Expanding the recombinant protein quality in Lactococcus lactis

Background: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. - Results : we have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. - Conclusions: metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators

Protocol d'accés a les tècniques de reproducció assistida

Reproducció humana assistida; Tècniques de reproducció; Sistema sanitariReproducción humana asistida; Técnicas de reproducción; Sistema sanitarioAssisted human reproduction; Reproduction techniques; Sanitary systemAquest document defineix les diferents proves diagnòstiques i els criteris clínics per poder accedir a les diverses tècniques i les activitats que podran dur a terme els diferents proveïdors a Catalunya

Expanding the recombinant protein quality in Lactococcus lactis

Background: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. - Results : we have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. - Conclusions: metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators

WHO test.

<p>A, B represents the change from baseline in the WHO test vs placebo by treatment and HIV-status. C, D represents the percentage of responders in the WHO test by treatment and HIV-status. Responders at each time point were defined as those subjects for whom the change from baseline induced by vaccination for the specific stimulus and time point was higher than the median of the change from baseline obtained in the placebo group (i.e. horizontal bar) at the same time point and stimulus. *Statistically significant change from baseline (p-value <0.05).</p

Consort chart representing the participant flow of the CT.

<p>From the 111 subjects included, ninety-five patients were randomised and included in both the safety and immunogenic analyses. Five patients withdrew the study, being a total of 90 patients who completed the trial.</p

TB ELISPOT.

<p>A–F represents the change from baseline in TB ELISPOT vs placebo by treatment in HIV-positive (A, B, C) and HIV-negative (D, E, F) groups. G–L represents the percentage of responders in TB ELISPOT by treatment in HIV-positive (G, H, I) and HIV-negative (J, K, L) subjects. Responders at each time point were defined as those subjects for whom the change from baseline induced by vaccination for the specific stimulus and time point was higher than the median of the change from baseline obtained in the placebo group (i.e. horizontal bar) at the same time point and stimulus. *Statistically significant change from baseline (p-value <0.05).</p

Safety, Tolerability, and Immunogenicity of the Novel Antituberculous Vaccine RUTI: Randomized, Placebo-Controlled Phase II Clinical Trial in Patients with Latent Tuberculosis Infection

<div><p>Objectives</p><p>To evaluate the safety, tolerability and immunogenicity of three different doses (5, 25 and 50 µg) of the novel antituberculous vaccine RUTI compared to placebo in subjects with latent tuberculosis infection.</p><p>Methods and Findings</p><p>Double-blind, randomized, placebo-controlled Phase II Clinical Trial (95 patients randomized). Three different RUTI doses and placebo were tested, randomized both in HIV-positive (n = 47) and HIV-negative subjects (n = 48), after completion of one month isoniazid (INH) pre-vaccination. Each subject received two vaccine administrations, 28 Days apart. Five patients withdrew and 90 patients completed the study. Assessment of safety showed no deaths during study. Two subjects had serious adverse events one had a retinal detachment while taking INH and was not randomized and the other had a severe local injection site abscess on each arm and was hospitalized; causality was assessed as very likely and by the end of the study the outcome had resolved. All the patients except 5 (21%) patients of the placebo group (3 HIV+ and 2 HIV−) reported at least one adverse event (AE) during the study. The most frequently occurring AEs among RUTI recipients were (% in HIV+/−): injection site reactions [erythema (91/92), induration (94/92), local nodules (46/25), local pain (66/75), sterile abscess (6/6), swelling (74/83), ulcer (20/11), headache (17/22) and nasopharyngitis (20/5)]. These events were mostly mild and well tolerated. Overall, a polyantigenic response was observed, which differed by HIV− status. The best polyantigenic response was obtained when administrating 25 µg RUTI, especially in HIV-positive subjects which was not increased after the second inoculation.</p><p>Conclusion</p><p>This Phase II clinical trial demonstrates reasonable tolerability of RUTI. The immunogenicity profile of RUTI vaccine in LTBI subjects, even being variable among groups, allows us considering one single injection of one of the highest doses in future trials, preceded by an extended safety clinical phase.</p><p>Trial Registration</p><p> ClinicalTrials.gov <a href="http://clinicaltrials.gov/show/NCT01136161" target="_blank">NCT01136161</a></p></div

Treatment emergent adverse events by treatment, HIV-status and System Organ Class.

<p>N: Number of subjects inoculated; n: Number of subjects with adverse events; E: Number of adverse events. Percentages calculated as the percentage of the total number of subjects inoculated in each treatment group.</p