Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

HipBST Toxin-antitoxin System of Prokaryotes / 中国生物化学与分子生物学报

Toxin-antitoxin system (TA) is a genetic element widely found in chromosomes and plasmids of bacteria, archaea and prophages. TA usually consists a toxin that inhibits the growth of bacteria and an antitoxin that neutralizes its toxicity. Since the discovery of the first CcdB / CcdA TA in the 1980s, TA has been proved to exist in almost all sequenced microorganisms and plays an important role in maintaining plasmid stability, anti-phage and promoting biofilm formation. At present, TA is divided into type I-VIII, among which type IITA is the most widely studied. HipBA is a type II TA. The toxin HipA in Escherichia coli HipBA is a serine / threonine kinase, which inhibits protein translation by phosphorylating bacterial Glutamyl tRNA synthetase (GltX), and its toxicity can be specifically neutralized by HipB. Recently, it has been found that Escherichia coli HipA homologous proteins exist widely in microorganisms, and they form a potential novel TA with genes of the same promoter, in which HipBST has been confirmed by experiments. The toxin HipT and the antitoxin HipS in this TA are similar to the C-terminal and N-terminal of E. coli HipA respectively, and the neutralization mechanism and the substrate of the toxin are different from that of E. coli toxin HipA. This study summarizes the recent discovery of special TA, especially the neutralization mechanism of HipBST which widely exists in prokaryotes.

Cryoelectron microscopy structures of a human neutralizing antibody bound to MERS-CoV spike glycoprotein.

Neutralizing monoclonal antibodies (mAbs) against highly pathogenic coronaviruses represent promising candidates for clinical intervention. Here, we isolated a potent neutralizing monoclonal antibody, MERS-S41, from a yeast displayed scFv library using the S protein as a bait. To uncover the neutralization mechanism, we determined structures of MERS-S41 Fab in complex with the trimeric spike glycoprotein by cryoelectron microscopy (cryo-EM). We observed four distinct classes of the complex structure, which showed that the MERS-S41 Fab bound to the "up" receptor binding domain (RBD) with full saturation and also bound to an accessible partially lifted "down" RBD, providing a structural basis for understanding how mAbs bind to trimeric spike glycoproteins. Structure analysis of the epitope and cell surface staining assays demonstrated that virus entry is blocked predominantly by direct competition with the host receptor, dipeptidyl peptidase-4 (DPP4).

Structure and function of a novel lineage-specific neutralizing epitope on H protein of canine distemper virus.

Canine distemper virus (CDV) infects many sensitive species worldwide and its host range is expanding. The hemagglutinin (H) protein, the major neutralizing target, binds to cellular receptors and subsequently triggers fusion for initial viral infection. So it's necessary to clarify the precise neutralizing epitopes of H protein and extend the knowledge of mechanisms of virus neutralization. In this study, a neutralizing monoclonal antibody (mAb) 2D12 against CDV H protein, which had different reactivity with different CDV strains, was generated and characterized. A series of truncated H proteins were screened to define the minimal linear epitope 238DIEREFD244 recognized by 2D12. Further investigation revealed that the epitope was highly conserved in America-1 vaccine lineage of CDV strains, but different substitutions in the epitope appeared in CDV strains of the other lineages and two substitutions (D238Y and R241G) caused the change of antigenicity. Thus, the epitope represents a novel lineage-specific neutralizing target on H protein of CDV for differentiation of America-1 vaccine lineage and the other lineages of CDV strains. The epitope was identified to localize at the surface of H protein in two different positions in a three-dimensional (3D) structure, but not at the position of the receptor-binding site (RBS), so the mAb 2D12 that recognized the epitope did not inhibit binding of H protein to the receptor. But mAb 2D12 interfered with the H-F interaction for inhibiting membrane fusion, suggesting that the mAb plays key roles for formation of H-F protein oligomeric structure. Our data will contribute to the understanding of the structure, function, and antigenicity of CDV H protein and mechanisms of virus neutralization.

Insights into the Neutralization and DNA Binding of Toxin-Antitoxin System ParESO-CopASO by Structure-Function Studies.

ParESO-CopASO is a new type II toxin-antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParESO severely inhibits cell growth, while CopASO functions as an antitoxin to neutralize ParESO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParESO-CopASO complex that adopted an open V-shaped heterotetramer with the organization of ParESO-(CopASO)2-ParESO. The structure showed that upon ParESO binding, the intrinsically disordered C-terminal domain of CopASO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParESO. Thermodynamics analysis showed the DNA-binding affinity of CopASO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParESO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParESO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParESO-CopASO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.

A New Surface Charge Neutralizing Nano-Adjuvant to Potentiate Polymyxins in Killing Mcr-1 Mediated Drug-Resistant Escherichia coli.

Resistance to polymyxins when treating multidrug-resistant (MDR) Gram-negative bacterial infections limit therapeutic options. Here, we report the synthesis of a nickel (Ni) doped Zinc oxide (NZO) combined with black phosphorus (BP) (NZB) nanocomposite and its synergistic action with polymyxin B (PolB) against polymyxin-resistant Escherichia coli harboring mobilized colistin resistance (mcr-1) gene. NZB and PolB combination therapy expressed a specific and strong synergy against Mcr-1 expressing E. coli cells. The underlying mechanism of the synergy is the charge neutralization of the E. coli cell surface by NZB, resulting in a more feasible incorporation of PolB to E. coli. The synergistic concentration of NZB with PolB was proved biocompatible. Thus, the NZB is the first biocompatible nano-adjuvant to polymyxins against polymyxin-resistant E. coli cells, recognizing the physical status of bacteria instead of known adjuvants targeting cellular gene products. Therefore, NZB has the potential to revive polymyxins as leading last-resort antibiotics to combat polymyxin-resistant Gram-negative bacterial infections.

Type VII Toxin/Antitoxin Classification System for Antitoxins that Enzymatically Neutralize Toxins.

Toxin/antitoxin (TA) systems are present in nearly all bacterial and archaeal strains and consist of a toxin that reduces growth and an antitoxin that masks toxin activity. Currently there are six primary classes for TA systems based on the nature of the antitoxin and the way that the antitoxin inactivates the toxin. Here we show that there now are at least three additional and distinct TA systems in which the antitoxin is an enzyme and the cognate toxin is the direct target of the antitoxin: Hha/TomB (antitoxin oxidizes Cys18 of the toxin), TglT/TakA (antitoxin phosphorylates Ser78 of the toxin), and HepT/MntA (antitoxin adds three AMPs to Tyr104 of the toxin). Thus, we suggest the type VII TA system should be used to designate those TA systems in which the enzyme antitoxin chemically modifies the toxin post-translationally to neutralize it. Defining the type VII TA system using this specific criterion will aid researchers in classifying newly discovered TA systems as well as refine the framework for recognizing the diverse biochemical functions in TA systems.

Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody.

Rabies virus (RABV), the etiological agent for the lethal disease of rabies, is a deadly zoonotic pathogen. The RABV glycoprotein (RABV-G) is a key factor mediating virus entry and the major target of neutralizing antibodies. Here, we report the crystal structures of RABV-G solved in the free form at ∼pH-8.0 and in the complex form with a neutralizing antibody 523-11 at ∼pH-6.5, respectively. RABV-G has three domains, and the basic-to-acidic pH change results in large domain re-orientations and concomitant domain-linker re-constructions, switching it from a bent hairpin conformation into an extended conformation. During such low-pH-induced structural transitions, residues located in the domain-linker are found to play important roles in glycoprotein-mediated membrane fusion. Finally, the antibody interacts with RABV-G mainly through its heavy chain and binds to a bipartite conformational epitope in the viral protein for neutralization. These structures provide valuable information for vaccine and drug design.

Neutralization sites of human papillomavirus-6 relate to virus attachment and entry phase in viral infection.

Human papillomavirus type 6 (HPV6) is the major etiologic agent of genital warts and recurrent respiratory papillomatosis. Although the commercial HPV vaccines cover HPV6, the neutralization sites and mode for HPV6 are poorly understood. Here, we identify the HPV6 neutralization sites and discriminate the inhibition of virus attachment and entry by three potent neutralizing antibodies (nAbs), 5D3, 17D5, and 15F7. Mutagenesis assays showed that these nAbs predominantly target surface loops BC, DE, and FG of HPV6 L1. Cryo-EM structures of the HPV6 pseudovirus (PsV) and its immune complexes revealed three distinct binding modalities - full-occupation-bound to capsid, top-center-bound-, and top-rim-bound to pentamers - and illustrated a structural atlas for three classes of antibody-bound footprints that are located at center-distal ring, center, and center-proximal ring of pentamer surface for 5D3, 17D5, and 15F7, respectively. Two modes of neutralization were identified: mAb 5D3 and 17D5 block HPV PsV from attaching to the extracellular matrix (ECM) and the cell surface, whereas 15F7 allows PsV attachment but prohibits PsV from entering the cell. These findings highlight three neutralization sites of HPV6 L1 and outline two antibody-mediated neutralization mechanisms against HPV6, which will be relevant for HPV virology and antiviral inhibitor design. HighlightsMajor neutralization sites of HPV6 were mapped on the pseudovirus cryo-EM structuremAb 15F7 binds HPV6 capsid with a novel top-rim binding modality and confers a post-attachment neutralizationmAb 17D5 binds capsid in top-centre manner but unexpectedly prevents virus from attachment to cell surface.

Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope.

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

The development of therapeutic antibodies against dengue virus.

Dengue virus, a positive-sense RNA virus, is one of the major human pathogens transmitted by mosquitoes. However, no fully effective licensed dengue vaccines or therapeutics are currently available. Several potent neutralizing antibodies against DENV have been isolated from mice and humans, and the characterization of their properties by biochemical and biophysical methods have revealed important insights for development of therapeutic antibodies. In this review, we summarize recently reported antibody-antigen complex structures, their likely neutralization mechanisms and enhancement propensities, as well as their prophylactic and therapeutic capabilities in mouse models. This article forms part of a symposium on flavivirus drug discovery in the journal Antiviral Research.