Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.     

Effect of temperature on the biology of Creontiades dilutus (Stål) (Heteroptera: Miridae)

The egg and nymphal development, fecundity and survival of the green mirid, Creontiades dilutus were examined at a range of temperatures and a modified day-degree model fitted to the data. Day degree (DD) requirements for egg and nymphal development, and threshold temperatures were calculated from the fitted lines. Female fecundity and longevity, egg and nymphal development, and survival of C. dilutus were significantly influenced by temperature. Eggs and nymphs failed to complete development at temperatures below 17 and at 38°C. Females also failed to produce any eggs at 11 and 38°C. The optimum temperature range for female fecundity was found to be 26–32°C. The optimum temperature for the development of eggs was calculated from the model as 30.5°C and for nymphs as 31.5°C. The threshold temperature for development was 15.8°C for egg and 15.1°C for nymph; 69.4 and 156.7 DD were required for completing the egg and the nymphal development, respectively. At the optimum temperature, it was estimated that development from egg to adult took 15 days. Survival was highest at 26°C for eggs and at 30–32°C for nymphs.     

Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism. RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.     

Evaluation of accuracy and applicability of protein models: retrospective analysis of biological and biomedical predictions

In order to study protein function and activity structural data is required. Since experimental structures are available for just a small fraction of all known protein sequences, computational methods such as protein modelling can provide useful information. Over the last few decades we have predicted, with homology modelling methods, the structures for numerous proteins. In this study we assess the structural quality and validity of the biological and medical interpretations and predictions made based on the models. All the models had correct scaffolding and were ranked at least as correct or good by numerical evaluators even though the sequence identity with the template was as low as 8%. The biological explanations made based on models were well in line with experimental structures and other experimental studies. Retrospective analysis of homology models indicates the power of protein modelling when made carefully from sequence alignment to model building and refinement. Modelling can be applied to studying and predicting different kinds of biological phenomena and according to our results it can be done so with success.     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of Maturity,Reproduction and Reproductive Potentials of Torpedo Scad, <Emphasis Type="Italic">Megalaspis cordyla</Emphasis> (Linnaeus, 1758) from Northern Arabian Sea Coast of Pakistan

Port landing specimens of torpedo scad, Megalaspis cordyla were collected at monthly intervals from August 2013 to June 2014 from the northern Arabian Sea coast of Pakistan. The life history characteristics revealed that torpedo scad has a prolonged breeding season which occurred with two peaks: from May to June and from November to February. The spawning seasonality was also validated with microscopic gonad examination.The highest GSI value was noted in May and January whereas the lowest in November was recorded for both sexes. The age at which individuals of both sexes become 50% mature (t₅₀) was estimated as 2.44 year. Life span (t_max) calculated as 3.96 years for male and females. The size of 270 and 265 mm at which 50% individuals attained sexual maturity (L₅₀) was determined for males and females respectively. The reproductive potential (fecundity)was made from ten ripe females ranged from 29–43.8 cm TL and weighing 220–540 g of IV–V developmental stage was enumerated as 32306–236677 eggs (CV = 2.135) with an average number of eggs 86544 counted per ripe female/season. This study contains fundamental data of an important fish species in the northern Arabian Sea coast of Pakistan. It provides vital information to the fisheries managers for implementation on sustainable use of the resource during spawning season.     

Structure of the bundle-forming pilus from enteropathogenic Escherichia coli

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization.     

9,10-seco-9,19-Cyclolanostane arabinosides from the roots of Actaea podocarpa

Seven 9,10-seco-9,19-cyclolanostane arabinosides, named podocarpasides A-G (1-7), were isolated from the roots of Actaea podocarpa DC., a species closely related to black cohosh (a well known dietary supplement). Their structures were determined with the help of spectroscopic data including extensive 2D NMR spectroscopy. The isolates were found inactive, when tested for cytotoxic, estrogenic, and antioxidant activities in cell based assays. They were also tested for anticomplement activity against the classical pathway of complement system and only podocarpaside C (3) inhibited modest complement activity with an IC50 value of 200 microM.     

Methyl cyanide induces α to β transition and aggregation at high concentrations in E-state of human serum albumin

We have studied the effect of 2,2,2-trifluoroethanol (TFE), an α-helix inducer, versus methyl cyanide (MeCN), a β-sheet inducer, on acid-denatured human serum albumin (HSA) using far-UV circular dichroism, intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate binding, and acrylamide quenching studies. Interestingly, at pH 2.0, where the recovery and resolution of the protein in reverse phase chromatography is high, its secondary structure remains unchanged even in the presence of very high concentration (76% v/v) of MeCN. Gain of 23 and 34% α-helicity was observed in the presence of 20 and 50% TFE, respectively. At pH 7.3, HSA aggregates in the presence of 40% MeCN, but it remains soluble up to 75% MeCN at pH 2.0. The results seem to be important for HSA isolation and purification.