Anti-gp120 Minibody Gene Transfer to Female Genital Epithelial Cells Protects against HIV-1 Virus Challenge In Vitro

Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb) that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV)-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc), or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal) in an organotypic human vaginal epithelial cell (VEC) model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles

Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

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 adeno-associated 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+ tumor-bearing 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 tumor-infiltrating Ly-6G+ FcγRIIIa(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

Binding and immunogenicity of MHC class-I specific peptides and glycopeptides

Short synthetic MHC class I (MHC-I) Db and Kb binding peptides were found to be strongly immunogenic and capable of inducing virus-specific cytotoxic Tlymphocyte (CTL) responses. Restimulation of primed splenocytes with a low peptide concentration generated optimal CTL responses. CTL responses were found to correlate with the capacity of peptides to bind to MHC class I measured as MHC-I upregulation (I-II). Earlier studies using 15-16 mer peptides have demonstrated that CD4+ helperT-cells were required to induce optimal CTL responses. We investigated if optimal-length peptides still required CD4+ T-cell help to generate CTL. The results clearly established that activation of CTL responses by short optimal peptides did not require CD4+ T-cell help (III). MHC-I heavy chains can appear on the cell surface in different configurations, including forms which are designated as functionally "empty". Such empty MHC-I molecules can directly bind synthetic peptides having the correct motifs. A method to measure the fraction of "empty " Db molecules on different cells was established by using monoclonal antibodies, staining either for Db-bound glycopeptides and for Db itself (IV). This method has been used to measure "empty" MHC-I molecules on cells from TAP and b2-M gene targeted mice as well as TAP1/b2-M double mutant mice. The influence of pH on glycopeptide binding to MHC-I molecules was also investigated (V). MHC-I chains are known to internalize and recycle, to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-Ibound peptides also can recycle was not known. We have investigated this bytwo different approaches. First, glycopeptide recycling was tested with Gal2specific monoclonal antibodies. By flow cytometry, a return of Gal2 epitopesto the surface was found, after cellular internalization and cell surface clearanceby pronase treatment (VI). Recycling may be important for MHC-I processingof exogenous antigens. Second, this was tested using two different forms ofthe H-2Db molecule expressed in transgenic mice, either transmembraneous (Db-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (Db-GPI). Only the tm form of Db was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus and tested for CTL responses against an immunodominant nucleoproteinepitope, only Db-tm mice were found to generate specific CTL responses (VII). We have found that some Kb binding glycopeptides generated CTL which could recognize the carbohydrate present in a glycolipid form, in an MHC-I unrestricted way. An interesting finding in the present investigation was that glycopeptide and glycolipid specific CTL were found to use different T cell receptors (TCR:s) to recognize and kill target cells (VIII)

Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases

Neuropathy of type 1 diabetes in the Arab world: A systematic review and meta-analysis

Abstract AimsAlthough type 1 diabetes (T1D) is a common disease in the Arab nations, there is no data available on the prevalence of peripheral neuropathy (PN) among T1D subjects in Arab countries. The aim of this study is to analyze the prevalence of PN in T1D subjects via published literature and to draw attention to the dearth of the published work in this serious complication of T1D. MethodsA meta-analysis was performed on studies representing different Arab countries with a total number of 2243 T1D subjects. ResultsThe pooled prevalence of PN among T1D subjects in the Arab region was estimated as 18% with 95% confidence intervals (CI): 0.09–0.34. The PN prevalence was significantly higher in the >16-yr age group, with 59.1% (95% CI: 0.45–0.72) compared to 9.5% (95% CI: 0.05–0.19) in the 16years) with T1D are affected more with PN than younger age Arab people (<16years). PN is more frequently present in Arab subjects with a longer duration of T1D diabetes than in those with shorter duration.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector

Immunogenicity of Influenza Virus Vaccine Is Increased by Anti-Gal-Mediated Targeting to Antigen-Presenting Cells▿

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing α-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fcγ receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in α1,3galactosyltransferase (α1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of α-Gal epitopes on carbohydrate chains of PR8 virus (PR8αgal) was catalyzed by recombinant α1,3GT, the glycosylation enzyme that synthesizes α-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8αgal displayed much higher numbers of PR8-specific CD8+ and CD4+ T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking α-Gal epitopes. Mice immunized with PR8αgal also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines

Retinopathy of Type 1 Diabetes in Arab Countries: Systematic Review and Meta-Analysis.

To conduct a systematic review and meta-analysis of retinopathy prevalence in patients with type 1 diabetes (T1D) in 22 Arab countries. We systematically searched 4 different literature databases (PubMed, Science Direct, Web of Science and Embase), from the date of inception until December 2017, to collect all the information about patients with T1D who developed retinopathy complications; for statistical analysis, we used MetaXL to evaluate the pooled prevalence estimate and the subgroup prevalence estimates employing double arcsine transformation and inverse variance heterogeneity models. Our search strategy returned 475 studies, of which 39 met our inclusion criteria; of those, 16 were eligible for meta-analysis that were captured only in 15 Arab countries, through 45 years (1969-2014). The number of retinopathy patients was 396 out of 1,931 patients with T1D. The prevalence of retinopathy was 19% (95% CI 10-28%). Substantial heterogeneity was observed (Q 240.78, p < 0.0001, I2 93.77%, 95% CI 91.35-95.52%); however, no single study considerably affected the overall pooled prevalence estimate. Almost one fifth of T1D patients in 15 Arab countries have diabetic retinopathy, therefore it is important to improve the care of patients with T1D and in Arab countries to avoid the development of such a devastating complication

Major histocompatibility complex class I binding glycopeptides for the estimation of 'empty' class I molecules

Different forms of major histocompatibility complex (MHC) class I heavy chains are known to be expressed on the cell surface, including molecules which are functionally 'empty'. Direct peptide binding to cells is obvious during sensitization of target cells in vitro for cytotoxic T lymphocyte killing and 'empty' MHC-I molecules are comparatively abundant on TAP- 1 2 peptide transporter mutant cells. In the present work we have estimated the fraction of 'empty' MHC class I molecules using glycosylated peptides and cellular staining with carbohydrate specific monoclonal antibodies. Synthetic Db and Kb binding peptides were coupled at different positions with different di- or trisaccharides, using different spacing between the carbohydrate and the peptide backbone. Binding of sugar specific mAbs was compared in ELISA and cellular assays. An optimal Db binding glycopeptide was used for comparative staining with anti-Db and anti-carbohydrate monoclonal antibodies to estimate fractions of 'empty' molecules on different T lymphoid cells. On activated normal T cells, a large fraction of Db molecules were found to be 'empty'. The functional cole of such 'empty' MHC class I molecules on T cells is presently unclear. However, on antigen presenting cells they might participate in the antigen presentation process