Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease.

The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform.

The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms.

The tobacco-related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. benthamiana is frequently hampered by undesired proteolysis. Here, we show that the expression of the human anti-HIV antibodies 2F5, 2G12, and PG9 in N. benthamiana leaves leads to the accumulation of discrete heavy chain-derived degradation products of 30-40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40-kDa 2F5 fragment, the 30-kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen-binding domain, the VH -CH 1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down-regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant-based expression platforms destined for the production of biopharmaceuticals.

Lessons learned from a double-blind randomised placebo-controlled study with a iota-carrageenan nasal spray as medical device in children with acute symptoms of common cold.

BACKGROUND: Common cold is caused by a variety of respiratory viruses. The prevalence in children is high, and it potentially contributes to significant morbidity. Iota-carragenan, a polymer derived from red seaweed, has reduced viral load in nasal secretions and alleviated symptoms in adults with common cold. METHODS: We have assessed the antiviral and therapeutic activity of a nasal spray containing iota-carrageenan in children with acute symptoms of common cold. A cohort of 153 children between 1-18 years (mean age 5 years), displaying acute symptoms of common cold were randomly assigned to treatment with a nasal spray containing iota-carrageenan (0.12%) as verum or 0.9% sodium chloride solution as placebo for seven days. Symptoms of common cold were recorded and the viral load of respiratory viruses in nasal secretions was determined at two consecutive visits. RESULTS: The results of the present study showed no significant difference between the iota carrageenan and the placebo group on the mean of TSS between study days 2-7. Secondary endpoints, such as reduced time to clearance of disease (7.6 vs 9.4 days; p = 0.038), reduction of viral load (p = 0.026), and lower incidence of secondary infections with other respiratory viruses (p = 0.046) indicated beneficial effects of iota-carrageenan in this population. The treatment was safe and well tolerated, with less side effects observed in the verum group compared to placebo. CONCLUSION: In this study iota-carrageenan did not alleviate symptoms in children with acute symptoms of common cold, but significantly reduced viral load in nasal secretions that may have important implications for future studies. TRIAL REGISTRATION: ISRCTN52519535, http://www.controlled-trials.com/ISRCTN52519535/

Prevalence and clinical course of viral upper respiratory tract infections in immunocompromised pediatric patients with malignancies or after hematopoietic stem cell transplantation.

BACKGROUND: Respiratory tract infections (RTI) in immunosuppressed pediatric patients with malignancies or after hematopoietic stem cell transplantation (HSCT) are associated with significant morbidity and mortality. Prospective data on the incidence and clinical role of infections by respiratory viruses in this population have been lacking. METHODS: In this prospective study, 191 children between 0 and 18 years of age were investigated by real-time polymerase chain reaction for the presence of 8 common respiratory virus types in transnasal aspirations. The study included 110 children with leukemia, lymphoma, or solid tumors (subgroup 1); 31 children after HSCT (subgroup 2); and 50 immunocompetent control patients. RESULTS: In comparison with the control group, immunocompromised children showed a significantly higher incidence of positive virus tests (subgroup 1: 53%; subgroup 2: 81%; controls: 24%; P<0.0001), and more frequently experienced ensuing viral infections in the lower respiratory tract (subgroup 1: 74%; subgroup 2: 88%; controls: 25%; P<0.0001). Sixteen percent of these children had coinfections by 2 or more viruses and revealed more severe respiratory illness. CONCLUSIONS: The present epidemiologic study on viral upper RTI in immunocompromised children revealed a high virus-associated morbidity which was particularly prominent in HSCT recipients. In these children, detection of viral coinfections was identified as a risk factor for a severe course of lower RTI.