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Background The importance of dementia, a condition of memory and intellectual impairment, is increasing along with the increase in the older population. The total number of people with dementia worldwide in 2010 was estimated at 35.6 million and is projected to nearly double every 20 years to 65.7 million in 2030 and 115.4 million in 2050. This condition produces a great impact on the healthcare budget and social care . Therefore, it has gained much attention.
Dementia, especially age-related dementia, is associated with many factors including forebrain and hippocampal atrophy [, ], acetylcholine (ACh) reduction , cholinergic hypofunction [, ], basal forebrain cholinergic neuron degeneration, neurotrophic signaling reduction  and excess oxidative stress . Based on the crucial role of hypocholinergic function on dementia mentioned earlier, current anti-dementia drugs are targeted at the enhancement of cholinergic function. However, the current therapeutic efficacy is still limited, and adverse effects are commonly experienced . Therefore, protection from dementia is required. Medicinal plants have long been used for longevity promotion, neuroprotection and memory enhancement in traditional folklore.
Both Cyperus rotundus, a plant in the Cyperaceae family, and Zingiber officinale, a plant in the Zingiberaceae family, are both reputed to exhibit longevity promotion. The phytochemical constituents of C.
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Rotundus and Z. Officinale have been studied extensively.
Rotundus contains quercetin, kaempferol, alkaloids, flavonoids, tannins, starch, glycosides, chalcones, gallic acid and p-coumaric acid [, ]. Officinale includes gingerol, paradols, and shogaol [, ]. Scientific data have demonstrated that C.
Rotundus and Z. Officinale possess antioxidant, acetylcholinesterase inhibitory (AChEI), neuroprotective and memory-enhancing effects [–]. Based on the crucial role of hypocholinergic function and oxidative stress in dementia, the beneficial effect of both plants in dementia is the focus of this study. To optimize the benefit of the plant extracts, the positive modulation effect from the interaction of both plants has gained attention. We hypothesized that the combination of the extracts from C. Rotundus and Z.
Officinale (CP1) could protect against age-related dementia. To test this hypothesis, we aimed to determine the antioxidant and AChEI effects of CP1. In addition, an in vivo study was also carried out to determine the neuroprotective effect of CP1 against age-related dementia in an animal model induced by a cholinotoxin, AF64A. Plant collection and extract preparation The aerial part of C. Rotundus and the rhizome of Z.
Officinale were harvested from Khon Kaen province, Thailand from September – November 2012. Rotundus was authenticated by Associate Professor Panee Sirisa-ard, from the Faculty of Pharmacy, Chiang Mai University, Thailand (voucher specimen No. 023159), and Z. Officinale was authenticated by the National Museum of THAI Traditional Medicine, Thailand (voucher specimen No. The plant materials were prepared as 95% alcoholic extracts.
The percent yield of the C. Rotundus and Z.
Officinale extracts were 7.41% and 10.48%, respectively. Based on our pilot in vitro study, a 1:5 ratio of C.
Rotundus to Z. Officinale was found to exhibit the highest potential to protect against neurodegeneration induced by oxidative stress and increased the levels of neurotransmitters such as acetylcholine and dopamine, which play important roles in learning and memory (see Additional file: Table S1). Therefore, this ratio was selected for developing a novel neuroprotectant “CP1”. To control the quality of the developed neuroprotectant, the finger print of CP1 and the concentrations of gingerol and quercetin, the major chemical constituents of Z. Officinale and C. Rotundus that were previously reported to produce neuroprotection and memory enhancement [, ], were analyzed using high-performance liquid chromatography. The HPLC-UV analysis indicated that CP1 comprises gingerol, quercetin and several other unidentified peaks (See Additional file: Figure S1 and S2).
In addition, semi-quantitative analysis revealed that the concentration of gingerol and quercetin was 65 and 7 mg/mL, respectively. The combined extract was kept at -20 °C in a dark bottle until use.
Determination of antioxidant activity Radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical of the combined extract of Z. Officinale and C. Rotundus (CP1) was determined spectrophotometrically . The principle of the assay is based on the color change of the DPPH solution from purple to yellow when the radical is quenched by the antioxidant. In brief, 2.96 mL of a 0.1 mM solution of DPPH in methanol was incubated with 40 μL of various concentrations of extract (1.0, 2.0, 5.0, 10.0, 20.0, 25.0 mg/mL) at room temperature for 30 min.
The decrease in DPPH radicals was evaluated by the optical density measurement at 515 nm. The stable free radical scavenging capacity is presented as the percentage of inhibition of DPPH radicals calculated according to the following equation:% inhibition of DPPH = (Abs control-Abs sample/Abs control) × 100.
Determination of antioxidant activity by ferric reducing antioxidant power (FRAP) The ferric reducing antioxidant power assay was performed according to the procedure previously described  with some modifications. Briefly, the working FRAP reagent was mixed with 25 mL of 300 mM acetate buffer (3.1 g C 2H 3NaO 2 3H 2O and 16 mL C 2H 4O 2), pH 3.6, 2.5 mL of 10 mM tripyridyltriazine (TPTZ) solution in 40 mM HCl, and 2.5 mL of 20 mM FeCl 3 6H 2O solution. Then, 1.8 mL of the FRAP solution was mixed with the CP1 extract (10 μL) in 1 mL distilled water. The absorbance of the reaction mixture at 593 nm was measured spectrophotometrically after incubation at 37 °C for 10 min. The results were expressed as μM ascorbic acid/100 g fresh weight. Determination of acetylcholinesterase (AChE) inhibition AChE inhibitory activity was measured by using Ellman's colorimetric method .
Briefly, in 96-well plates, 25 μL of 15 mM ATCI, 75 μL of 3 mM DTNB and 50 μL of 50 mM Tris–HCl, pH 8.0, containing 0.1% bovine serum albumin (BSA), and 25 μL of the tested phytochemicals were added. The absorbance was measured at 405 nm after a 5-min incubation at room temperature. Then, 25 μL of 0.22 U.ml -1 of AChE was added and incubated for 5 min at room temperature, and the absorbance was measured at 412 nm. Acetylcholinesterase (5–1,000 μM) was used as a reference standard. The percentage inhibition was calculated using the following equation: Inhibition (%) = 1 – (A sample/A control) × 100, where A sample is the absorbance of the sample extracts, and A control is the absorbance of the blank (50% aqueous methanol in buffer).
In addition to the in vitro assay of AChE mentioned earlier, we also determined AChE activity in the hippocampal homogenate. In brief, the hippocampus was isolated and homogenized in ice-cold 0.1 M phosphate-buffered saline (pH 8.0). The homogenate was centrifuged at 1,000 g for 10 min at 4 °C, and the supernatant was used as the source of the enzyme in the AChE assay. AChE activity in hippocampus was evaluated using Ellman's method with slight modifications . Animals Eight-week-old male Wistar rats weighing 180-220 g were used as experimental animals.
They were derived from the National Laboratory Animal Center, Salaya, Nakorn Pathom. They were housed 6 per cage, maintained in a 12: 12 light: dark cycle, and given a standard pellet diet and water ad libitum. The experiments were performed to minimize animal suffering, and the experimental protocols were approved by the Animal Ethics Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of National Research Council of Thailand (Confirmation No. AEKKU 41/2554). AF64A preparation The preparation of AF64A was performed according to the method described by Hanin.
In brief, an aqueous solution of acetylethylcholine mustard HCl (Sigma–Aldrich Co., USA) was adjusted to pH 11.3 with NaOH and stirred for 30 min. Then, the pH of the solution was adjusted to pH 7.4 with the gradual addition of HCl and stirred for 60 min at room temperature. The amount of AF64A was then adjusted to 2 nmol/2 μL.
Artificial cerebrospinal fluid (ACSF) or vehicle of AF64A was distilled water, which was prepared in the same manner as AF64A. Surgical procedures Sodium pentobarbital (Jagsonpal Pharmaceuticals LTD, Haryana, India) at a dose of 60 mg/kg BW was administered to the animals via the intraperitoneal route to induce anesthesia. The memory deficit was induced by the bilateral intracerebroventricular (i.c.v.) injection of AF64A (2 nmol/2 μL, 2 μL/side). Burr holes were made in the skull according to the following stereotaxic coordinates; posterior 0.8 mm, lateral ±1.5 mm, and ventral (from dura) 3.6 mm. AF64A was perfused via a 30-gauge needle that was inserted through the burr holes, and the perfusion rate was 1.0 μL/min. After being left at the injection site for 5 min, the needle was slowly withdrawn.
The animals were allowed to recover from anesthesia and then placed in their cages. Group V-VII CP1 + AF64A; rats were treated with CP1 at doses of 100, 200 and 300 mg.kg -1 BW for a period of 14 days after the administration of AF64A. Rats in all groups were orally given the assigned substances for a period of 14 days after the bilateral intracerebroventricular administration of AF64A. A memory assessment was performed every 7 days throughout the 14-day study period, whereas the measurements of the malondialdehyde (MDA) level and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and acetylcholinesterase (AChE) in the hippocampus were performed at the end of study. Moreover, the density of the surviving neurons in various subregions of the hippocampus, including CA1, CA2, CA3 and the dentate gyrus, was also determined. Determination of spatial memory Spatial memory was evaluated using the Morris water maze test. Rats were subjected to a metal pool (170 cm in diameter × 58 cm height) filled with tap water (25 °C, 40 cm deep).
This pool comprised 4 quadrants including a northeast, southeast, southwest, and northwest quadrant. The water surface was covered with non-toxic milk. The removable platform was immersed below the water level at the center of one quadrant. All rats were trained to memorize the location of the invisible platform by forming the association of their location and the location of the platform using external cues. The time that the animal took to reach the top of the hidden platform was recorded as the escape latency or acquisition time.
To determine the capability of the animals to retrieve and retain information, the platform was removed 24 hr later, and the rats were re-exposed to the same condition, except that the platform was removed. The time that each animal spent in the region that previously contained the platform was recorded as the retention time. Histological study Following induction of anesthesia with sodium pentobarbital (60 mg/kg BW), brain fixation was carried out by transcardial perfusion with a fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. After the perfusion, the brain was removed and stored overnight in the fixative solution that was used in the perfusion, infiltrated with 30% sucrose solution and kept at 4 ° C. The specimens were frozen rapidly, and 10-μM thick coronal sections were prepared using a cryostat. All sections were rinsed in phosphate buffer and placed on slides coated with a 0.01% aqueous solution of a high molecular weight poly L-lysine.
Morphological analysis Five coronal sections from each rat in each group were studied quantitatively. The evaluation of the neuronal density in the hippocampus was performed under a light microscope at 40x magnification. The observer was blind to the treatment at the time of analysis. Viable stained neurons were identified on the basis of a stained soma with at least two visible processes. Counts were made in five adjacent fields, and the mean number was calculated and expressed as density of neurons per 255 μm 2. Determination of oxidative stress markers Rats were perfused with a cold saline solution to get rid of the blood from the brain tissue.
Then, the hippocampus was isolated and prepared as a hippocampal homogenate, and the determination of the oxidative stress markers was performed. The malondialdehyde (MDA) level was indirectly estimated by determining the accumulation of thiobarbituric acid reactive substances (TBARS) . To determine the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), the hippocampus of each rat was weighed and homogenized with a buffer consisting of 10 mM sucrose, 10 mM Tris–HCl and 0.1 mM EDTA (pH 7.4).
Then, a hippocampal homogenate was centrifuged at 3000 g at 4 °C for 15 min. The supernatant was separated and used for bioassays. The activity of SOD was determined using a xanthine/xanthine oxidase system as the source of superoxide radical production and the subsequent measurement of cytochrome c as a scavenger of the radicals. Optical density was measured using a spectrometer (UV-1601, Shimadzu) at 550 nm .
SOD activity was presented as units per milligram of protein (U mg -1 protein). One unit of enzyme activity was defined as the quantity of SOD required to inhibit the reduction rate of cytochrome c by 50%. CAT activity in the supernatant was measured by recording the reduction rate of H 2O 2 absorbance at 240 nm .
The activity of CAT was expressed as μmol H 2O 2.min -1mg -1 protein. GSH-Px was determined using t-butyl hydroperoxide as a substrate. The optical density was spectrophotometrically recorded at 340 nm and expressed as U mg -1protein . One unit of the enzyme was defined as one micromole (μmol) of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized per minute.
Western blot analysis The hippocampus was removed and rapidly frozen at -80 °C. The frozen tissue samples were homogenized in ice-cold RIPA buffer with protease inhibitors. The dissolved proteins were collected after centrifugation at 10,000 g for 30 min, and the supernatant was then collected. Protein concentration was determined using the NANOdrop Spectrophotometers.
Equal amounts of protein (35 μg) were separated by SDS-PAGE (10% SDS-polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). After transferring to the membrane, the blots were incubated in a blocking buffer (5% skim milk in Tris-buffer saline with 0.05% Tween-20) for 1 hr at room temperature and incubated overnight with antibodies against either phospho-ERK1/2 (1:1,000, Cell Signaling Cell Signaling Technology, Inc., Boston, MA, USA) or total ERK1/2 (1:1,000, Cell Signaling Cell Signaling Technology, Inc. Daughters Of The Moon Series Pdf. , Boston, MA, USA).
After incubation, the membrane was subjected to several washing steps. An HRP-linked secondary antibody (1:2,000) was incubated with the membrane for 1 hr at room temperature, and signals were visualized by chemiluminescence using an ECL kit (Pierce, ThermoScientific). Images were evaluated by ImageQuant LAS 4000, GE Healthcare. Band densities were quantified with ImageQuant TL (IQTL) software, GE healthcare . Antioxidant activity and acetylcholinesterase (AChE) inhibition of CP1 In the first part of this study, we determined and compared the antioxidant effect of C. Officinale and the combined extract of C. Rotundus and Z.
Officinale (CP1) by using DPPH and FRAP assays. In addition, acetylcholinesterase (AChE) inhibition was also determined using Ellman's colorimetric method. The results are shown in Table.
Interestingly, our data clearly demonstrated that the combination of the C. Rotundus and Z.
Officinale extracts (CP1) had a lower IC 50 of FRAP (1.743 ± 0.003 mg/ml), DPPH (1.008 ± 0.001 mg/ml) and AChEI (0.100 ± 0.103 mg/ml) than those of the C. Rotundus or Z. Officinale extracts.
Effect of CP1 on spatial memory In this part, we mimicked the memory impairment condition observed in age-related dementia in humans by inducing a hypocholinergic condition via the bilateral administration of AF64A, a cholinotoxin, into the lateral ventricles. Figure and showed that vehicle + ACSF showed no significant changes in both escape latency and retention time. Our data showed that the administration of AF64A significantly enhanced escape latency ( p-value.
Effect of CP1 on hippocampal neurodegeneration Figure (see Additional file: Figure S3) shows the effect of CP1 on neuronal density in the hippocampus. The results showed that AF64A significantly decreased neuronal density in the CA1, CA2, CA3 and dentate gyrus ( p-value. Effect of CP1 on oxidative stress markers The effects of CP1 on oxidative stress markers, including the level of MDA and the activity of SOD, CAT and GSH-Px in the hippocampus, were also evaluated.
The results are shown in Table. AF64A injection was demonstrated to significantly increase the MDA level ( p.
Effect of CP1 on acetylcholinesterase (AChE) activity The effect of CP1 on cholinergic function was evaluated indirectly by using the activity of AChE as an indirect indicator reflecting the available acetylcholine in the hippocampus. The results are shown in Fig.. Rats exposed to AF64A showed an elevation in AChE ( p-value. Effect of CP1 on ERK1/2 activation Since the ERK cascade plays an important role in synaptic plasticity, long-term potentiation and cell survival, the effect of CP1 on ERK1/2 in the hippocampus was also assessed. The results are shown in Fig.. AF64A injection was found to significantly decrease phosphorylation of ERK1/2 (p-value.
Discussion Medicinal plants have long been used for treating various ailments either as single plants or as polyherbal recipes. However, the polyherbal recipes have been more widely used than the single plants based on the concept that the synergistic effect of multiple plants can provide more beneficial effects . However, less scientific evidence is available. In this study, we have clearly demonstrated that CP1, the combined extract of C. Rotundus and Z. Officinale, showed a lower IC 50 of both the antioxidant effect via DPPH and the AChEI effect. Therefore, our results confirmed the hypothesis that the interaction of both medicinal plants mentioned earlier could provide a greater benefit.
This was also in agreement with other studies that have demonstrated the beneficial effect of the combined extract [–]. The current results also demonstrated that CP1 significantly increased spatial memory, enhanced cholinergic function and decreased oxidative stress in the hippocampus. The current data revealed that CP1 at all doses in this study increased CAT activity, and the low dose of CP1 increased SOD activity. Therefore, the increase in CAT activity with SOD activity might involve the reduction of oxidative damage . In addition, CP1 also significantly enhanced the density of neurons in the CA1, CA2 and dentate gyrus and increased pERK1/2 levels in these same areas. ERK1/2, a subclass of mitogen-activated protein (MAP) kinases, has been reported to play a pivotal role in neurodegeneration via the mitochondrial apoptotic mechanism [–].
Neurodegeneration in the hippocampus, an important area for learning and memory, is associated with memory deficits [, ]. Therefore, the memory-enhancing effect of CP1 may occur partly via decreased oxidative stress by enhancing the activity of antioxidant enzymes in the hippocampus, which, in turn, could induce an increase in pERK1/2 , giving rise to an increased neuronal density in the CA1, CA2 and dentate gyrus, leading to improvements in the encoding, retrieval and consolidation processes resulting in enhanced spatial memory . Although the decreased oxidative stress could increase the phosphorylation of ERK1/2, resulting in an anti-apoptotic effect and leading to enhanced neuronal density in the hippocampus, no close relationship between the increase in pERK1/2 and the decrease in oxidative stress was observed, especially at the low concentration of CP1. Since decreased oxidative stress in rats with AF64A–induced memory deficits can increase the neuronal density in the hippocampus and can improve memory impairment [, ], we suggested that the antioxidant effect of CP1 might decrease oxidative stress status in the hippocampus, which in turn would decrease neurodegeneration induced by the attack of free radicals, resulting in increased neuronal density in this area.
In addition, the activation of ERK1/2 gives rise to the phosphorylation of ERK1/2, which in turn plays an important role in the function of acetylcholine via the nicotinic receptor . Therefore, it is also possible that CP1 at all doses used in this study may suppress AChE, leading to an increase in the available acetylcholine (ACh), which, in turn, may bind to the nicotinic receptor, resulting in the activation and phosphorylation of ERK1/2 and finally leading to improved spatial memory. These effects have been shown in Fig.. Schematic diagram shows the possible underlying mechanism of CP1 Our results also showed differential vulnerability to CP1. The CA3 region showed less vulnerability among the various subregions assessed in this study.
The possible explanation may be due to differences in the distribution of signal molecules and growth factors that play important roles in cell survival . Our data failed to show dose-dependent effects. The possible explanation might be related to the masking effect of non-active ingredients. In addition, the relationship between the concentration of CP1 and the observed parameters might not be a simple linear relationship, and the active ingredient may also exert the beneficial effect indirectly via other signal transduction process such as ERK1/2. Since no significant differences among doses were observed, we suggested that the medium dose would be the most appropriate dose for application based on its benefit in all parameters, including the effect on the ERK signal pathway. Since this dose could effectively exert a positive modulation effect on multiple targets, it could also provide a greater benefit.
In addition, the medium dose also provides a lower risk for toxicity than the high dose of CP1. A limitation of this study is that all ingredients of the combined extract are not determined.
Based on previous studies, it has been demonstrated that gingerol  and quercetin  exert protective effects against oxidative stress-related neurodegeneration. Therefore, we measured the concentrations of the mentioned substances in the combined extract.
Since both substances were also found in the combined extract, and the observed effect was similar to the effect of both substances, we suggested that they might be partly responsible for the neuroprotective effect of CP1 in this study. In addition to the direct effect of both substances mentioned earlier, interaction effects of various ingredients, including the interaction of both ingredients and the effect of other constituents, are still possible. However, further investigations are necessary to provide better understanding concerning the possible active ingredients. Conclusion CP1, the combined extract of C. Rotundus and Z. Officinale, is a potential supplement to improve neurodegeneration and memory impairment.
The possible mechanism for its beneficial effects may be through improving oxidative stress status, which in turn would increase pERK1/2 in the hippocampus, leading to improvement in memory impairment. In addition, CP1 can also suppress AChE activity in the hippocampus, giving rise to increased available ACh and increased function of ACh via the nicotinic receptor, resulting in enhanced memory performance.
However, further studies are necessary to investigate the precise active ingredients and subchronic toxicity of CP1 and its interaction with drugs that are commonly used in elderly patients to assure safe consumption.
Background The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies.
We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. Results A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45.
The recombinant protein induced high levels of functional TB antibodies in rats. Background The transmission of Plasmodium falciparum malaria from one person to another requires the production of male and female gametocytes in the human host that can be taken up by blood-feeding mosquitoes. Recent studies have indicated that a large proportion of individuals in malaria endemic areas carry gametocytes and may contribute to malaria transmission . Control strategies that confer prolonged protection including vaccines, are therefore needed to effectively block malaria transmission at the population level .
Falciparum Pfs48/45 is one of the leading transmission blocking vaccine (TBV) candidates that has entered the pipeline of clinical development (for a review see ). This protein is relatively cysteine-rich with multiple disulfide bonds that result in antibody epitopes which are dependent on properly folded tertiary structures rather than linear amino acid sequences. Functional in vitro studies have identified the C-terminal portion of Pfs48/45 (6C) containing three disulfide bonds as major target of transmission blocking (TB) antibodies .
This Pfs48/45 region is targeted by a monoclonal antibody (mAb) mAb45.1 which promotes strong TB activity in the standard membrane feeding assay (SMFA), the gold standard for assessing transmission blockade ex vivo [–]. The production of Pfs48/45 in bacterial and eukaryotic expression systems has been problematic due to insufficient protein folding capabilities of these systems. Proper folding of cysteine-rich Pfs48/45 depends on correct formation of disulfide bridges. Since eukaryotic cells possess a sophisticated machinery for disulfide bond formation, recombinant Pfs48/45 has been produced in a range of Baculovirus ( Spodoptera frugiperda Sf9) cells , Vaccinia virus , Saccharomyces cerevisiae , and Pichia pastoris , Chlamydomonas reinhardtii , and Nicotiana benthamiana . However, protein yields were rather low and in those cases where expression was successful, there was limited reactivity with mAbs against conformational TB epitopes suggesting misfolding.
The bacterial Lactococcus lactis expression system represents a significant advancement in the production of recombinant Pfs48/45 [, ]. Lactis cell factories generally recognized as safe (GRAS status) are well suited for the production of heterologous proteins and used for a wealth of food applications.
In the recent years, L. Lactis has also been used in modern biotechnology within the fields of mucosal delivery  generation of self-adjuvanting bacterium-like particles  and recombinant proteins (reviewed in . Lactis do not produce endotoxins or extracellular proteases. Moreover, gene expression can be controlled by a set of tightly regulated promoters in a simple and scalable fermentation process from a few ml up to thousands of liters. Recombinant proteins can be secreted into the culture medium in the absence of spore formation which clearly facilitates downstream processing. Accordingly, L. Lactis has been used for the manufacturing of the GMZ2 malaria vaccine candidate [–].
To advance development of a protein-vaccine based on Pfs48/45, we established a manufacturing process for R0.6C in L. The product was characterized by mass spectrometry (MS)-based and by HPLC-based methods and tested for functional immunogenicity in rats. The specificity of induced antibodies was assessed by ELISA, and functional capacity for transmission blockade in the standard membrane-feeding assay (SMFA). Molecular design and expression of chimeric GLURP- Pfs48/45 fusion proteins We designed a set of fusion proteins (Fig. a) consisting of the Pfs48/45 6-Cys domain to address whether expression levels of secreted R0.6C was affected by: (1) codon optimization, (2) different signal peptides, and (3) the presence and/or position of a range of affinity-tags. All constructs were transformed into L. Lactis MG1363 and grown in 5 ml of LAB medium at 30 °C without shaking.
Firstly, the codon optimized construct generated the same amount of recombinant R0.6C fusion protein as did the non-optimized construct (Fig. b, compare lanes 1 and 2). Secondly, we found that protein yields were similar between constructs with and without a His-tag (Fig. b, compare lanes 1 and 3), suggesting that the His-tag per see does not affect production yields of R0.6C. Thirdly, fusion proteins containing tags that can be used for various conjugation strategies including the SpyTag-spyCatcher technology [, ] and Streptavidin-mSA mediated conjugation to bacterial outer membrane vesicles  were explored.
The addition of these tags to the N- or C-terminal end of R0.6C did not affect over all expression levels (Fig. b, lanes 5, 6, and 7). Finally, we showed that a native USP45 signal peptide derived from an abundantly secreted L. Lactis protein did not increase protein yields in culture supernatants (Fig. b, lanes 1 and 8). Production of recombinant R0.6C in bioreactor Since all constructs tested gave similar yields, we choose R0.6C with a C-terminal His-tag (Fig. a, construct no. 1) for optimization of fermentation in lab-scale bioreactors. The generation of R0.6C showed a substantial accumulation in the culture medium at 10–15 h post inoculation (Fig. a).
Recombinant R0.6C was produced as an intact fusion protein as indicated by Coomassie staining (Fig. b upper panel) and immune blotting with an antibody against the C-terminal his-tag (Fig. b, middle panel). The secreted protein was properly folded as indicated by immune blotting with the conformation dependent mAb45.1 (Fig. b lower panel). Subsequently, a robust workflow for production was developed by growing L. Lactis MG1363 expressing R0.6C in a 1 l stirred bioreactor for 15 h at 30 °C (Fig. a). The non-oxidative fermentation resulted in rapid acidification due to the production of lactate. Acidification eventually inhibits cell growth but also induces protein expression by activating the P170 promoter . In order to optimize both cell growth and promoter activity, the fermenter was equipped with a pH electrode to monitor and control pH by addition of 2 M NaOH.
The culture medium was also supplemented with 5 mM cysteine and 0.5 mM cystine which, together with the micro-aerobic milieu, is essential for high yield production of disulfide-bonded recombinant protein. Purification of recombinant R0.6C Supernatants were concentrated and buffer exchanged for phosphate buffered saline (PBS) pH 7.4 supplemented with 15 mM imidazole. R0.6C was captured on a HisTrap HP column and bound protein was eluted with a linear imidazole gradient (Fig. b). Fractions containing recombinant R0.6C were analyzed by SDS-PAGE and by mAb45.1 sandwich ELISA (Fig. b).
Fractions containing high concentration of immune reactive protein were pooled and loaded on an anion ion-exchange chromatography column, to separate protein species with native and non-native disulfide bonds (Fig. c). Fractions (P1) containing mAb45.1 reactive monomer were pooled with a major band of monomeric protein strongly reactive to mAb45.1 (Fig. d). This R0.6C fraction contained >80% properly folded Pfs48/45 relative to immune purified reference material (Fig. e and Additional file ).
The same work-flow for R0.6C constructs tagged with SpyTag, SpyCatcher, and mSA at their N-terminal ends resulted in similar yields of properly folded R0.6C. Mass spectrometry analysis of R0.6C The mass of non-reduced and reduced full-length R0.6C was 71325.4 and 71331.3 Da as determined by LC–MS respectively (Additional file a, b). This molecular weight corresponds well to the predicted value of 71331.01 Da assuming that the fusion protein contains the vector-encoded amino acid residues A-E-R-S at the N-terminal end and a 6xHis-tag at the C-terminal end.
Reduction of R0.6C resulted in a shift in the measured mass of the intact protein by approximately 6 Da, consistent with the presence of 3 disulfide bonds. The correct primary structure of R0.6C was further verified through MS/MS sequencing of 39 tryptic peptides derived from reduced R0.6C (Additional file c). These peptides cover 85% of the protein confirming that the purified R0.6C fusion protein was intact with the predicted amino acid residues corresponding to a full-length GLURP 27–500– Pfs48/45 291–428 fusion protein. The disulfide connectivity of R0.6C was characterized by analyzing tryptic peptides by LC–MS/MS. Four peptides were not found in non-reduced R0.6C sample, indicating their involvement in cross-linking, whereas, two peptides were exclusively present in non-reduced R0.6C, referred to as XL-peptides I and II (Fig. a, b, respectively). The identity of these cross-linked peptides was established by manual inspection of masses of peptide precursors and the corresponding fragment ions (Fig. d for peptide I). In XL-peptide I, C492 formed a disulfide bond with C521.
In XL-peptide II, C604 was disulfide bonded to C546 or C538, whereas C606 formed a disulfide bond with the other cysteine residue. Due to the close proximity of C604 and C606 it was only possible to resolve the disulfide connectivity to either of the two cysteine residues. Several control peptides without cysteine residues were monitored simultaneously with an example shown in (Fig. d).
A diagram of the disulfide connectivity of the correctly folded Pfs48/45-6C region is shown in (Fig. e). Size-exclusion and reversed-phase HPLC analysis of purified R0.6C A Pfs48/45-based vaccine antigen should consist of properly folded monomer. The final R0.6C batch elutes as a single peak at 42.5 min by analytical size exclusion chromatography (SEC), representing the active monomer state (Fig. a). Subsequent reversed-phase chromatography analysis showed a main peak with a retention time of 17.0 min and a smaller peak at approximately 16.5 min (Fig. b). The latter was absent in reference material containing immune-purified R0.6C (data not shown) suggesting that this peak corresponds to host cell proteins or a minor fraction of product-related impurities.
The overall purity of R0.6C as determined by RP-HPLC was 87%. Immunogenicity of recombinant R0.6C To ascertain that R0.6C elicit adequate levels of gametocyte-specific antibodies with the capacity to inhibit parasite fertilization, Wistar rats (N = 8) were immunized 3 times at 3-week intervals with increasing doses (2.5, 10, and 25 μg) of R0.6C inducing similar levels of vaccine-specific (Fig. a), epitope I-specific (Fig. b), and 6C-specific (Fig. c) antibodies. There was a strong correlation between 6C- and epitope I specific antibody levels (Fig. d) suggesting that epitope I is the main epitope in the 6-Cys domain of Pfs48/45. Collectively, these results demonstrate that R0.6C gives an adequate presentation of conformational epitope(s) in Pfs48/45. Serum pools from each dosing group were tested for functional activity in the SMFA showing >99% TB activity at a 1/9 dilution. Discussion Production of recombinant protein is important for subunit vaccine development. Since there is no single universal host, which is perfect for production of all desired recombinant proteins, the selection of the right expression system is pivotal for development of a manufacturing process.
The production host is not only important for the correct protein folding (protective antibodies often target conformational epitopes) but also process economics are key to keeping the price of the vaccine as low as possible, especially for vaccines targeting low-income countries. From a manufacturing perspective, the major challenge with recombinant Pfs48/45 is production of correctly folded protein with sufficient yield (reviewed in ). One way to increase the amount of properly folded protein has been to produce Pfs48/45 in the L. Lactis expression system genetically fused to the GLURP-R0 region [, ].
The efficient expression of disulfide-bonded protein in L. Lactis was unexpected as this organism is low in its cysteine content and lacking known disulfide bond forming enzymes . Thus, disulfide bond formation of recombinant Pfs48/45 most likely occurs spontaneously after secretion and is not only dependent on the primary structure but also on environmental factors that determine cysteine oxidation. By carefully optimizing the ratio of different redox couples in the fermentation broth, we have identified conditions, which render the extracellular milieu more oxidizing allowing formation of structural disulfide bonds in the Pfs48/45-6C domain of the fusion protein. These conditions seem to favor the formation of intramolecular disulfide bonds as secreted R0.6C was mainly present in the monomer state. However, the finding that crude R0.6C shows reduced reactivity with mAb45.1 compared to the reference material consisting of mAb45.1 immune-purified R0.6C  suggests that the monomer fraction contains a mixture of conformers with native and non-native cysteine connectivity.
A work-flow has therefore been developed using batch fermentation in lab-scale stirred bioreactor to produce discreet batches of recombinant R0.6C purified by a simple 2-step purification process. The first step captures the recombinant protein from the culture supernatant and the second step aims to separate correctly folded and misfolded protein species. The SEC–HPLC analysis demonstrates that the final batch of R0.6C mainly contains monomers with all six cysteine residues in the oxidized state as shown by mass spectrometry analysis. The established disulfide connectivity of R0.6C is in excellent agreement with the disulfide bond connectivity of another recombinant protein containing the Pfs48/45-10C region (Mistarz et al., unpublished). The purity of the R0.6C batch is 87% as determined by RP-HPLC.
Impurities in the final R0.6C batch are likely, at least in part, product-related consisting of misfolded protein species or degradation products. Although the final yield of purified recombinant R0.6C is almost 25 mg per L culture broth, we suggest that further improvements are possible. Such improvements may include procedures to increase the biomass through systems where lactate is removed from the culture broth during fermentation using for example the REED™ (Reverse Electro-Enhanced Dialysis) technology . Alternatively, product yield may be increased through adjusting the fermentation process, which is very easy to scale up to 200–1000 L since there is no requirement for oxygen and heavy stirring during fermentation. Finally, product yields can be increased through performing multiple fermentations that can be pooled before downstream purification.
Previously we have investigated the immunogenicity of Pfs48/45-based vaccine candidates using immune purified protein [,, ]. Here, we showed that Pfs48/45 fusion protein purified by conventional chromatographic procedures together with Montanide ISA 720 VG elicits TB antibodies at the lowest dose of 2.5 µg. Previously, doses below 4.7 µg failed to elicit TB antibodies when adjuvanted in Alhydrogel . Improved formulations may contain immune modulators [, ] or may be based on novel delivery systems including virus like particles (VLP). This concept has been demonstrated recently for another TBV candidate, Pfs25, which was covalently coupled to VLPs [, ]. In this context, it is clearly important that the L.
Lactis expression system is able to accommodate a range of affinity tags added to either end of the recombinant protein. In conclusion, we have demonstrated that L. Lactis microbial factories are highly suited for the production of Pfs48/45. A manufacturing process for soluble and correctly folded R0.6C was developed and the resulting vaccine antigen induces high levels of functional antibodies. The high yield of correctly folded protein and the straightforward production process offers the possibility to investigate the vaccine potential of Pfs48/45 in human clinical trials. Preparation of constructs All the constructs are based on the L.
Lactis pSS1 plasmid vector . The R0.6C construct with a C-terminal His tag has been described . Non-tagged R0.6C was generated through PCR with forward primer (5′-GAAT GGA TCC TAC AAG TGA GAA TAG AAA TAA ACG) and reverse primer (5′-GAAT AGA TCT TTA TGC TGA ATC TAT AGT AAC TGT CAT ATA AGC) using R0.6C.6H gene as template. Codon optimized R0.6C.6H and N-terminal SpyCatcher and SpyTag  containing BamHI- BglII was synthesized by (GeneArt ® Life Technologies, Germany) and inserted into pSS1. For construction of chimeric N-terminal (mSA-R0.6C.6H) or C-terminal (R0.6C-mSA.6H) R0.6C, a synthetic DNA fragment encoding the monomeric Streptavidin (mSA) (GeneBank: 4JNJ_A) with a small Gly–Gly-Ser linker was synthesized by (GeneArt ® Life Technologies) and cloned into R0.6C.6H.
The USP45-R0.6C.6H construct was generated by replacing the mutD310 signal peptide in pSS1 with the USP45 signal peptide  synthesized by (GeneArt ® Life Technologies) and cloned into R0.6C. All the constructs were verified by sequencing and subsequently transformed into L. Lactis MG1363 by electroporation as described .
Screening, fermentation and protein purification L. Lactis MG1363, containing R0.6C constructs was grown overnight at 30 °C in 5 ml LAB medium  supplemented with 4% glycerol–phosphate, 5% glucose and 5 µg/ml erythromycin. Culture supernatants were clarified by centrifugation at 9000 g for 20 min and analysis of all the constructs was performed by Coomassie stained SDS-PAGE gel and Western blotting with mAb45.1 against conformational epitope I. Fermentation of L.
Lactis MG1363/R0.6C.6H was performed in a 1 l lab scale bioreactor at 30 °C with gentle stirring (150 rpm). For the time-course experiment 10 ml samples were withdrawn and used for analysis. Optical density at 600 nm (OD600) was used to assess cell density. Cell-free culture-filtrates were concentrated tenfold and buffer exchanged into phosphate buffered saline (PBS) pH 7.4 supplemented with 15 mM imidazole) using a QuixStand Benchtop system (Hollow fiber cartridge with cutoff at 50,000 Da, surface area 650 cm 2, GE Healthcare, Sweden) followed by filtration through a Durapore filter (PVDF, 0.22 µm, Millipore) and applied to a 5 ml HisTrap HP column (GE Healthcare, Sweden). Bound protein was step gradient eluted with 500 mM imidazole in tris buffer pH 8.0 (55 mM tris, 21 mM NaCl) at a flow rate of 4 ml/min and fractions containing the desired protein were applied to a 5 ml HiTrap Q HP column (GE Healthcare, Sweden). Bound protein was eluted through step gradient elution in tris buffer pH 8.0 (55 mM tris, 1 M NaCl) and fractions containing monomers with the highest amount of mAb45.1-reactive protein were concentrated by a VIVA spin column 30 kDa cutoff (GE Healthcare, Sweden), and kept in 55 mM tris, 300 mM NaCl, 0.025% Tween 80 and 1 mM EDTA, pH 8.0 at −80 °C until use. Immune purification of R0.6C was performed as described earlier [, ].
Analysis of all the fractions was performed by SDS-PAGE. Immunoblotting was performed with mAb45.1 against conformational epitope I. Protein concentration was measured using either the BCA protein assay (Thermo Fisher Scientific) or by densitometric analysis of Coomassie stained SDS-PAGE gel using Image Quant TL (IQTL) software (GE Healthcare). SEC–HPLC and RP-HPLC analysis of R0.6C Native size exclusion high-performance liquid chromatography (SEC–HPLC) or reversed-phase HPLC (RP-HPLC) of intact R0.6C were performed using an Agilent 1100 Series HPLC System (Agilent Technologies, USA) equipped with a TSKgel G3000SWXL SEC column, 5 µm, 7.8 × 300 mm (Tosoh Bioscience, Japan) or equipped with a Vydac 214TP C4 reversed-phase column, 5 µm, 4.6 × 250 mm (The Separations Group, CA, US), respectively. 210 pmol of protein was loaded on the SEC column and eluted with a 0.15 ml/min flow of elution buffer (200 mM phosphate, 0.65 g/l l-arginine, 0.05% NaN 3, pH 6.7) at room temperature. For RP-HPLC, 210 pmol of protein was loaded on the RP-column and eluted with a linear gradient of 3–95% of 0.1% trifluoroacetic acid (TFA), 20% isopropanol and 70% acetonitrile over 30 min.
The absorbance was measured at 280 or 215 nm for SEC–HPLC or RP-HPLC, respectively, and chromatographic peaks were integrated by HPLC ChemStation (Agilent Technologies, CA, US). Mass spectrometry Accurate molecular mass of full-length R0.6C was measured by LC–ESI–MS. 30 pmol of protein were loaded on a C4 pre-column (Acquity UPLC Protein BEH C4 Vanguard, 1.7, 2.1 × 5 mm, Waters, USA) and eluted onto a Q-TOF mass spectrometer (Synapt G2 HDMS, Waters, UK) with a chromatographic gradient. LC–MS data where recorded and analyzed by MassLynx software (Waters, UK). Mass spectra were deconvoluted using the MaxEnt 1 algorithm in the MassLynx software. Peptide mapping of R0.6C was performed by LC–MS/MS of tryptic digests.
Guanidine hydrochloride, dithiothreitol and iodoacetamide were added to 100 pmol of protein. This was followed by addition of iodoacetamide and the protease trypsin. Tryptic peptides were desalted and chromatographically separated over 40 min on a UHPLC C18 pre column and column, respectively (Acquity UPLC BEH column, Waters, USA) using a nanoACQUITY UPLC system (Waters, USA). The eluate ionized by electrospray ionization and data were acquired on the same mass spectrometer software as described above.
LC–MS/MS data were analyzed using PLGS (waters, USA) and peptide maps were created using DynamX (Waters, USA). Cysteine connectivity characterization was done with identical UPLC conditions as for peptide mapping. Tryptic digestion was additionally performed under similar conditions, with and without addition of guanidine hydrochloride, dithiothreitol or iodoacetamide to produce peptides without and without intact cysteine bonds, respectively. Collision induced dissociation (CID) fragmentation was performed on the two identified cross-linked peptides to produce MS/MS spectra used for manual analyzing the MS/MS fragments by MassLynx software for characterization of cysteine connectivity.
Animals and immunogenicity studies Female Wistar Hannover rats (Taconic, Denmark) and kept in the Laboratory Animal Facility Centre at Panum, University of Copenhagen, Denmark for 7 days before the first immunization. All procedures regarding animal immunizations complied with European and National regulations.
Groups of eight rats were immunized by the s.c. Route 3 times at 3-week intervals with increasing doses (2.5, 10, and 25 μg) of R0.6C. Protein preparations were emulsified in Montanide ISA720 VG (Seppic) immediately before use. Responses were measured using sera taken three weeks after the third immunization (Day 63). Enzyme-linked immunosorbent assay (ELISA) The mAb45.1 sandwich ELISA was performed as previously described [, ]. The coating concentration of recombinant Pfs48/45 (6C) and R0.6C was 0.5 µg/ml and bound antibodies were detected with HRP-conjugated rabbit-anti rat IgG-HRP (DAKO, Denmark) as described in detail .
The mAb45.1 competition ELISA was performed as follows; Briefly, ELISA MaxiSorp microtitre plates (Sterilin ® ELISA plates, The Netherlands) were coated at 4 °C overnight with 100 µl per well of 5 µg/ml of mAb45.1 in PBS pH 7.4. Wells were blocked with 150 µl of 5% non-fat skimmed milk powder in PBS for 1 h followed by three washings with PBS containing 0.05% Tween 20 (PBST). Sera at threefold dilutions (50 µl) were mixed with 50 µl R0.6C (1 µg/ml) in PBST, added to the wells and incubated for 4 h at room temperature.
Wells were washed with PBST and bound antigen was revealed with anti-His-HRP (1/6000 in PBST) for 2 h at room temperature. Midpoint (EC50) values were calculated using GraphPad Prism 7, (GraphPad Software, USA).