Assessment of software methods for estimating protein-protein relative binding affinities.

A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG < -0.5 kcal/mol) with high (83-98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.

Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines.

The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.

Differential expression analysis for subolesin in Rhipicephalus microplus infected with Anaplasma marginale.

Rhipicephalus microplus (formerly Boophilus microplus) ticks are potential vectors of several pathogens of livestock especially in tropical and subtropical regions where may have substantial effects on economic development. Among tick-borne pathogens, Anaplasma marginale is considered one of the most important in domestic and wild ruminants worldwide. Different molecular mechanisms have been employed by both ticks and these intracellular pathogens, in order to be able to adapt and survive. Subolesin, originally called 4D8, is an evolutionarily well-preserved protein among ixodid tick species. This new antigen was found to be protective against tick infestations when used as a vaccine, as it has an essential role in tick blood digestion, development and infection of host cells by A. marginale. Recent studies have demonstrated that infection of both tick and vertebrate host cells with this microorganism changed gene expression. Therefore, the main objective of this study was to investigate subolesin expression in uninfected and A. marginale-infected R. microplus salivary glands by real-time reverse transcriptase (RT)-PCR. To analyze the differential expression of the recombinant protein subolesin, the gene was previously expressed from ticks infected with A. marginale. Results from this study revealed that, the expression of subolesin was significantly higher in salivary glands of infected R. microplus in comparison to uninfected ones.

High-Density Antigen Microarrays for the Assessment of Antibody Selectivity and Off-Target Binding.

With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.

Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus.

BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 µM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 µM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. CONCLUSIONS: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.

Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use.

Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens.

Vaccination is a feasible strategy for controlling the haematophagous poultry red mite Dermanyssus gallinae. A cDNA library enriched for genes upregulated after feeding was created to identify potential vaccine antigens. From this library, a gene (Dg-CatD-1) encoding a 383 amino acid protein (Dg-CatD-1) with homology to cathepsin D lysosomal aspartyl proteinases was identified as a potential vaccine candidate. A second gene (Dg-CatL-1) encoding a 341 amino acid protein (Dg-CatL-1) with homology to cathepsin L cysteine proteinases was also selected for further study. IgY obtained from naturally infested hens failed to detect Dg-CatD-1 suggesting that it is a concealed antigen. Conversely, Dg-CatL-1 was detected by IgY derived from natural-infestation, indicating that infested hens are exposed to Dg-CatL-1. Mortality rates 120 h after mites had been fed anti-Dg-CatD-1 were significantly higher than those fed control IgY (PF<0·01). In a survival analysis, fitting a proportional hazards model to the time of death of mites, anti-Dg-CatD-1 and anti-Dg-CatL-1 IgY had 4·42 and 2·13 times higher risks of dying compared with controls (PF<0·05). Dg-CatD-1 and L-1 both have potential as vaccine antigens as part of a multi-component vaccine and have the potential to be improved as vaccine antigens using alternative expression systems.

Assessment of Anopheles salivary antigens as individual exposure biomarkers to species-specific malaria vector bites.

BACKGROUND: Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. METHODS: For this purpose, salivary gland proteins 6 (SG6) and 5'nucleotidases (5'nuc) from An. gambiae (gSG6 and g-5'nuc) and An. funestus (fSG6 and f-5'nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). RESULTS: The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5'nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5'nucleotidase protein. CONCLUSION: This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.

Designing the next generation of vaccines for global public health.

Vaccine research and development are experiencing a renaissance of interest from the global scientific community. There are four major reasons for this: (1) the lack of efficacious treatment for many devastating infections; (2) the emergence of multidrug resistant bacteria; (3) the need for improving the safety of the more traditional licensed vaccines; and finally, (4) the great promise for innovative vaccine design and research with convergence of omics sciences, such as genomics, proteomics, immunomics, and vaccinology. Our first project based on omics was initiated in 2000 and was termed reverse vaccinology. At that time, antigen identification was mainly based on bioinformatic analysis of a singular genome. Since then, omics-guided approaches have been applied to its full potential in several proof-of-concept studies in the industry, with the first reverse vaccinology-derived vaccine now in late stage clinical trials and several vaccines developed by omics in preclinical studies. In the meantime, vaccine discovery and development has been further improved with the support of proteomics, functional genomics, comparative genomics, structural biology, and most recently vaccinomics. We illustrate in this review how omics biotechnologies and integrative biology are expected to accelerate the identification of vaccine candidates against difficult pathogens for which traditional vaccine development has thus far been failing, and how research will provide safer vaccines and improved formulations for immunocompromised patients in the near future. Finally, we present a discussion to situate omics-guided rational vaccine design in the broader context of global public health and how it can benefit citizens in both developed and developing countries.

Opportunities for recombinant antigen and antibody expression in transgenic plants--technology assessment.

Plants are now gaining widespread acceptance as a general platform for the large-scale production of recombinant proteins. The principle has been demonstrated by the success of a diverse repertoire of proteins, with therapeutic molecules showing the most potential for added value. Over the past 10 years, several efficient plant-based expression systems have emerged. However, a number of issues remain to be addressed before plant bioreactors can be accepted and adopted widely in preference to the established microbial and mammalian platforms. Overcoming bottlenecks imposed by low yields, poor and inconsistent product quality and difficulties with downstream processing are the most important goals for researchers working in this field. The achievement of these goals is conditional on the development of extraction and processing steps that comply with GMP standards, including extensive quality assurance and control procedures. Such rigorous and validated standards should be combined with measures applied earlier in production to ensure product sustainability and quality, such as the use of master seed banking procedures. Moreover, there are several further challenges concerning topics of environmental impact, biosafety and risk assessment, which reflect the release of transgenic plants, as well the safety of the plant-derived products themselves. We are facing a growing demand for protein diagnostics and therapeutics, but lack the capacity to meet those demands using established facilities. A shift to plant bioreactors may, therefore, become necessary within the next few years, making it more imperative that the technical and regulatory limitations are addressed and solved. The production of pharmaceutical proteins in plants will only realize its huge potential if the products are provided at consistent high quality levels, allowing the delivery of clinical grade proteins that will gain regulatory approval and which can be used routinely in clinical trials.

Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis.

The aim of this study was to test the capacity of recombinant phages to deliver antigens for vaccination against porcine cysticercosis. Thus, three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7, previously proven to induce high levels of protection against pig cysticercosis, were expressed on the surface of the M13 bacteriophage at multiple copies. The pool of these four recombinant phages induced high levels of protection against an experimental murine cysticercosis. The immunogenicity of the phage vaccine preparation was therefore, tested in pigs, the natural host of Taenia solium. Subcutaneous or oral vaccination with these phages induced antigen-specific cellular immune responses in pigs. Preliminary data also points to the protective capacity of this recombinant phage vaccine against pig cysticercosis. The immunogenicity of these recombinant phages, together with the low cost of their production, make them a realistic candidate to be tested in pigs as an anti-cysticercus phage vaccine for field trials. This is the first report describing the application of a filamentous bacteriophage as a vaccine in large animals such as pigs, the only intermediate hosts of T. solium, a parasite of major medical importance in developing countries. The potential application of phages as a modern platform for vaccines for human and animal diseases is discussed.