Rational design of heat stable lyophilized rotavirus vaccine formulations.

To develop a safe and efficacious heat-stable rotavirus vaccine, new lyophilized formulations were developed using rotavirus serotypes constituting RotaTeq®. A series of formulation compositions, differing in buffering agents, bulking agents, cryoprotectants, amino acids and divalent cations, were screened for their ability to provide stability to rotavirus serotypes during lyophilization and when stored under elevated temperatures for extended periods. Lead formulations and lyophilization cycles were further optimized. Stability profiles of thus optimized formulations showed their ability to retain the potency of rotavirus for > 36 months at 5°C, 20 months at 37°C, and 7 months at 45°C. The heat-stable lyophilized rotavirus formulations developed met the all critical quality attributes for appearance, heat-stability during storage, moisture content as well as pH, viability and stability after reconstitution and has great potential to be used as vaccine candidates for improving access in low-income countries.

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity.

The human eosinophil granule ribonuclease, eosinophil-derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus-B (RSV-B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine-residue insertion in its carboxy-terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN.

Oestrogen causes degradation of KLF5 by inducing the E3 ubiquitin ligase EFP in ER-positive breast cancer cells.

KLF5 (Krüppel-like factor 5) is a multifunctional transcription factor involved in cell proliferation, differentiation and carcinogenesis. In addition to frequent inactivation in different types of human cancers, including breast cancer, KLF5 has been identified as an essential co-factor for the TGF-ß (transforming growth factor ß) tumour suppressor. In our previous study demonstrating a negative regulation of ER (oestrogen receptor α) function by KLF5 in breast cancer cells [Guo, Dong, Zhao, Sun, Li and Dong (2010) Int. J. Cancer 126, 81-89], we noticed that oestrogen reduced the protein level of KLF5. In the present study, we have tested whether and how oestrogen/ER signalling regulates KLF5 protein. We found that oestrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors, but not other inhibitors. The oestrogen-inducible E3 ligase EFP (oestrogen-responsive finger protein) was identified as a key player in oestrogen-mediated degradation of KLF5, as knockdown and overexpression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. Although only unubiquitinated EFP interacted with KLF5, overexpression of EFP appeared to prevent the ubiquitination of KLF5, while resulting in heavy ubiquitination of the E3 itself. Furthermore, ubiquitination of EFP interrupted its interaction with KLF5. Although the mechanism for how EFP degrades KLF5 remains to be determined, the results of the present study suggest that oestrogen causes the degradation of KLF5 protein by inducing the expression of EFP in ER-positive breast cancer cells.

Role of catalytic and non-catalytic subsite residues in ribonuclease activity of human eosinophil-derived neurotoxin.

Human eosinophil-derived neurotoxin (EDN), a secretory protein from eosinophils, is a member of the RNase A superfamily. The ribonucleolytic activity of EDN is central to its biological activities. EDN binds RNA in a cationic cleft, and the interaction between EDN and RNA substrate extends beyond the scissile bond. Based on its homology with RNase A, putative substrate binding subsites have been identified in EDN. The B1 and B2 subsites interact specifically with bases, whereas P0, P1, and P2 subsites interact with phosphoryl groups. In this study, we evaluated the role of putative residues of these subsites in the ribonucleolytic activity of EDN. We demonstrate that of the two base binding subsites, B1 is critical for the catalytic activity of EDN, as the substrate cleavage was dramatically reduced upon substitution of B1 subsite residues. Among the phosphate-binding subsites, P1 is the most crucial as mutations of its constituting residues totally abolished the catalytic activity of EDN. Mutation of P0 and P2 subsite residues only affected the catalytic activity on the homopolymer Poly(U). Our study demonstrates that P1 and B1 subsites of EDN are critical for its catalytic activity and that the other phosphate-binding subsites are involved in the activity on long homopolymeric substrates.

Mycobacterium tuberculosis ClpC1: characterization and role of the N-terminal domain in its function.

Caseinolytic protein, ClpC is a general stress protein which belongs to the heat shock protein HSP100 family of molecular chaperones. Some of the Clp group proteins have been identified as having a role in the pathogenesis of many bacteria. The Mycobacterium tuberculosis genome demonstrates the presence of a ClpC homolog, ClpC1. M. tuberculosis ClpC1 is an 848-amino acid protein, has two repeat sequences at its N-terminus and contains all the determinants to be classified as a member of the HSP100 family. In this study, we overexpressed, purified and functionally characterized M. tuberculosis ClpC1. Recombinant M. tuberculosis ClpC1 showed an inherent ATPase activity, and prevented protein aggregation. Furthermore, to investigate the contribution made by the N-terminal repeats of ClpC1 to its functional activity, two deletion variants, ClpC1Delta1 and ClpC1Delta2, lacking N-terminal repeat I and N-terminal repeat I along with the linker between N-terminal repeats I and II, respectively were generated. Neither deletion affected the ATPase activity. However, ClpC1Delta1 was structurally altered, less stable and was unable to prevent protein aggregation. Compared with wild-type protein, ClpC1Delta2 was more active in preventing protein aggregation and displayed higher ATPase activity at high pH values and temperatures. The study demonstrates that M. tuberculosis ClpC1 manifests chaperone activity in the absence of any adaptor protein and only one of the two N-terminal repeats is sufficient for the chaperone activity. Also, an exposed repeat II makes the protein more stable and functionally more active.

Ribosome inactivating protein saporin induces apoptosis through mitochondrial cascade, independent of translation inhibition.

Ribosome inactivating proteins (RIPs) are toxic translation inhibitors that kill eukaryotic cells by arresting protein synthesis at the translocation step. Saporin-6, expressed in the seeds of Saponaria officinalis plant, is a type I RIP comprising of a single polypeptide chain. Saporin is a specific RNA N-glycosidase and it removes a specific adenine residue from a conserved loop of the large rRNA of eukaryotic cells. Saporin-6 is one of the most potent of several isoforms of saporin, obtained from different tissues of the Saponaria plant. In addition to potently inhibiting translation, saporin has been also shown to induce cell death by apoptosis in different cellular models. To elucidate the mechanism of apoptosis induction by saporin, we have investigated the apoptotic pathway triggered by saporin. We have also analyzed whether the inhibition of protein synthesis by the toxin is the trigger for induction of apoptosis. We demonstrate that saporin-6 induces caspase-dependent apoptosis in U937 cells via the mitochondrial or intrinsic pathway. Unlike many other toxins the catalytic N-glycosidase activity of saporin is not required for apoptosis induction, and the apoptosis onset occurs before any significant inhibition of protein synthesis ensues.

Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis.

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P(-1). Although RNase A also contains a P(-1) subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P(-1) subsite residues Arg(36), Asn(39), and Gln(40) of EDN in its catalytic activity. The individual mutation of Arg(36), Asn (39), and Gln(40) resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P(-1) subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates.

Transformation of an edible crop with the pagA gene of Bacillus anthracis.

Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here.