Functional Assays and Disease Models

The third module of the MIMIC® platform is the Functional Assay or Disease Model module. This module measures the effect of a vaccine on the immune response to an infection or an infectious disease. Thus, one could measure the effectiveness of a vaccine candidate to neutralize or clear a pathogen. The module uses standard or proprietary functional assays to help define correlates of protection, or engineered tissue constructs that directly measure the impact of the immune response on the pathogen. This module enables the “Clinical Trial in a Test Tube”™ application of the MIMIC® technology. To date, this module has been used for seasonal and pandemic influenza, Ebola, Marburg, Yellow Fever, Venezuelan equine encephalitis virus, and latent tuberculosis (Mtb) as model diseases.

An example of functional assays is the Hemagglutination inhibition (HAI) assay for influenza. We used a a sensitive version of an HAI functional assay to measure hemagglutinin inhibition (a standard measure of protective response to influenza) in a MIMIC® study of influenza vaccine. We stimulated the dendritic cells with a commercially available seasonal influenza vaccine (Fluvirin®). The stimulated dendritic cells were transferred to the Lymphoid Tissue Equivalent module. The LTE module was exposed to the vaccine. We collected the LTE fluid (supernatant) and tested for the presence of antibodies that would prevent influenza infection ("neutralizing antibodies") using a hemagglutinin inhibition (HAI) assay. In this study, the antibodies by the MIMIC® System showed detectable levels of HAI antibodies especially following vaccination. A boost in HAI titer is expected following vaccination, thus the MIMIC® results are consistent with in vivo findings.

Bar chart showing pre and post FV challenge titer among 19 donors, SA_HAI for MIMIC_3 with Brisbane Virus

In addition to standard functional assays, VaxDesign has developed proprietary functional assays that have advantages in cost, time, and sensitivity.

One of our proprietary functional assays is a surface-assisted HAI (SA-HAI) assay that resembles the traditional HAI assay in spirit, i.e., the capacity of specific antibody to prevent attachment of the virus to the erythrocytes provides a measure of its protective efficacy, but the mechanisms of interaction in the revised assay are quite unique at the molecular level. By visual inspection, the readout of the SA-HAI very much resembles a traditional HAI assay, with the formation of halos and buttons that can be registered by eye or short-focus camera. This robust and simple assay strongly correlates with the traditional HAI assay. The SA-HAI assay works with avian, human, and guinea pig erythrocyte populations and, unlike the traditional HAI assay, works with influenza B, H5, and H7 strains with high sensitivity. Finally, the SA-HAI assay has 20 times greater sensitivity than the traditional HAI assay. This superior sensitivity might enable us to discern various cross-reactivity and cross-blocking relationships that are not apparent in the conventional assay.

We have further improved the HAI assay with a proprietary image processing platform that reads out the HAI titer with greater precision and eliminates subjectivity introduced by the researcher in the evaluation process. The platform includes a software graphical user interface to facilitate loading, viewing, and processing HA and HAI assay well images.

Additionally, we have developed other assays that can be used to evaluate the functional efficacy of specific antibodies in MIMIC® supernatants and donor sera. Such assays include highly sensitive cell-based fluorescent microneutralization (fmNt) and fluorescent adherence inhibition (fADI) assays that evaluate the ability of antibodies to inhibit the interaction of inactivated viruses with various target cells.

Although functional correlates of protection, like HAI assays, are known for some pathogens, many others have none. In these more difficult cases, researchers have to measure the progress of an infection more directly by looking at viral or bacterial counts or whether a pathogen enters a cell to cause infection. VaxDesign develops Disease Model modules to examine the effect of the immune response directly to a pathogen, which determines the effectiveness of the response. In addition, Disease Model modules may enable researchers to dissect the immune response and find correlates of protection. Often correlates of protection are easier to measure than direct effects on pathogens.

As an example, VaxDesign in collaboration with the University of Central Florida has developed a novel Disease Model module for latent tuberculosis (TB) in humans. TB infects macrophages in the lung to form granulomatous structures. Granulomas are hallmark of tuberculosis infection and are considered an equilibrium condition between host and pathogen. Formation of granulomas is a protective response by the host to prevent spread of infection to healthy tissue. TB is said to go into a dormant state in granulomas until resuscitated by a suppressed immune system as in AIDS patients and elderly subjects.

The TB Disease Model module recapitulates the formation of granulomas in the lung with an in vitro engineered tissue construct. The construct uses PBMC cells from healthy human donors with different ratios of TB in an extracellular matrix. Uninfected macrophages do not show granuloma formation; infected macrophages induce granuloma formation. The TB model is a close analog to many biodefense infectious disease such as VEEV, burkholderia, Junin, tularemia, and yersinia pestis.

Disease Model module - Granuloma Formation

TB induced granuloma formation in three dimensional extracellular matrix. Left (200× magnification): Macrophages infected with TB. Right (100× magnification): Uninfected macrophages under same experimental conditions do not show granuloma formation.

VaxDesign can create other disease modules for the MIMIC® platform, as new commercial applications are developed for other diseases. We plan to develop disease models for HIV and VEEV, among others.