Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

1H-NMR Metabolomic Study of the Mushroom Pleurotus djamor for the Identification of Nematocidal Compounds.

Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (1H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC50 290.8 µg/mL) and basidiomes (EC50 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the 1H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the 1H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC50 of 237.7 µg/mL. The results obtained showed that chemometric analyses of the 1H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

In Vitro and In Vivo Cysticidal Effects of Carica Papaya Cell Suspensions.

Parasitic diseases are a major public health problem worldwide. Plant-derived products appear to be ideal candidates from a biotechnological perspective, being sustainable and environmentally friendly. The antiparasitic properties of Carica papaya have been attributed to some of its components, including papain and other compounds that are concentrated in the latex and seeds. This study demonstrated in vitro a high and insignificantly different cysticidal activity of soluble extract that was obtained after the disruption of nontransformed wild-type (WT) cells as well as transformed papaya calluses (PC-9, PC-12, and PC-23) and papaya cell suspensions (CS-9, CS-12, and CS-23). In vivo, cell suspensions of CS-WT and CS-23 that had been previously lyophilized were tested with respect to their cysticidal effects, compared with those of three commercial antiparasitic drugs. CS-WT and CS-23 together reduced the number of cysticerci, the number of buds, and the percentage of calcified cysticerci in a similar extent to albendazole and niclosamide, whereas ivermectin was less effective. Mice were then orally immunized with CS-23 that expressed the anti-cysticercal KETc7 antigen (10 µg/mouse), CS-WT (10 mg/mouse), or both together to evaluate their preventive properties. CS-23 and CS-WT significantly reduced the expected parasite and increased the percentage of calcified cysticerci as well as recovery, being more effective when employed together. The results reported in this study support the feasibility of the development of an anti-cysticercosis vaccine from cells of C. papaya in in vitro cultures, as they are a source of an anthelmintic, natural, and reproducible product.

Improvement of cell suspension cultures of transformed and untransformed Carica papaya cell lines, towards the development of an antiparasitic product against the gastrointestinal nematode Haemonchus contortus.

Parasitic diseases have a major impact on human and animal health worldwide. Despite the availability of effective anti-parasitic drugs, their excessive and uncontrolled use has promoted the emergence of drug resistance, severely affecting ecosystems and human health. Thus, developing environmentally friendly antiparasitic treatments is urgently needed. Carica papaya has shown promising effects against infectious diseases. C. papaya embryogenic calluses were genetically modified by our research team to insert immunogenic peptides with the goal of developing an oral anti-cysticercosis vaccine. Among these callus cell lines, one labeled as CF-23, which expresses the KETc7 immunogenic peptide, induced the highest protection levels against experimental cysticercosis. In the process of designing a natural antiparasitic product based on C. papaya that simultaneously induced immunity against cysticercosis, both transformed (SF-23) and untransformed (SF-WT) suspension cultures were produced and optimized. Our results showed a better duplication time (td) for SF-23 (6.9 days) than SF-WT (13.02 days); thus, the SF-23 line was selected for scale-up in a 2-L airlift bioreactor, reaching a td of 4.4 days. This is the first time that a transgenic line of C. papaya has been grown in an airlift bioreactor, highlighting its potential for scale-up cultivation in this type of reactor. Considering the previously reported nematocidal activity of C. papaya tissues, their activity against the nematode Haemonchus contortus of aqueous extracts of SF-WT and SF-23 was explored in this study, with promising results. The information herein reported will allow us to continue the cultivation of the transgenic cell suspension line of C. papaya under reproducible conditions, to develop a new anti-parasitic product.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures.

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.