Dengue virus neutralizing antibody: a review of targets, cross-reactivity, and antibody-dependent enhancement.

Dengue is the most common viral infection spread by mosquitoes, prevalent in tropical countries. The acute dengue virus (DENV) infection is a benign and primarily febrile illness. However, secondary infection with alternative serotypes can worsen the condition, leading to severe and potentially fatal dengue. The antibody raised by the vaccine or the primary infections are frequently cross-reactive; however, weakly neutralizing, and during subsequent infection, they may increase the odds of antibody-dependent enhancement (ADE). Despite that, many neutralizing antibodies have been identified against the DENV, which are thought to be useful in reducing dengue severity. Indeed, an antibody must be free from ADE for therapeutic application, as it is pretty common in dengue infection and escalates disease severity. Therefore, this review has described the critical characteristics of DENV and the potential immune targets in general. The primary emphasis is given to the envelope protein of DENV, where potential epitopes targeted for generating serotype-specific and cross-reactive antibodies have critically been described. In addition, a novel class of highly neutralizing antibodies targeted to the quaternary structure, similar to viral particles, has also been described. Lastly, we have discussed different aspects of the pathogenesis and ADE, which would provide significant insights into developing safe and effective antibody therapeutics and equivalent protein subunit vaccines.

Chlorpyrifos and parathion regulate oxidative stress differentially through the expression of paraoxonase 2 in human neuroblastoma cell.

Organophosphate (OP) compounds are frequently linked to both chronic and acute forms of nervous system disorders. Chlorpyrifos (CPF) and parathion (PA) are two of the most widely used OP insecticides throughout the world. These compounds are acetylcholinesterase inhibitors and cause a cholinergic crisis. However, there are other non-cholinergic effects of the OP compounds as well. The role of Paraoxonase 1 (PON1) in the metabolism of OP compounds is well established owing to its significant organophosphatase activity. Since PON2 has no paraoxonase activity and the level of its expression is 20-40 fold higher in the brain, in this article the role of PON2 in response to CPF and PA exposure concerning both cholinergic and non-cholinergic effects are explored. The effect of these OPs on cell viability, reactive oxygen species (ROS), PON2 gene expression, and function was studied. Glutathione level, esterase activity, and paraoxonase activity were also measured in CPF- and PA-treated IMR-32 cells. At these levels, both CPF and PA showed different impacts on IMR-32 cells. PA at higher concentrations (50-200 µM) proved to be less toxic than CPF. Interestingly, induction of ROS was also lower in the case of PA-treated cells as compared to the CPF. However, PON2 protein expression was increased with the increasing concentration of PA and decreased with the increasing concentration of CPF. To explore the possible mechanism of the differential regulation of PON2 gene expression by CPF and PA, we investigated the possible binding and signaling through the human M2 muscarinic acetylcholine receptor (M2AChR). Since M2AChRs are similar to G-protein coupled receptors and function through cAMP signalling, we measured the cAMP level after CPF and PA treatment. CPF- and PA-treated IMR-32 cells can be used as a model to study the mechanism by which PON2 acts as a ROS scavenger in response to xenobiotics stimulation in the brain.

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein.

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.

Production and immunogenicity of Fubc subunit protein redesigned from DENV envelope protein.

Dengue virus (DENV) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (DWS+) and severe dengue (SD). Several studies have shown that fusion (Fu) and bc loop of DENV envelope domain II are highly conserved and consist some of the most dominant antigenic epitopes. Therefore, in this study, Fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole DENV envelope protein, and its immunogenic potential as fusion peptide was estimated. For de novo designing of the antigen, Fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in DENV envelope protein. The redesigned Fubc protein was expressed in E. coli and purified. Subsequently, structural integrity of the purified protein was verified by CD spectroscopy. To characterise immune responses against recombinant Fubc protein, BALB/c mice were subcutaneously injected with emulsified antigen preparation. It was observed by ELISA that Fubc fusion protein elicited higher serum IgG antibody response either in the presence or in absence of Freund's adjuvant in comparison to the immune response of Fu and bc peptides separately. Furthermore, the binding of Fubc protein with mice antisera was validated by SPR analysis. These results suggest that Fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against DENV.

Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process.

BACKGROUND: The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein. RESULTS: It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer. CONCLUSIONS: Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.

Designing antibody against highly conserved region of dengue envelope protein by in silico screening of scFv mutant library.

Dengue being one of the deadliest diseases of tropical regions, enforces to put continuous efforts for the development of vaccine and effective therapeutics. Most of the antibodies generated during dengue infection are non-neutralizing and cause antibody dependent enhancement. Hence, making a potent neutralizing antibody against all four dengue serotypes could be very effective for the treatment. However, designing a single antibody for all serotypes is difficult due to variation in protein sequences. Therefore, the objective is to identify conserved region of dengue envelope protein and then develop an antibody against that conserved region. Before advancing to the development of such an antibody, it is desirable to validate the interactions between antibody and dengue envelope protein. In silico analysis of such interactions provides a good platform to find out a suitable region to design and construct an antibody against it by analyzing antigen-antibody interaction before synthesizing the antibody. In this study, two highly conserved regions of dengue envelope protein were identified and an scFv was constructed against it. Both scFv and FuBc proteins were expressed in bacterial expression system and binding efficiency was analyzed by SPR analysis with KD value 2.3 µM. In order to improve binding efficiency, an in silico scFv mutant library was created which was virtually screened for higher binding efficiency. Six mutants with high binding efficiency were selected for further analysis. The binding ability of these mutants were predicted using simulation analysis which shows these mutations were stabilizing scFv-FuBc complex.

Recent Developments Toward Antibody Engineering and Affinity Maturation.

BACKGROUND: Monoclonal antibodies have been proven to deliver significant contribution in health industry for the development of both therapeutics and diagnostics. Efforts have been made to achieve immunoglobulin with high antigen specificity and stability. In this regard, smaller fragment of antibody has been constructed as an alternative of full immunoglobulin molecules due to the feasibility of recombinant production in various host cells. Antibody fragments are that part of an immunoglobulin which can form a complete epitope binding site and also retain the binding efficiency and accuracy of a whole antibody. However, the effector functions cannot be accomplished by antibody fragments alone as they lack Fc region. Hence, full antibody is constructed by fusing Fc domain of a human antibody. Nevertheless, to find an antibody with high antigen specificity and stability is still a big challenge. CONCLUSION: Recent protein engineering techniques have enabled many options of modification and tailoring of antibody fragments for better stability, specificity and pharmacokinetic properties. This review focuses on the latest techniques applied for the construction of antibody fragments, recent developments toward affinity maturation and applications of recombinant antibodies.