1a.Objectives (from AD-416):
This collaborative research project seeks to analyze the events and cell type requirements leading to the production of Foot-and-Mouth Disease Virus (FMDV)-specific bovine immunoglobulins in vitro. Antibody production in vitro will be quantified and characterized by virus seroneutralizing, ELISA and ELISPOT to evaluate the requirements necessary to induce adaptive responses after FMDV infection in the bovine model.
Specific objectives include:
1. Detect and characterize the in vitro production of FMDV-specific antibodies in the presence of different immune cell types.
2. Study the role of the different immune cell types in the onset of the humoral adaptive responses against FMDV.
3. Assess the thymus independent characteristic of the elicited antibody response by using a multi-meric protein containing 40 copies of a critical Foot-and-Mouth Disease Virus (FMDV) neutralizing epitope.
1b.Approach (from AD-416):
1. Bovine immune cells will be cultured with live or inactivated viruses. FMDV-specific antibody production will be quantified and characterized from the culture supernatants and FMDV-specific antibody secreting cells will also be determined. Cell cultures will be sorted for specific surface cell markers to determine FMDV-specific antibodies.
2. The levels of interferon will be measured to determine antibody concentrations. Specific antibody detection will be performed to quantify the amount of stimulated cells and measure the magnitue of response multiple bond connection strenght. Activation markers will be detected through flow cytometery and to determine activation methodologies.
3. A recombinant antigen comprising 40 tandem copies (a multi-peptide construct) of the FMDV viral protein (VP)1 site A will be assessed as thymus (T)-cell independent antibody inducer to study on the structural features of FMDV T-cell independent antigens. Specifically, we will produce the recombinant antigen and will study the cellular requirement to induce the antibody response against it. If positive results are obtained, the immunogenicity of the recombinant antigen will be assessed in experimental model (mice) and natural host (cattle).
This collaborative research project is focused on the detection and characterization of the in vitro production of Foot-and-Mouth Disease Virus (FMDV)-specific antibodies in the presence of different immune cell types and the study of the in vivo mechanisms and cells involved in the induction of FMDV-specific B-cell memory after vaccination in cattle. Specifically, we will 1. determine the role of the different immune cell types in the onset of primary and secondary humoral adaptive responses against FMDV and 2. Identify the antigen structural features which are required to elicit these antibody responses.
In objective 1, the isolation and culture of different immune cell types from naïve cattle in different combination, conditions and periods of time under the stimulation with recombinant FMDV antigens, live or inactivated virus particles will be conducted. During FY 2013 a protocol was established to induce anti-FMDV antibodies in vitro, using peripheral blood mononuclear cells (PBMC) from naïve cattle.
In objective 2, we aim to determine if the local FMDV-specific memory cells are locally elicited or if they are produced in other lymphoid tissues and further migrate to the mucosal lymph nodes. In addition, we will assess the presence of viral antigens in these mucosal lymph nodes to stimulate the production of FMDV-specific B-lymphocytes. During FY 2013 an assay to detect FMDV-specific memory B-cells was developed utilizing PBMC from FMD-vaccinated cattle.
No technologies were transferred in FY2013. No publications were produced in FY 2013.