intervals during Mycobacterium tuberculosis subunit vaccine boosting contributes to eliciting immunity mediated by central memory-like T cells

It is believed that central memory T cells (TCM) provide long-term protection against tuberculosis (TB). However, the effects of TB subunit vaccine immunization schedule, especially the vaccination intervals, on T cell immune memory is still unclear. In this study, mice were immunized with fusion protein ESAT6-Ag85B-MPT64 (190-198)-Mtb8.4-Rv2626c (LT70) based subunit vaccine three times according to the following schedules: ① 0, 3rd and 6th week respectively (0-3-6w), ② 0, 4th and 12th week (0-4-12w), and ③ 0, 4th and 24th week (0-4-24w). We found that both schedules of 0-4-12w and 0-4-24w induced higher level of antigen specific IL-2, IFN-γ and TNF-α than 0-3-6w immunization. Among them, 0-4-12w induced the highest level of IL-2, which is a key cytokine mainly produced by TCM. Moreover, by cultured IFN-γ ELISPOT and cell proliferation assay etc., we found that the vaccination schedule of 0-4-12w elicited higher numbers of TCM like cells, stronger TCM - mediated immune responses and higher protective efficacy against M. bovis BCG challenge than 0-3-6w did. It suggests that prolonging the vaccination interval of TB subunit vaccine to some extent contributes to inducing more abundant TCM like cells and providing stronger immune protection against mycobacteria infection.

Constituent and effects of polysaccharides isolated from Sophora moorcroftiana seeds on lifespan, reproduction, stress resistance, and antimicrobial capacity in Caenorhabditis elegans

Sophora moorcroftiana (S. moorcroftiana) is an endemic leguminous dwarf shrub in Tibet, China. Decoctions of the seeds have been used in Chinese folk medicine for dephlogistication, detoxication, and infectious diseases. The present study aimed to investigate the constituent and biological effects of polysaccharides from S. moorcroftiana seeds in Caenorhabditis elegans (C. elegans). Polysaccharides from S. moorcroftiana seeds (SMpol) were extracted with 60% ethanol and constituent was analyzed by GC-MS. SMpol was composed of glucose, galactose and inositol in the molar ratio of 35.7 : 1.3 : 17.0. Synchronized worms were treated with SMpol and then lifespan, motility, reproduction, stress resistance and antimicrobial activity were examined. Compared with the control group, the lifespan was increased to the average of 27.3 days and the number of laying eggs showed a 1.3-fold increase in nematodes treated with SMpol (4 mg·mL-1). In SMpol (4 mg·mL-1) treated worms, there was a 1.1-fold increase in 24-h survival of acute heat stress and a 1.6-fold increase in 2-h survival of oxidative stress The colonization of the bacteria in the SMpol treated nematode was significantly lower than that of the untreated group by 68.3%. In vivo studies showed SMpol significantly extended the life span, improved reproduction, increased stress resistance and antimicrobial capacity of C. elegans. In conclusion, those results indicated that the polysaccharides from S. moorcroftiana seeds were involved in a variety of biological activities leading to its modulatory effects on C. elegans which may be developed as a natural supplement agent.

Anti-parasitic effects of water-soluble alkaloid fractions from ethanolic extracts of Sophora moorcroftiana seeds in Caenorhabditis elegans

Parasite infections of humans and animals remain a major global health problem, with limited choice of drugs being available to the treatment of parasitosis in the clinic. Sophora moorcroftiana (S. moorcroftiana) is a shrub that grows in Tibet Plateau of China. Decoction of the seeds has been used as a traditional Tibetan medicine to treat parasitosis for years. But the anti-parasitic effects of water-soluble fractions in the seeds need further investigation. In the present study, the water-soluble alkaloid fractions (E2) were obtained from S. moorcroftiana seeds by refluxing extraction with 60% ethanol and low polarity fraction (E2-a) and high polarity fraction (E2-b) were subsequently isolated from E2 using column chromatography. As a parasite model, Caenorhabditis elegans (C. elegans) were treated with different fractions and their survivals were recorded. The results showed that that E2-a induced a lower survival rate in C. elegans than E2-b and E2. The protoscoleces of Echinococcus granulosus (E. granulosus) were cultured in the presence of E2-a. Compared with E2-b and E2, protoscoleces exhibited decreased survival rate following E2-a treatment. Furtherly, the effects of E2-a on the behavior, brood size, and lifespan of the worms were investigated. Body bend frequencies of the worms treated with the high concentration of E2-a were reduced by two-thirds compared with the control group (P < 0.01). Compared with non-E2-a-treated group, exposure of nematodes to E2-a led to a decrease in head thrashes and pharyngeal pumps frequency (P < 0.01). E2-a treatment resulted in a significantly lower brood size (P < 0.01). Additional E2-a treatment induced a significantly shortened lifespan, compared with the control (P < 0.05). These findings indicated that water-soluble fraction E2-a from S. moorcroftiana seeds was a potential helminthic agent.