Antibody-binding Regions Research Articles

Crystal structure of the CTLD7 domain of human M-type phospholipase A2 receptor

M-type phospholipase A2 receptor (PLA2R) is a member of the mannose receptor family. Recent evidence shows that PLA2R is a major autoantigen causing idiopathic membranous nephropathy (IMN), which is an autoimmune disease and one of the most common causes for nephrotic syndrome in adults. The epitope mapping data suggest that the major epitopes of PLA2R locate at the CysR, CTLD1 and CTLD7 domains. However, due to the lack of the high-resolution structural information, it is unclear how the autoantibodies interact with PLA2R. Here we determine the crystal structure of the CTLD7 domain of PLA2R at 1.8 Å, showing that it adopts a typical CTLD fold, and the structural alignments also provide hints for the potential antibody binding regions. In addition, the high-resolution structural information of CTLD7 could be applied to identify the epitopes for autoantibodies, which would facilitate the therapeutic strategies against IMN.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding.

DNA aptamers were developed against five different peptides from the known binding regions of anti-Cytomegalovirus and anti-Herpes Simplex Virus-2 antibodies and the aptamers were ranked by relative affinity based on an ELISA-like (ELASA) microplate assay. The secondary structures of the top five highest affinity aptamers were studied for stem-loop commonalities and the most probable peptide binding sites. Two of these stem-loop structures were converted into beacons by addition of TYE 665 dye on the 5' end and Iowa Black quencher on the 3' end. When competed against increasing concentrations of each of the five peptides, only three of the possible ten interactions demonstrated "lights on" fluorescence beacon responses. When modeled by generation of PDB files, after passage through PATCHDOCK and YASARA, two of the aptamer beacon-peptide interactions showed no theoretical evidence of separating the G-C stem-loop region, despite clear empirical evidence of separation of the fluorophore and quencher beyond the Förster distance leading to abundant fluorescence. And in the second beacon's case, YASARA modeling suggested that the beacon was always open despite clear empirical evidence that it was not (no fluorescence response) and only opened in the presence of one of the five peptides. These results are interpreted as a demonstration that 3-dimensional docking software such as PATCHDOCK and YASARA, which are based on rigid receptor-ligand shape complementarity may not reflect the "induced-fit" interactions between aptamers and their cognate targets. Therefore, for the most complete and accurate picture of aptamer-peptide binding, several theoretical and empirical (e.g., beacon fluorescence) analysis methods may be needed.

Abstract 1777: REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer

Abstract Advanced ovarian cancer has a high rate of recurrence, thus there is a need for therapies that can produce durable responses and extend overall survival. Infiltrating CD3+ T cells are reported to correlate with improved clinical outcome in stage III/IV ovarian cancer. Bispecific antibodies that engage T cells via CD3 and a tumor antigen on ovarian tumor cells may be able to exploit these tumor-infiltrating T cells and have drawn interest as a novel strategy of antitumor immunotherapy. Mucin16 (MUC16) is a well described antigen highly expressed in ovarian cancer and several other tumors. We generated a human bispecific antibody (REGN4018) that binds MUC16 on tumor cells and CD3, bridging MUC16-expressing cells with CD3+ T cells. REGN4018 demonstrates MUC16-directed T cell activation and polyclonal T cell killing of MUC16-expressing tumor cells in vitro and in vivo. Binding and cytotoxicity are minimally affected by high concentrations of CA-125, the shed form of MUC16 that is used as a biomarker for ovarian cancer. Several murine tumor models were developed to determine the in vivo anti-tumor effects of REGN4018. As a xenogenic tumor model, human OVCAR-3 cells were grown as ascites in immunodeficient mice [NOD-SCID-IL2Rgamma deficient (NSG)] pre-implanted with human PBMC. Significant tumor inhibition by REGN4018 was observed at doses of ≥ 0.1 mg/kg. To enable investigation of clinical bispecific antibodies in tumor-bearing immune competent mice, mice were genetically engineered to humanize both CD3 and a part of MUC16 covering the antibody binding region. ImmunoPET imaging demonstrated localization of REGN4018 in both T cell-rich organs such as the spleen and lymph nodes as well as in MUC16-expressing tumors. REGN4018 inhibited growth of murine tumors transfected with human MUC16 in syngeneic tumor models at doses ≥ 0.05 mg/kg. The safety and tolerability of REGN4018 were evaluated in cynomolgous monkey studies. REGN4018 administration resulted in a minimal and transient increase in serum cytokines and C-reactive protein with no overt toxicity. Collectively, these data show the potent anti-tumor activity and tolerability of REGN4018 and provide strong support for the clinical testing of REGN4018 in patients with MUC16-expressing ovarian cancers. Citation Format: Alison Crawford, Lauric Haber, Kristin Vazzana, Lauren Canova, Priyanka Ram, Jennifer Principio, Arpita Pawashe, Curtis Colleton, Marcus Kelly, Sosina Makkonen, Carlos Hickey, Paurene Duramad, Stephen Godin, John Lin, Eric Smith, Gavin Thurston, Jessica R. Kirshner. REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1777.

Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens.

Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens.

Research on Preparation of Human Immune Cell <em>in vitro</em> with Response to Shrimp Allergen

Shrimp is one of the most important food allergens. Tropomyosin is its major allergen. Wherein Pen a 1, contains five antibody binding regions, has been identified as the only major shrimp allergen. However, the study on IgE with response to shrimp allergen is still a serious lack, compared with the allergenic proteins. Particularly in the aspects of the preparation of IgE in vitro, it is restricted and can only obtain the complete IgE molecules by polyclonal or monoclonal technology. As for the preparation of small molecule IgE to the shrimp allergen has not yet been reported. This study attempts to carry out research on obtaining of cell materials that are used to clone. It sets up a convenient and efficient immune system in vitro which combines dendritic cell differentiation, allergens immune, mixed lymphocyte culture and so on. Finally the system successfully activates the proliferation of specific B cells and the secretion of a large number of specific IgE antibodies to shrimp allergen.

MOLECULAR CHARACTERIZATION ON THE BASIS OF HA, NA AND M GENE REVEALED CHANGES IN CRITICAL AMINO ACID POSITIONS OF INFLUENZA A (H3N2) VIRUS CIRCULATING IN INDIA DURING 2011-2013

Influenza a (H1N1) virus is responsible for acute respiratory infection in human, which occasionally causes epidemic and rarely a pandemic. Full length DNA sequencing of HA, NA and M gene of H3N2 virus from year 2011-2013 was performed for determination of the circulating strains in India along with monitoring status of various mutations associated with drug resistance and virulence. Genetic analysis of HA gene of study samples in comparison to reference strain showed maximum amino acid changes in epitopes of high neutralization efficiencies (antigenic sites A, B and D) along with gain of glycosylation sites at position 45 (except single sample) and loss at position 126 (7 samples). NA gene has four amino acid changes in antibody binding region at position S367N, K369T, S332F and S334I along with gain and loss of glycosylation site at position 367 and 402 respectively whereas no change was observed in its active site. While comparison of deduced amino acid sequences of study samples with Indian strains from earlier years showed six amino acid changes in three antigenic sites on HA protein and three amino acid changes in NA protein epitope sequence. All these changes indicate that the H3N2 virus is undergoing antigenic drift and which may result in the emergence of new antigenic variants. We want to report that our study provided for the first time full length matrix gene sequence of influenza a (H3N2) virus from India.

The Importance of Intergenic Recombination in Norovirus GII.3 Evolution

Norovirus genotype II.3 (GII.3) strains are a major cause of sporadic gastroenteritis. Intergenic recombination between the capsid and RNA-dependent RNA polymerase (RdRp) genes is common and results in the acquisition of an alternative RdRp genotype. This study aimed to explore the evolution of the GII.3 capsid gene, focusing on the influence of intergenic recombination. The capsid genes from six GII.3 norovirus strains, collected from Australian children between 2001 and 2010, were sequenced and aligned with 66 GII.3 capsid sequences from GenBank, spanning 1975 to 2010. The GII.3 capsid gene evolved at a rate of 4.16 × 10(-3) to 6.97 × 10(-3) nucleotide substitutions/site/year from 1975 to 2010 and clustered into five temporally sequential lineages. Clustering of the GII.3 capsid gene sequences was associated with intergenic recombination and switches between RdRp genotypes GII.3, GII.a, GII.b, GII.12, and an undefined ancestral RdRp. Comparison of the substitution rate of the GII.3 and GII.b RdRps suggested that RdRp switching allows a higher evolutionary rate, leading to increased genetic diversity and adaptability. Alignment of GII.3 capsid sequences revealed 36 lineage-specific conserved amino acid substitutions, four of which were under positive selection. Many conserved substitutions were within predicted antibody binding regions and close to host attachment factor binding sites. In conclusion, evolution of GII.3 noroviruses was primarily driven by intergenic recombination. The acquisition of new RdRps may lead to a faster mutation rate and increased genetic diversity, improving overall GII.3 fitness.

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5×10−13M and 4.8×10−10M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75°C while the scFv melts at ∼65°C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1μgL−1. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.

All major prion types recognised by a multiplex immunofluorometric assay for disease screening and confirmation in sheep

Prion diseases or transmissible spongiform encephalopathies (TSEs) in small ruminants are presented in many forms: classical scrapie, Nor98/atypical scrapie, CH1641 scrapie and bovine spongiform encephalopathy (BSE). We previously described a multiplex immunofluorometric assay (mIFMA), based on a bead array flow cytometry technology, which provided, in a single assay, discrimination between BSE (in cattle and sheep) and classical scrapie (Tang et al., 2010). In this study, we extended the mlFMA to differentiate classical scrapie, atypical scrapie, BSE (experimentally infected sheep and naturally infected cattle) and CH1641 (both experimental and natural CH1641-like infections in sheep). Three capture antibodies were used, two distinct PrP N-terminus specific antibodies 12B2 and 9A2, and a PrP core specific antibody 94B4. All three antibodies were shown to bind classical scrapie PrPres strongly, whereas in Nor98/atypical scrapie PrPres only 12B2 and 9A2 binding was observed. PrPres binding of 12B2 was low for both BSE and CH1641, as expected.Furthermore, analysis of serially diluted samples indicated that the assay provided a similar level of sensitivity for atypical scrapie as that found using a well established commercial test. Unexpectedly, 9A2 binding to CH1641 PrPres was reduced by 2.1 fold both for experimental CH1641 and CH1641-like scrapie when compared with BSE, suggesting that major cleavage of the N-terminus occurs further towards the C-terminus in CH1641 than in BSE. The ratios of 12B2/94B4 and 9A2/94B4 were similar between experimental CH1641 and CH1641-like cases, although two CH1641-like subjects displayed slightly elevated ratios of both 12B2/94B4 and 9A2/94B4. To verify this finding for PrPres, mass spectrometry based quantification was used to determine the absolute abundance of the peptides associated with all three antibody binding regions. There was a 2.2 fold reduction of peptides containing the 9A2 epitope for experimental CH1641 PrPres in comparison to BSE PrPres. Observation of reduced PrPres may serve as a new marker for CH1641. This mIFMA may thus provide the basis for simplified TSE diagnosis with capability for simultaneous screening and differential diagnosis.

Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.

Mapping of IgE and IgG4 Antibody-Binding Epitopes in Cyn d 1, the Major Allergen of Bermuda Grass Pollen

Background: Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG₄ antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. Methods: Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG_1–4 antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. Results: Four major IgE-binding epitopes (peptides 15^–1, 21, 33^–2 and 35⁺¹, corresponding to amino acids 70–79, 101–110, 159–167 and 172–181) and 5 major IgG₄-binding epitopes (peptides 15^–1, 30^–2, 33^–2, 35⁺¹ and 39, corresponding to amino acids 70–79, 144–153, 159–167, 172–181 and 192–200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG₄. Their critical amino acids were all characterized. Major epitopes for human IgG₁ to IgG₄ are almost identical. Conclusions: This is the first study to map the sequential epitopes for human IgE and IgG₄ subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis.

Fast photochemical oxidation of proteins for epitope mapping.

The growing use of monoclonal antibodies as therapeutics underscores the importance of epitope mapping as an essential step in characterizing antibody-antigen complexes. The use of protein footprinting coupled with mass spectrometry, which is emerging as a tool in structural biology, offers opportunities to map antibody-binding regions of antigens. We report here the use of footprinting via fast photochemical oxidation of proteins (FPOP) with OH radicals to characterize the epitope of the serine protease thrombin. The data correlate well with previously published results that determined the epitope of thrombin. This study marks the first time oxidative labeling has been used for epitope mapping.

Identification of an antibody-binding epitope on the rotavirus A non-structural protein NSP2 using phage display analysis

The non-structural protein 2 (NSP2) of rotavirus has important roles in rotavirus replication associated with RNA binding, hydrolysis of NTPs and RNA, and helix destabilizing properties. A cell-culture assay using an NSP2-specific mAb and polyclonal antiserum to block virus replication showed a 73 and 96 % reduction in the amount of virus produced during replication, respectively. Phage display technology was used to identify the antibody-binding region on the NSP2 protein with the motif (244)T-(Y/F)-Ø-Ø-Ø-X-K-Ø-G(252), where Ø is a hydrophilic residue and X is any amino acid. This region was mapped to the three-dimensional NSP2 crystal structure to visualize the epitope. Analysis revealed identity to a region on NSP2 that mapped to a site exposed on the surface of the protein, which could possibly interfere with a functionally important region of the protein. Antibody binding to this region could disrupt the essential roles of NSP2, such as the formation of viroplasms with NSP5 or the interaction with viral RNA, thereby indicating a possible mechanism for the observed inhibition of virus replication. Genetic analysis of the putative binding region of NSP2 revealed a high level of conservation, suggesting that the region is under strict control.

The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC 50 values: 212–310 nM), whereas E6 and G8 inhibited PKCɛ activity (IC 50 values: 103–233 nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.

Regions of botulinum neurotoxin A light chain recognized by human anti-toxin antibodies from cervical dystonia patients immunoresistant to toxin treatment. The antigenic structure of the active toxin recognized by human antibodies

This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141–159), L14 (183–201) and L18 (239–257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365–383) and L29 (379–397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation.

Characterization of single chain antibody targets through yeast two hybrid

BackgroundDue to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.ResultsWe describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.ConclusionsOur method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.

Erratum to: Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

Erratum to: Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

In previous studies, we showed that certain peptides of the H(N) and H(C) domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505-523/519-537/533-551/547-565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799-817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785-803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H(C) domain, the overlap C23/C24 (1163-1181/1177-1195) provided no protection. Peptide C31 (1275-1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051-1069/1065-1083) or peptides C18/C19/C20 (1093-1111/1107-1125/1121-1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.