Development of transformed lactobacilli as a microbicide

转化乳酸杆菌作为杀菌剂的开发

• 批准号: 7800351
• 负责人: Richard B. Markham
• 金额: $ 24.64万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-07 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800351
• 关键词: Address; Adverse effects; Affinity; Alpaca; Anti-Retroviral Agents; Antibiotics; Antibodies; Appearance; Bacteria; Bacterial Chromosomes; Binding; Biological Assay; Biological Models; Biological Preservation; Blood group antibody D; Bone Marrow; Bone Marrow Stem Cell; CD34 gene; Cell Adhesion Molecules; Cells; Clinical; Coitus; Collaborations; Detergents; Development; Dissociation; Effectiveness; Engineering; Epithelium; Epitopes; Face; Failure; Female; Fetal Liver; Frequencies; Generations; Genetic Variation; Genital system; Genitourinary system; Glycoproteins; Growth; HIV-1; HIV-1 vaccine; Heel; Herpes Simplex Infections; Human; Human Herpesvirus 2; ITGB2 gene; Immunoglobulin Variable Region; In Situ; In Vitro; Inbred BALB C Mice; Infection; Infection prevention; Inflammatory; Integrin beta Chains; Integrins; Intercellular adhesion molecule 1; International; Laboratories; Lactobacillus; Lactobacillus casei rhamnosus; Libraries; Life Cycle Stages; Ligands; Liquid substance; Liver; Lymphocyte Function-Associated Antigen-1; Macaca; Measures; Methods; Modeling; Movement; Mus; Mutation; NOD/SCID mouse; Non obese; Nonoxynol 9; Organism; Peripheral Blood Mononuclear Cell; Phage Display; Phase III Clinical Trials; Plague; Plasmids; Play; Production; Progesterone; Prophylactic treatment; Research Personnel; Resistance; Role; SCID Mice; Seminal Plasma; Series; Serum; Sexually Transmitted Diseases; Specificity; Streptococcus gordonii; System; Techniques; Technology; Tertiary Protein Structure; Testing; Therapeutic; Thymus Gland; Toxic effect; Transplantation; Urinary tract; Vagina; Vaginal Douching; Viral; Viral Proteins; Virulent; Virus; Virus Receptors; Woman; base; cellulose sulfate; design; diabetic; fetal; glycoprotein D-herpes simplex virus type 2; in vitro Model; in vivo Model; microbicide; mouse model; prevent; receptor; rectal; small molecule; tool; transmission process; Address; Adverse effects; Affinity; Alpaca; Anti-Retroviral Agents; Antibiotics; Antibodies; Appearance; Bacteria; Bacterial Chromosomes; Binding; Biological Assay; Biological Models; Biological Preservation; Blood group antibody D; Bone Marrow; Bone Marrow Stem Cell; CD34 gene; Cell Adhesion Molecules; Cells; Clinical; Coitus; Collaborations; Detergents; Development; Dissociation; Effectiveness; Engineering; Epithelium; Epitopes; Face; Failure; Female; Fetal Liver; Frequencies; Generations; Genetic Variation; Genital system; Genitourinary system; Glycoproteins; Growth; HIV-1; HIV-1 vaccine; Heel; Herpes Simplex Infections; Human; Human Herpesvirus 2; ITGB2 gene; Immunoglobulin Variable Region; In Situ; In Vitro; Inbred BALB C Mice; Infection; Infection prevention; Inflammatory; Integrin beta Chains; Integrins; Intercellular adhesion molecule 1; International; Laboratories; Lactobacillus; Lactobacillus casei rhamnosus; Libraries; Life Cycle Stages; Ligands; Liquid substance; Liver; Lymphocyte Function-Associated Antigen-1; Macaca; Measures; Methods; Modeling; Movement; Mus; Mutation; NOD/SCID mouse; Non obese; Nonoxynol 9; Organism; Peripheral Blood Mononuclear Cell; Phage Display; Phase III Clinical Trials; Plague; Plasmids; Play; Production; Progesterone; Prophylactic treatment; Research Personnel; Resistance; Role; SCID Mice; Seminal Plasma; Series; Serum; Sexually Transmitted Diseases; Specificity; Streptococcus gordonii; System; Techniques; Technology; Tertiary Protein Structure; Testing; Therapeutic; Thymus Gland; Toxic effect; Transplantation; Urinary tract; Vagina; Vaginal Douching; Viral; Viral Proteins; Virulent; Virus; Virus Receptors; Woman; base; cellulose sulfate; design; diabetic; fetal; glycoprotein D-herpes simplex virus type 2; in vitro Model; in vivo Model; microbicide; mouse model; prevent; receptor; rectal; small molecule; tool; transmission process

DESCRIPTION (provided by applicant): The major hypothesis of this proposal is that bacteria capable of sustained vaginal colonization of a broad cross-section of women can be engineered to produce alpaca- derived beta integrin-specific variable region antibodies (VHH) that will inhibit infection with HIV-1. This approach to preventing HIV-1 transmission offers the unique advantage of targeting receptor-ligand interactions that are completely independent of mutable viral proteins. A similar delivery system targeting Glycoprotein D of herpes simplex type 2 will also prevent infection with that virus, which enhances transmission of HIV-1 infection. This hypothesis will be pursued by demonstration of the efficacy of bacterially-delivered VHH using three different mouse model systems and two different bacterial strains, one of which, Streptococcus gordonii, is selected specifically for the purpose of demonstrating proof of principle in mouse model systems that require pre-treatment of mice with progesterone. Lactobacillus rhamnosus GR-1 will be used in a mouse model that does not require progesterone pre-treatment, as this strain of lactobacillus has been demonstrated to be effective in colonizing a broad cross-section of women. Lactobacilli are appealing as a microbicide vehicle because they are "generally regarded as safe" organisms by the FDA, constitute the primary bacterial component of the normal flora of the female genitourinary tract, intrinsically inhibit the growth of more virulent bacteria, would be transparent to users, and would allow dissociation of microbicide application from coitus. Alpaca-derived antibody binding domains (VHH) are developed from a class of camelid antibodies consisting solely of heavy chains and the binding determinants of which are single protein domains that are much smaller than any which might be generated from classical antibodies. Using this technology we will: 1) Raise antibodies in alpacas against the ectodomain of CD18, the beta-chain component of the CD11a/CD18 heterodimer that is LFA-1. From circulating PBMC of the immunized alpacas we will generate a library of VHH screened by phage display for binding affinity. Those VHH will then be expressed from plasmids and/or the bacterial chromosome of Lactobacillus rhamnosus GR-1 and Streptococcus gordonii. 2) Using the methods described in Aim 1 generate VHH targeting HSV-2 glycoprotein D. 3) Evaluate the potential toxicity of anti-CD18 and anti-GpD by measuring transepithelial resistance in two different in vitro model systems. 4) Test the ability of the CD18 and GpD-specific VHH to block transmission or neutralize infection in transwell or in vitro neutralization assays. 5) Evaluate colonization of the mouse vagina and secretion of CD18-specific VHH by transformed S. gordonii in progesterone-treated Hu-PBL-SCID mice and by L. rhamnosus in non-progesterone-treated humanized bone marrow liver thymus NOD- SCID (BLT) mice. Evaluate the ability of colonized mice to resist infection with cell- associated and cell-free HIV-1. 6) Evaluate colonization of the mouse vagina and production of Glycoprotein D-specific VHH by transformed S. gordonii progesterone- treated BALB/c mice. Evaluate in these mice the appearance of anti-Glycoprotein D and anti-CD18 VHH antibodies in vaginal lavage fluid and serum and examine vaginal lavage fluid for the appearance of inflammatory cells induced by the VHH. The passive protective ability of in situ-produced HSV-2 specific VHH will also be assayed in this mouse vaginal challenge model. and 7) If dictated by results of the previous studies, develop a plasmid-based expression system that does not depend on antibiotic selection. At the completion of these studies, we will have proved the potential efficacy of this approach using in vivo model systems and will have developed the VHH constructs that could be used in the clinical setting as well as established their production from a lactobacillus species that also may be applicable to the clinical setting.

描述（由申请人提供）：该提案的主要假设是，可以设计出能够持续的妇女阴道殖民化的细菌，可以设计出可产生脂肪蛋白整合蛋白特异性可变区域抗体（VHH）的细菌，这些抗体会抑制HIV-1的感染。这种防止HIV-1传播的方法为靶向受体配体相互作用完全独立于可突变的病毒蛋白提供了独特的优势。针对单纯疱疹2类糖蛋白D的类似递送系统也将防止感染该病毒，从而增强HIV-1感染的传播。该假设将通过使用三种不同的小鼠模型系统和两个不同的细菌菌株证明细菌递送VHH的功效来提出这一假设，其中一种是为了证明需要在小鼠模型系统中证明其原理的证明，而小鼠模型系统中的原理证明。鼠尾草乳杆菌GR-1将用于不需要孕激素预处理的小鼠模型中，因为这种乳酸杆菌的菌株已被证明可以有效地定居于广泛的女性横截面。乳酸乳杆菌作为一种杀菌剂具有吸引力，因为它们通常被FDA“被视为安全”的生物，这构成了女性泌尿生殖道正常植物的主要细菌成分，本质上抑制了更具毒性细菌的生长，对使用者的使用态度固定，并且会允许从COITUS的Microbobicies Opplycipiation。羊驼源性抗体结合结构域（VHH）是由一类骆驼抗体开发的，这些抗体仅由重链组成，其结合决定因素是单个蛋白质结构域，它们比从经典抗体产生的任何抗体小得多。使用此技术我们将：1）在羊驼中对CD18的外生域（CD11A/CD18异二聚二聚体的β链成分）提出抗体。从免疫羊驼的循环PBMC中，我们将生成一个通过噬菌体显示筛选的VHH库，以进行结合亲和力。然后，这些VHH将从质粒和/或rhamnosus gr-1和Gordonii链球菌的细菌染色体中表达。 2）使用AIM 1中描述的方法生成靶向HSV-2糖蛋白D的VHHD。3）通过测量在两种不同的体外模型系统中测量transepithelial耐药性，评估抗CD18和抗GPD的潜在毒性。 4）测试CD18和GPD特异性VHH阻止传播或中和感染的能力。 5）评估孕激素治疗的HU-PBL-SCID小鼠中转化的小肠链球菌对小鼠阴道的定殖和CD18特异性VHH的分泌，以及在未经培养基酮的人性化骨髓乳毛乳腺肝乳腺癌（BLT）中，在未经培养基酮治疗的人性化骨骨髓乳mi骨中，鼠李乳杆菌在非乳腺癌中的分泌。评估定植小鼠抵抗细胞相关和无细胞HIV-1感染的能力。 6）评估小鼠阴道的定殖和通过转化后的Gordonii孕酮治疗的BALB/C小鼠的糖蛋白D特异性VHH的定植。在这些小鼠中评估抗糖蛋白D和抗CD18 VHH抗体在阴道灌洗液和血清中的出现，并检查阴道灌洗液，以表现出VHH诱导的炎性细胞的出现。该小鼠阴道挑战模型也将测定原位生产的HSV-2特异性VHH的被动保护能力。 7）如果由先前研究的结果决定，则开发不依赖抗生素选择的基于质粒的表达系统。这些研究完成后，我们将使用体内模型系统证明这种方法的潜在功效，并将开发可在临床环境中使用的VHH构建体，并从乳杆菌物种中确定其生产，这些物种也可能适用于临床环境。