Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions.
We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:biology_diss-1117 |
| Date | 05 May 2012 |
| Creators | Nguyen, Doan C |
| Publisher | Digital Archive @ GSU |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Biology Dissertations |
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