Date Awarded

2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Virginia Institute of Marine Science

Advisor

Stephen L. Kaattari

Abstract

The study of antibody responses in prominent aquaculture species such as the rainbow trout, Oncorhynchus mykiss, can facilitate vaccine development and contribute to producing useful paradigms of adaptive immunity in lower vertebrates. Thus, it is essential to identify genes responsible for antibody responses. In the mouse model, hybridoma technology allows for the association of monoclonal antibodies possessing various affinities for antigen with specific VH sequences, gene family utilization, and other molecular events (i.e. somatic hypermutation) that occur during the specific immune response. The absence of a comparable hybridoma technology in piscine systems has limited similar studies of fish immunogenetics to date. Molecular and serological experiments were performed in an attempt to obtain information regarding somatic mutation and VH gene utilization for trout antibodies without reliance on hybridoma technology. PCR primers recognizing consensus sequences of FR1 and FR3 were used to amplify antibody VH sequences from panned, antigen-specific B cells. to follow the development of the expressed VH repertoire, lymphocytes were obtained at weeks 0, 5, 10, and 20 post primary immunization with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) or infectious hematopoietic necrosis virus (IHNV). Lymphocytes were also collected 10 weeks post secondary immunization (week 35). These studies were conducted in parallel with serological analyses of plasma antibodies obtained from the same sample in order to correlate molecular data with serological data from individual trout. Antigen-specific lymphocytes were processed to isolate RNA templates to produce cDNA which was cloned and sequenced. This sequence analysis allowed us to report, for the first time, the temporal accumulation of potential somatic variants that correlate to the development of new, high affinity antibody subpopulations during the immune response, some with the emergence of new antibody heavy chain isoelectropherotypes as identified by 2D-IEF/SDS-PAGE. Southern analysis and gene titration using various antigen-specific cDNA probes allowed us to correlate trout antibodies possessing various affinities for antigen with specific VH sequence and gene family utilization. Thus, trout Ig VH gene family utilization appears to follow the mouse model of differential use for specific immune response. These results reveal a capability for fine-tuning the piscine immune response previously not recognized.

DOI

https://dx.doi.org/doi:10.25773/v5-qvmw-xc70

Rights

© The Author

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