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Interleukin 17 (IL-17) and its main producer, T cell receptor γδ cells, have neurotoxic effects in the pathogenesis of intracerebral hemorrhage (ICH), aggravating brain injuries. To investigate the correlation between IL-17 and ICH, we dynamically screened serum IL-17 concentrations using enzyme-linked immunosorbent assay and explored the clinical values of IL-17 in ICH patients. There was a significant negative correlation between serum IL-17 level and neurological recovery status in ICH patients (r = -0.498, P < 0.01). To study the neurotoxic role of IL-17, C57BL/6 mice were used to establish an ICH model by injecting autologous blood into the caudate nucleus. Subsequently, the mice were treated with mouse neural stem cells (NSCs) and/or IL-17 neutralizing antibody for 72 hours. Flow cytometry, brain water content detection, Nissl staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling results indicated that NSC transplantation significantly reduced IL-17 expression in peri-hematoma tissue, but there was no difference in T cell receptor γδ cells. Compared with the ICH group, there were fewer apoptotic bodies and more Nissl bodies in the ICH + NSC group and the ICH + NSC + IL-17 group. To investigate the potential effect of IL-17 on directional differentiation of NSCs, we cultured mouse NSCs (NE-4C) alone or co-cultured them with T cell receptor γδ cells, which were isolated from mouse peripheral blood mononuclear cells, for 7 days. The results of western blot assays revealed that IL-17 secreted by T cell receptor γδ cells reduced the differentiation of NSCs into astrocytes and neurons, while IL-17 neutralization relieved the inhibition of directional differentiation into astrocytes rather than neurons. In conclusion, serum IL-17 levels were elevated in the early stage of ICH and were negatively correlated with outcome in ICH patients. Animal experiments and cytological investigations therefore demonstrated that IL-17 probably has neurotoxic roles in ICH because of its inhibitory effects on the directional differentiation of NSCs. The application of IL-17 neutralizing antibody may promote the directional differentiation of NSCs into astrocytes. This study was approved by the Clinical Research Ethics Committee of Anhui Medical University of China (For human study: Approval No. 20170135) in December 2016. All animal handling and experimentation were reviewed and approved by the Institutional Animal Care and Use Committee of Anhui Medical University (approval No. 20180248) in December 2017.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1β, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as “quasi-enveloped” particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2S results from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2S secretion via the secretory pathway. Unlike ORF2C, ORF2S is glycosylated and exists as a dimer. Nonetheless, ORF2S exhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

Anthropogenic transmission of SARS-CoV-2 to pet cats highlights the importance of monitoring felids for exposure to circulating variants. We tested cats in the United Kingdom for SARS-CoV-2 antibodies; seroprevalence peaked during September 2021-February 2022. The variant-specific response in cats trailed circulating variants in humans, indicating multiple human-to-cat transmissions over a prolonged period.

Broadly neutralizing antibodies (bNAbs) can prevent a new HIV-1 infection and can at least temporarily suppress an established infection. However, antibody-resistant viruses rapidly emerge in infected persons treated with any single bNAb. Several bispecific antibodies have been developed to increase the breadth of these antibodies, but typically only one arm of these bispecific constructs binds the HIV-1 envelope glycoprotein trimer (Env). Here, we develop and characterize bispecific constructs based on well-characterized V2-glycan and V3-glycan bNAbs and show that at least one member of this class is more potent than its parental antibodies, indicating that they can simultaneously bind both of these epitopes of a single Env trimer. These data show that bispecific antibody-like proteins can achieve greater neutralization potency than the bNAbs from which they were derived. Broadly neutralizing antibodies (bNAbs) can prevent and control an HIV-1 infection, but their breadth is invariably too limited for use as monotherapy. To address this problem, bi- and trispecific antibody-like constructs have been developed. These engineered antibodies typically have greater breadth than the native bNAbs from which they were derived, but they are not more potent because they do not, in most cases, simultaneously engage more than a single epitope of the HIV-1 envelope glycoprotein (Env). Here, we describe a new class of bispecific antibodies targeting the V2-glycan (apex) and V3-glycan regions of the HIV-1 envelope glycoprotein (Env). Specifically, bispecific antibodies with a single-chain (scFv) form of the CAP256.VRC26.25 V2-glycan (apex) antibody on one antibody arm and a full V3-glycan Fab on the other arm neutralizes more HIV-1 isolates than the bNAbs from which they were derived. Moreover, these bispecific antibodies are markedly more potent than their parental bNAbs, likely because they simultaneously engage both the apex and V3-glycan epitopes of Env. Our data show that simultaneous engagement of two critical epitopes of a single Env trimer can markedly increase the potency of a bispecific antibody. IMPORTANCE Broadly neutralizing antibodies (bNAbs) can prevent a new HIV-1 infection and can at least temporarily suppress an established infection. However, antibody-resistant viruses rapidly emerge in infected persons treated with any single bNAb. Several bispecific antibodies have been developed to increase the breadth of these antibodies, but typically only one arm of these bispecific constructs binds the HIV-1 envelope glycoprotein trimer (Env). Here, we develop and characterize bispecific constructs based on well-characterized V2-glycan and V3-glycan bNAbs and show that at least one member of this class is more potent than its parental antibodies, indicating that they can simultaneously bind both of these epitopes of a single Env trimer. These data show that bispecific antibody-like proteins can achieve greater neutralization potency than the bNAbs from which they were derived.

Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.

•Neutralization antibody were measured against SARS-CoV-2 variants after vaccination.•Low neutralization antibody titer was observed against beta and delta variants.•Neutralization antibody titer against R.1 lineage was equivalent to original Wuhan.•The weakened humoral response might not be caused solely by the E484K mutation. The reduced vaccine efficacy against the SARS-CoV-2 variant lineage B. 1.351 (beta variant) containing the E484K and N501Y mutations is well known. The E484K mutation in SARS-CoV-2 is thought to be responsible for weakened humoral immunity. Vaccine efficacy against the R.1 lineage, which contains the E484K mutation but not the N501Y mutation, is uncertain. Serum samples were collected from 100 healthy Japanese participants three weeks after receiving the second dose of the BNT162b2 vaccine, and serum neutralization antibody titers were measured against five SARS-CoV-2 variants. The geometric mean neutralization titers measured for the original and R.1 lineages were equivalent (91.90 ± 2.40 and 102.67 ± 2.28, respectively), whereas a low titer was measured for the beta variant (18.03 ± 1.92). Although further investigations with other variant strains and serum samples are essential, our results imply that the weakened humoral response is not caused solely by the E484K mutation. (UMIN000043340).

Striking number of mutations found in the spike protein of recently emerged SARS-CoV-2 Omicron subvariants BA.1, BA.2, BA.3 and BA.4/5 has raised serious concerns regarding the escape from current antibody therapies and vaccine protection. Here, we conducted comprehensive analysis on the extent of two major Omicron lineages BA.1/BA.1.1 and BA.2 to escape neutralization from the therapeutic antibodies approved by the regulatory authorities and convalescent plasma from SARS-CoV-2 patients infected during initial wave of pandemic in early 2020. We showed that Omicron BA.1/BA.1.1 were the most resistant in both magnitude and breadth against antibodies and convalescent plasma, followed by Beta, BA.2, Gamma, Delta and Alpha. While the majority of therapeutic antibodies lost binding and neutralization to Omicron variants, BRII combo (BRII-196 + BRII-198), S309, and AZ combo (COV2-2196 + COV2-2130) maintained neutralization despite of reduction due to either conserved epitope or combinational effect between the two designated antibodies. A single intraperitoneal injection of BRII combo as a prophylactic treatment protected animals from Omicron infection. Treated animals manifested normal body weight, survived infection up to 14 days, undetectable levels of infectious viruses in the lungs, and reduced lung pathology compared to the controls. Analyzing ACE2 from diverse host species showed that Omicron variants acquired ability to use mouse ACE2 for entry. These results demonstrate major antigenic shifts and potentially broadening the host range of two major Omicron lineages BA.1/BA.1.1 and BA.2, posing serious challenges to current antibody therapies and vaccine protection as well as increasing danger of spillover into the wildlife.

Evolution in blockade antibody epitopes in human norovirus GII.4 2015 strains resulted in virus with antigenicity and ligand-binding properties that are significantly distinct from those for GII.4 2012 strains, potentially accounting for the endemic spread of this virus. Abstract Background Human noroviruses are the leading cause of acute gastroenteritis. Strains of the GII.4 genotype cause pandemic waves associated with viral evolution and subsequent antigenic drift and ligand-binding modulation. In November 2015, a novel GII.4 Sydney recombinant variant (GII.P16-GII.4 Sydney) emerged and replaced GII.Pe-GII.4 Sydney as the predominant cause of acute gastroenteritis in the 2016–2017 season in the United States. Methods Virus-like particles of GII.4 2012 and GII.4 2015 were compared for ligand binding and antibody reactivity, using a surrogate neutralization assay. Results Residue changes in the capsid between GII.4 2012 and GII.4 2015 decreased the potency of human polyclonal sera and monoclonal antibodies. A change in epitope A resulted in the complete loss of reactivity of a class of blockade antibodies and reduced levels of a second antibody class. Epitope D changes modulated monoclonal antibody potency and ligand-binding patterns. Conclusions Substitutions in blockade antibody epitopes between GII.4 2012 and GII.4 2015 influenced antigenicity and ligand-binding properties. Although the impact of polymerases on fitness remains uncertain, antigenic variation resulting in decreased potency of antibodies to epitope A, coupled with altered ligand binding, likely contributed significantly to the spread of GII.4 2015 and its replacement of GII.4 2012 as the predominant norovirus outbreak strain.