Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.     

Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.     

Evaluation of a New Tumor Necrosis Factor-α-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen

Background: Tumor necrosis factor (TNF)‐α‐inducing protein (Tipα) is a newly identified carcinogenic factor present in Helicobacter pylori. Tipα has the unique function of inducing TNF‐α production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tipα against H. pylori infection and analyzed the immune responses. Methods: C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tipα + CpG, and recombinant del‐Tipα (a mutant of Tipα) + CpG. Eight weeks after the mice were infected with H. pylori (5 × 10⁷ CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tipα‐specific IgG and IgA antibodies in mouse serum were measured by an enzyme‐linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)‐10, TNF‐α and Interferon (IFN)‐γ in gastric mucosa was also measured by real time‐PCR. Results: Levels of Tipα‐specific antibodies were significantly higher in Tipα‐immunized and del‐Tipα‐immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tipα‐immunized mice (4.29 × 10⁵ CFU/g) and del‐Tipα immunized mice (2.5 × 10⁵ CFU/g) compared with infection control mice (5.7 × 10⁶ CFU/g). The levels of IFN‐γ and IL‐10 were significantly higher in del‐Tipα‐immunized mice than the infection control group. Conclusion: Vaccinations with Tipα and del‐Tipα were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell‐mediated immunity.     

Characterization of recombinant scFv antibody reactive with an apical antigen of Eimeria acervulina

The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding V _H and V _L genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l^–1 and exhibited virtually identical antigen reactivity as the original mAb.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric γ‐lactols, lasiolactols A and B ( 1 and 2 ), were characterized as (2S*,3S*,4R*,5R*,2′S*,3′S*,4′R*,5′R*)‐ and (2R*,3S*,4R*,5R*,2′R*,3′S*,4′R*,5′R*)‐(5‐(4‐hydroxymethyl‐3,5‐dimethyl‐tetrahydro‐furan‐2‐yloxy)‐2,4‐dimethyl‐tetrahydro‐furan‐3‐yl]‐methanols by IR, 1D‐ and 2D‐NMR, and HR‐ESI‐MS. Other four metabolites were identified as botryosphaeriodiplodin, (5R)‐5‐hydroxylasiodiplodin, (–)‐(1R,2R)‐jasmonic acid, and (–)‐(3S,4R,5R)‐4‐hydroxymethyl‐3,5‐dimethyldihydro‐2‐furanone ( 3  –  6 , resp.). The absolute configuration (R) at hydroxylated secondary C‐atom C(7) was also established for compound 3 . The compounds 1  –  3 , 5, and 6 , tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens.     

Cellular Aspects of the Resistance of Chickens to Eimeria tenella Infections*

SYNOPSIS. Sporozoites of Eimeria tenella were injected into the peritoneal cavity of normal chickens and chickens immunized against E. tenella. In some experiments normal scrum and serum from resistant chickens were injected prior to the injection of sporozoites. After 15 or 30 minute periods of intraperitoneal incubation, exudates were harvested and the occurrence of intracellular sporozoites was determined. Only macrophages and degranulated granulocytes were observed to contain sporozoites. There was no significant difference between the number of macrophages obtained from normal chickens (normal macrophages) which contained sporozoites and the number of macrophages obtained from immune chickens (immune macrophages) which contained sporozoites. Significantly fewer immune macrophages treated with immune serum contained sporozoites than untreated normal or immune cells, normal macrophages treated with either serum, or immune macrophages treated with normal scrum. Sporozoites in untreated normal macrophages did not appear to be harmed by the intracellular environment, based on structural observations. The majority of sporozoites in macrophages from all other groups were difficult to distinguish within the cytoplasm and were visibly distorted. It is hypothesized that the presence of fewer infected macrophages in exudates of immune chickens and serum-treated normal chickens was caused by an enhanced ability of these cells to destroy the parasite. Similar observations were noted in the case of sporozoites within degranulated granulocytes of experimental groups. The lack of understanding of the degranulation phenomenon makes it difficult to interpret these findings.     

Development and characterization of a recombinant chicken single-chain Fv antibody detecting <Emphasis Type="Italic">Eimeria acervulina</Emphasis> sporozoite antigen

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V_H and V_L genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l^–1. The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V_L genes suggested that the gene conversion with V pseudogenes might contribute to the diversification of V_L genes in chickens.     

The viability and survival of sporozoites of Eimeria in vitro

Some factors affecting excystation and viability of sporozoites of several species of Eimeria from chickens were examined in vitro. Chicken embryos or cultured kidney cells were inoculated with sporozoites in order to assess viability.Sporozoites of E. tenella survived in phosphate buffer (P.B.S.) containing 0·9 per cent NaCl for 14 days. Some sporozoites survived in solutions containing up to 16 per cent NaCl for 3 days at +4°C. Sporozoites of E. maxima and E. acervulina survived for only 27 h in phosphate buffer containing 1 or 2 per cent NaCl.Sporozoites of E. brunetti, E. maxima, and E. acervulina var: mivati were released rapidly from sporocysts in vitro, but survived for relatively short periods in PBS at 4°C. However, the addition of serum or gelatine to these solutions increased survival to at least 96 h.The viability of sporozoites after freezing and storing in liquid nitrogen was best when 12 per cent dimethyl sulphoxide (DMSO) was added to the sporozoite suspensions. P.B.S. with DMSO was less suitable than the other solutions used and serum or gelatine with the DMSO, was needed to increase survival. Increasing the density of sporozoites in the frozen stabilates did not increase survival.     

Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus)

Aims: To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac‐D1ae) and/or D4 (Lac‐D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). Methods and Results: The polymerase chain reaction (PCR)‐amplified 250‐ and 750‐bp sequences coding for domains D1 and D4 of aerolysin were individually cloned into pNZ8048 and electrotransformed into L. lactis. The recombinant vaccine candidates were then either orally fed or injected intraperitoneally into tilapia. The development of antibodies in sampled fish compared to control groups implied that the recombinant epitopes expressed in L. lactis were able to elicit an immunogenic response in tilapia. Interestingly, the lower doses of both Lac‐D1ae and Lac‐D4ae gave higher antibody levels over the study period. Fish immunized with Lac‐D1ae and Lac‐D4ae together showed the highest level of protection, and the mortality was reduced significantly compared to control strains in both modes of vaccination. Conclusions: The recombinant L. lactis strain expressing D1 and D4 produced aerolysin‐specific serum IgM in tilapia. Both D1 and D4 promoted 55–82% relative per cent survival (RPS) against Aeromonas infection through intraperitoneal injection, whereas the RPS following oral feeding of the vaccine was 70–100%. Significance and Impact of the Study: The D1 and D4 regions of the aerolysin protein have been successfully identified as immunogenic regions that can elicit antibody production in tilapia and protect against challenge with Aer. hydrophila. A promising oral vaccine using L. lactis harbouring the D1 and D4 regions has been developed to control Aer. hydrophila.     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 ( 1 ), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D ( 2 ), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and HBsAg‐induced splenocyte proliferation in HBsAg‐immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL‐2 and IFN‐γ) and Th2 (IL‐4 and IL‐10) cytokines from splenocytes in the HBsAg‐immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg‐specific IgG2a and IgG2b antibody response, as well as Th1‐cytokine secretion from splenocytes in the HBsAg‐immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1‐ and Th2‐potentiating activity.     

Utility of the P19 suppressor of gene‐silencing protein for production of therapeutic antibodies in Nicotiana expression hosts

To study how the P19 suppressor of gene‐silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co‐expressed with P19 in an RNAi‐based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102–106) containing different 5′ and 3′ UTRs, designated as vector sets 102–106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15‐fold (to about 2.3% of TSP) when co‐expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co‐expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I‐64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19‐induced responses.     

Plant‐derived recombinant immune complexes as self‐adjuvanting TB immunogens for mucosal boosting of BCG

Progress with protein‐based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency‐associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self‐polymerization of the complexes. TB‐RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen‐presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB‐RICs developed antigen‐specific serum IgG antibody responses with mean end‐point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN‐γ, IL‐2 and TNF‐α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG‐immunized mice with TB‐RICs led to a reduced M. tuberculosis infection in their lungs from log₁₀ mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant‐expressed TB‐RICs represent a novel molecular platform for developing self‐adjuvanting mucosal vaccines.