Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights.

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein.

BACKGROUND: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. METHODS: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. RESULTS: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. CONCLUSION: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Dynamics of lipid displacement inside the hydrophobic cavity of a nonspecific lipid transfer protein from Solanum melongena.

Nonspecific lipid transfer proteins are multifunctional and multispecific seed proteins with a characteristic hydrophobic cavity that runs form N-terminal to the C-terminal end. They are capable of binding and transferring different lipid molecules by means of their hydrophobic cavity. Apart from the cavity, lipid molecules bind and interact at key positions on the nsLTP surface as well. The plasticity of the hydrophobic cavity is an unusual property, considered as the primary lipid binding site. Here, we report a crystal structure of nsLTP from Solanum melongena with two lauric acid molecules bound inside the cavity. It has been observed that the extent of the N-terminal entry point and plasticity of the cavity can be extended, upon binding of one or two lipid molecules inside the cavity. The MD simulation further revealed that the lipid molecule shows high mobility inside the cavity and interestingly, was able to change its orientation. An alternate lipid entry site adjacent to the N-terminal end was uncovered during simulation and Arg-84 was implicated to be a potential regulatory residue aside from Tyr-59. Collectively, this study helps to understand that changes in orientation of the lipid inside the cavity could occur intermittently besides entering the cavity via tail-in-mechanism.Communicated by Ramaswamy H. Sarma.

Antibody multispecificity: A necessary evil?

Antibodies represent key effectors of the adaptive immune system. The specificity of antibodies is an established hallmark of the immune response. However, a certain proportion of antibodies exhibit limited promiscuity or multireactivity. Germline antibodies display plasticity which imparts multispecificity to enhance the antibody repertoire. Surprisingly, even affinity matured antibodies display such plasticity and multireactivity enabling their binding to more than one antigen. We propose that antibody multispecificity is a physiological requirement to expand the antibody repertoire at the germline level and to tolerate plasticity in antigens at the mature level. This property of the humoral immune response may attenuate the ability of infectious RNA viruses such as influenza, HIV and SARS-CoV-2 to acquire mutations that render resistance to neutralizing antibodies.

Dynamic Alteration in the Vaginal Secretory Proteome across the Early and Mid-Trimesters of Pregnancy.

Pregnancy is characterized by intense physiological and structural alterations in the vagina, cervix, and overlying fetal membranes. High vaginal fluid (HVF) is a proximal fluid that covers the lower part of the female reproductive system and the severity of vaginal pathology often adversely affects pregnancy outcomes. To identify the correlation of vaginal fluid proteome dynamics and physiological changes during the progression of pregnancy, a longitudinal study was performed on 20 pregnant women who delivered a baby in >37 weeks without any complications. SWATH-MS-based label-free quantitative proteomics was performed to profile the HVF proteome at three time points defined as V1 (7-12 weeks), V2 (18-20 weeks), and V3 (26-28 weeks). Linear mixed-effect models were used to estimate protein abundance as a function of the period of gestational age. In this study, we identified 1015 HVF proteins and 61 of them were significantly altered until late second trimester. Our result demonstrates that the HVF proteins reveal gestational age-specific expression patterns and the function of these proteins is associated with tissue remodeling, organ development, and microbial defense. Our study provides an opportunity to monitor the underlying physiology of pregnancy that may be further probed for the biomarker identification in pregnancy-related adverse outcomes. Data are available via ProteomeXchange with identifiers PXD014846 and PXD021811.

Exploring the different states of wild-type T-cell receptor and mutant conformational changes towards understanding the antigen recognition.

Recognition of proteolytic peptide fragments presented by major histocompatibility complex (MHC) on target cells by T-cell receptor (TCR) is among the most important interactions in the adaptive immune system. Several computational studies have been performed to investigate conformational and dynamical properties of TCRs for enhanced immunogenicity. Here, we present the large-scale molecular dynamics (MD) simulation studies of the two comprehensive systems consisting of the wild-type and mutant IG4 TCR in complex with the tumor epitope NY-ESO peptide (SLLMWITQC) and analyzed for mapping conformational changes of TCR in the states prior to antigen binding, upon antigen binding and after the antigen was released. All of the simulations were performed with different states of TCRs for each 1000 ns of simulation time, providing six simulations for time duration of 6000 ns (6µs). We show that rather than undergoing most critical conformational changes upon antigen binding, the high proportion of complementarity-determining region (CDR) loops change by comparatively small amount. The hypervariable CDRα3 and CDRß3 loops showed significant structural changes. Interestingly, the TCR ß chain loops showed the least changes, which is reliable with recent implications that ß domain of TCR may propel antigen interaction. The mutant shows higher rigidity than wild-type even in released state; expose an induced fit mechanism occurring from the re-structuring of CDRα3 loop and can allow enhanced binding affinity of the peptide antigen. Additionally, we show that CDRα3 loop and peptide contacts are an adaptive feature of affinity enhanced mutant TCR.Communicated by Ramaswamy H. Sarma.

Salivary proteome signatures in the early and middle stages of human pregnancy with term birth outcome.

The establishment and maintenance of pregnancy in humans proceed through a continuous change of biochemical and biophysical processes. It requires a constant interaction between the fetus and the maternal system. The present prospective study aims to elucidate changes in salivary proteome from the early to middle stages of term pregnancy, and establishing an expressional trajectory for modulated proteins. To date, a comprehensive characterization of the longitudinal salivary proteome in pregnancy has not been performed and it is our immediate interest. In the discovery phase, maternal saliva (N = 20) at 6-13, 18-21, and 26-29 weeks of gestation was analyzed using level-free proteomics (SWATH-MS) approach. The expression levels of 65 proteins were found to change significantly with gestational age and distributed into two distinct clusters with a unique expression trajectory. The results revealed that altered proteins are involved in maternal immune modulation, metabolism, and host defense mechanism. Further, verification of 12 proteins was employed using targeted mass spectrometry (MRM-MS) in a separate subset of saliva (N = 14). The MRM results of 12 selected proteins confirmed a similar expression pattern as in SWATH-MS analysis. Overall, the results not only demonstrate the longitudinal maternal saliva proteome for the first time but also set the groundwork for comparative analysis between term birth and adverse pregnancy outcomes.

Publisher Correction: High resolution structural and functional analysis of a hemopexin motif protein from Dolichos.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The structure of MP-4 from Mucuna pruriens at 2.22†Šresolution.

The structure of the MP-4 protein was previously determined at a resolution of 2.8 Å. Owing to the unavailability of gene-sequence information at the time, the side-chain assignment was carried out on the basis of a partial sequence available through Edman degradation, sequence homology to orthologs and electron density. The structure of MP-4 has now been determined at a higher resolution (2.22 Å) in another space group and all of the structural inferences that were presented in the previous report of the structure were validated. In addition, the present data allowed an improved assignment of side chains and enabled further analysis of the MP-4 structure, and the accuracy of the assignment was confirmed by the recently available gene sequence. The study reinforces the traditional concept that conservative interpretations of relatively low-resolution structures remain correct even with the availability of high-resolution data.

Comparative study of 7S globulin from Corylus avellana and Solanum lycopersicum revealed importance of salicylic acid and Cu-binding loop in modulating their function.

The plant seed proteins referred to as vicilins belong to a structurally common superfamily. While some of them are reported to exhibit superoxide dismutase activity, vicilins from other sources do not possess this activity. Vicilin from Corylus avellana (HZ.1) and Solanum lycopersicum (SL80.1) were purified and subjected to structure-function analysis. The superoxide dismutase activity assays were performed to understand the functional differences between them. While SL80.1 has the superoxide dismutase activity, HZ.1 was enzymatically inactive. Crystal structure followed by mass spectrometry analysis of both the proteins revealed that while SL80.1 has bound salicylic acid, HZ.1 does not. Comparison of C-terminal binding pocket of both the structures revealed that a point mutation at residue 321 in HZ.1 (Gly→Cys) leads to obstruction in binding of salicylic acid in the pocket. Similarly, copper-binding loop of HZ.1 was reportedly found to be intact and shorter than the loops reported in SL80.1. The copper-binding loop of SL80.1 is rich in polar residues and the absence of these residues in HZ.1 copper-binding loop possibly indicates deficiency in channeling of oxygen radicals to the active center of the enzyme. Difference in the enzymatic activity of vicilin from two evolutionarily distinct sources is due to mutations in its co-factor binding pocket and copper-binding loop.

Structural insights into the lipid transfer mechanism of a non-specific lipid transfer protein.

The non-specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo-state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid-dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface-bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.

High resolution structural and functional analysis of a hemopexin motif protein from Dolichos.

It is increasingly evident that seed proteins exhibit specific functions in plant physiology. However, many proteins remain yet to be functionally characterized. We have screened the seed proteome of Dolichos which lead to identification and purification of a protein, DC25. The protein was monomeric and highly thermostable in extreme conditions of pH and salt. It was crystallized and structure determined at 1.28 Å resolution using x-ray crystallography. The high-resolution structure of the protein revealed a four-bladed ß-propeller hemopexin-type fold containing pseudo four-fold molecular symmetry at the central channel. While the structure exhibited homology with 2S albumins, variations in the loops connecting the outermost strands and the differences in surface-charge distribution may be relevant for distinct functions. Comparative study of the protein with other seed hemopexins revealed the presence of four conserved water molecules in between the blades which cross-link them and maintain the tertiary structure. The protein exhibited intrinsic peroxidase activity, which could be inhibited by binding of a heme analog. The identification of redox-sensitive cysteine and inhibition of peroxidase activity by iodoacetamide facilitated characterization of the possible active site. The determined peroxidase activity of DC25 may be responsible for rescuing germinating seeds from oxidative stress.

Cross-clade antibody reactivity may attenuate the ability of influenza virus to evade the immune response.

Vaccines developed against influenza lose efficacy primarily due to the ability of the virus to generate variants that escape recognition of the immune system. Frequent accumulation of mutations in the virus surface proteins is believed to be responsible for immune evasion. Surprisingly, despite the high mutation rate, the appearance of new viral strains through antigenic drift is slow. This delay in the emergence of new strains has been explained by several different hypotheses over the past decade. In the present study, we have probed the antibody response against multiple clades of influenza neutralizing epitope in the context of antigenic drift. Both, the serum IgG and the monoclonal antibodies raised against the epitope showed strong predisposition against different variants even with non-conservative mutations. The physiologically relevant binding with hemagglutinin protein and its variants revealed multi-reactive recognition potential of human single-chain variable fragments (scFvs). Differential scope for antibody cross-reactivity was evident among different clades that could counterbalance the effect of antigenic drift. Our findings reveal that the majority of epitope variants, which could manifest as single or double amino acid substitutions, would not escape immune surveillance. However, mutations beneficial for the virus do appear causing effective antigenic changes. It is suggested that inherent antibody promiscuity could reduce the deleterious effects of natural mutations on antigen recognition and may be responsible for the delay in the appearance of new antigenic variants of the fast-mutating viruses.

Antibody specificity and promiscuity.

The immune system is capable of making antibodies against anything that is foreign, yet it does not react against components of self. In that sense, a fundamental requirement of the body's immune defense is specificity. Remarkably, this ability to specifically attack foreign antigens is directed even against antigens that have not been encountered a priori by the immune system. The specificity of an antibody for the foreign antigen evolves through an iterative process of somatic mutations followed by selection. There is, however, accumulating evidence that the antibodies are often functionally promiscuous or multi-specific which can lead to their binding to more than one antigen. An important cause of antibody cross-reactivity is molecular mimicry. Molecular mimicry has been implicated in the generation of autoimmune response. When foreign antigen shares similarity with the component of self, the antibodies generated could result in an autoimmune response. The focus of this review is to capture the contrast between specificity and promiscuity and the structural mechanisms employed by the antibodies to accomplish promiscuity, at the molecular level. The conundrum between the specificity of the immune system for foreign antigens on the one hand and the multi-reactivity of the antibody on the other has been addressed. Antibody specificity in the context of the rapid evolution of the antigenic determinants and molecular mimicry displayed by antigens are also discussed.

A Pregnancy Cohort to Study Multidimensional Correlates of Preterm Birth in India: Study Design, Implementation, and Baseline Characteristics of the Participants.

Globally, preterm birth is a major public health problem. In India, 3.6 million of the 27 million infants born annually are preterm. Risk stratification of women based on multidimensional risk factors assessed during pregnancy is critical for prevention of preterm birth. A cohort study of pregnant women was initiated in May 2015 at the civil hospital in Gurugram, Haryana, India. Women are enrolled within 20 weeks of gestation and are followed until delivery and once postpartum. The objectives are to identify clinical, epidemiologic, genomic, epigenomic, proteomic, and microbial correlates; discover molecular-risk markers by using an integrative -omics approach; and generate a risk-prediction algorithm for preterm birth. We describe here the longitudinal study design, methodology of data collection, and the repositories of data, biospecimens, and ultrasound images being created. A total of 4,326 pregnant women, with documented evidence of recruitment before 20 weeks of gestation, have been enrolled through March 2018. We report baseline characteristics and outcomes of the first 2,000 enrolled participants. A high frequency of preterm births (14.9% among 1,662 live births) is noteworthy. The cohort database and the repositories will become global resources to answer critical questions on preterm birth and other birth outcomes.

Deciphering evolution of immune recognition in antibodies.

BACKGROUND: Antibody, the primary effector molecule of the immune system, evolves after initial encounter with the antigen from a precursor form to a mature one to effectively deal with the antigen. Antibodies of a lineage diverge through antigen-directed isolated pathways of maturation to exhibit distinct recognition potential. In the context of evolution in immune recognition, diversity of antigen cannot be ignored. While there are reports on antibody lineage, structural perspective with respect to diverse recognition potential in a lineage has never been studied. Hence, it is crucial to evaluate how maturation leads to topological tailoring within a lineage enabling them to interact with significantly distinct antigens. RESULTS: A data-driven approach was undertaken for the study. Global experimental mouse and human antibody-antigen complex structures from PDB were compiled into a coherent database of germline-linked antibodies bound with distinct antigens. Structural analysis of all lineages showed variations in CDRs of both H and L chains. Observations of conformational adaptation made from analysis of static structures were further evaluated by characterizing dynamics of interaction in two lineages, mouse VH1-84 and human VH5-51. Sequence and structure analysis of the lineages explained that somatic mutations altered the geometries of individual antibodies with common structural constraints in some CDRs. Additionally, conformational landscape obtained from molecular dynamics simulations revealed that incoming pathogen led to further conformational divergence in the paratope (as observed across datasets) even while maintaining similar overall backbone topology. MM-GB/SA analysis showed binding energies to be in physiological range. Results of the study are coherent with experimental observations. CONCLUSIONS: The findings of this study highlight basic structural principles shaping the molecular evolution of a lineage for significantly diverse antigens. Antibodies of a lineage follow different developmental pathways while preserving the imprint of the germline. From the study, it can be generalized that structural diversification of the paratope is an outcome of natural selection of a conformation from an available ensemble, which is further optimized for antigen interaction. The study establishes that starting from a common lineage, antibodies can mature to recognize a wide range of antigens. This hypothesis can be further tested and validated experimentally.

Structure-guided identification of function: role of Capsicum annuum vicilin during oxidative stress.

Proteins belonging to cupin superfamily are known to have critical and diverse physiological functions. However, 7S globulins family, which is also a part of cupin superfamily, were undermined as only seed storage proteins. Structure determination of native protein - Vic_CAPAN from Capsicum annuum - was carried out, and its physiological functions were explored after purifying the protein by ammonium sulfate precipitation followed by size exclusion chromatography. The crystal structure of vicilin determined at 2.16 Šresolution revealed two monomers per asymmetric unit which are juxtaposed orthogonal with each other. Vic_CAPAN consists predominately of ß-sheets that folds to form a ß-barrel structure commonly called cupin fold. Each monomer of Vic_CAPAN consists of two cupin fold domains, N-terminal and C-terminal, which accommodate two different ligands. A bound ligand was identified at the C-terminal cupin fold in the site presumably conserved for metabolites in the crystal structure. The ligand was confirmed to be salicylic acid through mass spectrometric analysis. A copper-binding site was further observed near the conserved ligand-binding pocket, suggesting possible superoxide dismutase activity of Vic_CAPAN which was subsequently confirmed biochemically. Vicilins from other sources did not exhibit this activity indicating functional specificity of Vic_CAPAN. Discovery of bound salicylic acid, which is a known regulator of antioxidant pathway, and revelation of superoxide dismutase activity suggest that Vic_CAPAN has an important role during oxidative stress. As salicylic acid changes the redox state of cell, it may act as a downstream signal for various pathways involved in plant biotic and abiotic stress rescue.

Multiple target sites for designing candidate drugs.

Rational drug discovery strategy requires a design of small molecules as candidate drugs which can specifically inhibit a target protein or any other macromolecule and effectively interfere in a defined physiological process. One of the important bacterial protein targets aimed toward developing new antibiotics is peptidyl-tRNA hydrolase (Pth). The discovery that cytarabine, a known anticancer drug, binds to Pth from Acinetobacter baumannii in a cleft located away from the catalytic site of this enzyme, published in Biochemical Journal, opens up interesting new avenues for drug design. An approach involving crystallographic identification of multiple ligand-binding sites on a target protein surface could enable iterative optimization of multiple high-affinity ligands, which may synergistically interfere in the target function with enhanced effect.