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Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV., Previously, a broadly neutralizing antibody, 3I14, active against groups 1 and 2 influenza A viruses was isolated from human memory B cells and showed protection in mice from lethal viral challenge. Here, Harshbarger and Deming et al. provide the crystal structure of 3I14 Fab in complex with H3, H6, and H10.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV− ethanol-fed, or HIV− pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Characterization of the human antibody (Ab) repertoire in mouse models of the human immune system is essential to establish their relevance in translational studies. Single human B cells were sorted from bone marrow and periphery of humanized NOD/SCID γc(null) (hNSG) mice at 8-10 months post engraftment with human cord blood-derived CD34(+) stem cells. Human IG variable heavy (V(H)) and kappa (V(κ)) genes were amplified, cognate V(H)-V(κ) gene-pairs assembled as single-chain variable fragment-Fc Abs (scFvFcs) and functional studies were performed. Although overall distribution of V(H) genes approximated the normal human Ab repertoire, analysis of the V(H)-third complementarity-determining regions in the mature B-cell subset demonstrated an increase in length and positive charges, suggesting autoimmune characteristics. Additionally, >70% of V(κ) sequences utilized V(κ)4-1, a germline gene associated with autoimmunity. The mature B-cell subset-derived scFvFcs displayed the highest frequency of autoreactivity and polyspecificity, suggesting defects in checkpoint control mechanisms. Furthermore, these scFvFcs demonstrated binding to recombinant HIV envelope corroborating previous observations of poly/autoreactivity in anti-HIVgp140 Abs. These data lend support to the hypothesis that anti-HIV broadly neutralizing antibodies may be derived from auto/polyspecific Abs that escaped immune elimination and that the hNSG mouse could provide a new experimental platform for studying the origin of anti-HIV-neutralizing Ab responses.

Background: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). Methodology/Principal Findings: In this study, a xenograft model of CTCL was established and a recombinant adenoassociated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or ‘‘minibody’’) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4 + tumorbearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumorinfiltrating Ly-6G + FccRIIIa(CD16A) + murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4 + tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A + CD56 + NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. Conclusions/Significance: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A + immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

Background Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles. Methodology/Principal Findings Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 106-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. Conclusions/Significance This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

Expression of SH2-Homology-Containing Protein-Tyrosine Phosphatase-1 (Shp-1), a candidate tumor suppressor, is repressed in HTLV-1 transformed lymphocyte cell lines, leukemic cells of Adult T-cell Leukemia (ATL) patients and other hematologic malignancies. However, the mechanisms underlying regulation and repression of Shp-1 remain unclear. Herein, we cloned the putative full-length, hematopoietic cell specific Shp-1 P2 promoter and identified a 277 bp "core" promoter region. In a dose-dependent manner, wild type HTLV-1 Tax and its M22/M47 mutant derivatives profoundly inhibited Shp-1 P2 promoter activity. NF-kB, not CBP or p300, was found to markedly stimulate basal P2 promoter activity and reverse Tax-induced Promoter Silencing (TIPS) in Jurkat cells. Mutagenesis of two NF-kB binding sites in the core promoter region led to only modest inhibition of basal promoter activity but to complete loss of TIPS. Further studies show that TIPS is potentiated by the histone deacetylase-1 (HDAC1) and in co-immunoprecipitation studies NF-kB could compete with HDAC1 for association with Tax protein. In addition, chromatin immunoprecipitation (ChIP) studies showed that Tax recruits HDAC1 to the core promoter and displaces NF-kB binding. We propose that in TIPS, Tax recruits HDAC1 to the Shp-1 P2 promoter which results in deacetylation and dissociation of NF-kB from the promoter leading to attenuation of Shp-1 expression, thus providing a possible first step toward leukemogenesis through its silencing of this key immediate early negative regulator of IL-2 signaling.