Sanofi Pasteur Vaxdesign Corporation

Massachusetts Institute of Technology

The Scripps Research Institute

Mount Sinai School of Medicine

Vaxdesign Corporation

Virginia Commonwealth University

Whitehead Institute for Biomedical Research

Whitehead Institute

Sanofi Pasteur Vaxdesign Corp. Et Al.

New York University

Anatoly Kachurin

William L Warren

Russell Higbee

Heather Fahlenkamp

Guzman Sanchez-Schmitz

Conan Li

Donald Drake

Robert Parkhill

Mike Nguyen

Darrell J Irvine

Eric Mishkin

John G Tew

Gwendalyn J Randolph

Mohey Eldin M El Shikh

Luis Mosquera

Selva Sukumar

Bruce Torbett

Vaughan Wittman

Tenekua Tapia

Olga Kachurina

Nir Hacohen

Michael N Nguyen

Nir Harcohen

David Moe

Ayesha Mahmood

Alexander Karol

Vipra Dhir

Conan K N Li

G Sanchez

The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the normal tissues that interact with vaccines in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interaction with the immune system.

Artificial immune system: methods of use

The present invention comprises artificial tissue constructs that serve as in vitro models of mammalian lung tissue. The artificial tissue constructs of the present invention comprise functionally equivalent in vitro tissue scaffolds that enable immunophysiological function of the lung. The constructs can serve as novel platforms for the study of lung diseases (e.g., interstitial lung diseases, fibrosis, influenza, RSV) as well as smoke- and smoking-related diseases. The artificial tissue constructs of the present invention comprise the two components of alveolar tissue, epithelial and endothelial cell layers.

Artificial tissue constructs comprising alveolar cells and methods for using the same

The present invention comprises rugged, inexpensive, reliable, and sensitive laboratory assays of antibody-based viral neutralization activity and antibody-based viral adherence inhibition activity. The assays use inactivated, fluorescently-labeled virus, allowing the tests to be performed without extensive safety precautions. The interaction of the labeled virus with target cells is monitored using flow cytometric methods. A preferred embodiment uses simple and inexpensive flow cytometry methodologies and equipment, such as bead array readers used as simplified flow cytometers. The assays are rapid, taking no longer than a few hours and are readily conducted by a trained technician. The assays are sensitive because they use labeled viruses at low concentrations and determine neutralizing and blocking capacity of sera and antibody at low concentrations. The methods are appropriate for high-throughput screening of large panels of samples.

Fluorescent neutralization and adherence inhibition assays

Hemagglutination (HA) and hemagglutination inhibition (HAI) functional assays remain important instruments of analysis of virus-cell interaction and protecting efficacy of virus-specific antibodies and sera. However, they demonstrate limited sensitivity towards many viruses, and require significant volumes of viruses, erythrocytes, sera, and antibodies. The present invention comprises new and significantly more sensitive versions of the HA and HAI assays based on observing agglutination on activated surfaces of specifically opsonized plates and ELISA plates rather than in solution. A version of the new assay that uses ELISA plates additionally allows characterizing the affinity of functional antibodies in the tested sera and fluids, which is not possible in the classical HAI assay. The methods of the present invention can also be used to improve the sensitivity of agglutination methods based on latex beads and to develop agglutination methods using target cells other than erythrocytes.

Surface-assisted hemagglutination and hemagglutination inhibition assays

The present invention incorporates germinal centers (GCs) into three-dimensional (3D) engineered tissue constructs (ETCs). In an embodiment, we have incorporated the GC in the design of an artificial immune system (AIS) to examine immune responses to vaccines and other compounds. Development of an in vitro GC adds functionality to an AIS, in that it enables generation of an in vitro human humoral response by human B lymphocytes that is accurate and reproducible, without using human subjects. The invention also permits evaluation of, for example, vaccines, allergens, and immunogens, and activation of human B cells specific for a given antigen, which can then be used to generate human antibodies. In an embodiment of the present invention the function of the in vitro GC is enhanced by placing FDCs and other immune cells in a 3D ETC; FDCs appear more effective over a longer time (antibody production is sustained for up to about 14 days.

Methods for assaying responses to vaccines

The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the normal tissues that interact with vaccines in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interactions with the immune system.

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Orlando, FL, United States of America

Anatoly Kachurin