Location: Foreign Animal Disease Research
Project Number: 8064-32000-061-11-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2012
End Date: May 31, 2017
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. Amendment 1: Provides for the additional study of the mechanisms behind the induction of memory responses against FMDV by vaccination in cattle. Specific objectives include: 4. Identification of the presence of FMDV-specific memory B-cells in lymph nodes draining from the vaccination site or located in the respiratory track, and in peripheral circulation from vaccinated cattle. 5. Detect the presence of FMDV antigens derived from vaccination and located in lymph nodes far from the vaccination site.
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). Amendment 1: To determine the role that FMDV vaccination plays on the induction of memory cells and antigenic production, naive cattle will be vaccinated with inactivated FMDV O1/Campos strain vaccine at INTA. Sera will be collected and mononuclear cell cultures prepared and tested for memory B-cell presence. FMDV-specific antibody secreting-cells (ASC) will be detected by ASC ELISpot assay developed by INTA. Vaccinated cattle will be euthanized and necropsied to obtain lymph nodes and tissue from the inoculation site. Mononuclear cell suspensions will be prepared to perform the detection of FMDV ASC by ELISpot. Serum samples from vaccinated cattle will be taken before each immunization and 15 days after the last booster and analyzed by the presence of FMDV-specific antibodies in an indirect sandwich ELISA. The ELISAs will be developed to assess anti-FMDV humoral responses and determine the presence of FMDV antigens in the processed cattle lymph nodes.