The proposed study will elucidate mechanisms of immune responses to the infused factor VIII (FVIII) used to arrest bleeding in hemophilia A (HA) patients, in which ~25% of patients develop """"""""inhibitors"""""""" (neutralizing antibodies) that can greatly complicate their treatment by causing life- and limb-threatening bleeds. The projects seek explanations for the observation that inhibitor incidence in black HA patients is twice that of white patients. Clinical collaborators include 15 U.S. hemophilia treatment centers (HTCs) with patient populations that include large numbers of African Americans, and the National HTC network in South Africa. DNA and plasma collected as part of the Hemophilia Growth and Development Study (HGDS) funded by the NIH/NICHD, and also from a Canadian Repository, will also be studied. Laboratory collaborators -- based in Los Angeles (CA), Seattle (WA), San Antonio (TX), and Burlington (VT) -- will investigate the genetic basis for inter-individual variability in inhibitor formation, and humoral and cellular aspects of immune responses to FVIII, using established assays that they developed. High-throughput DNA sequencing and other PCR-based assays will identify the F8 mutations causing hemophilia as well as the alleles of all F8 nonsynonymous-singlenucleotide polymorphisms (ns-SNPs), which are alterations in a single coding DNA base that result in amino acid changes in the FVIII protein. A previous analysis of F8 haplotypes (Viel et al, NEJM 2009) will be expanded and refined. In that study, black HA patients whose endogenous (self) FVIII proteins were allelically mismatched, at sites corresponding to ns-SNPs, with commonly infused FVIII proteins had a significantly higher frequency of inhibitors than black patients whose endogenous FVIII matched the therapeutic FVIII. The proposed study greatly increases the number of subjects, which will provide improved control for covariates and adequate statistical power to perform subgroup analyses. The study utilizes sensitive, haplotype-specific tests capable of identifying even low-titer (concentration) and non-neutralizing anti-FVIII antibodies, which are not detectable by conventional Bethesda assays. Microarrays will be used to genotype almost 2,000,000 SNPs and copy number variants for genome-wide analyses, concentrating initially on the subset located in known immune response genes. Finally, HLA-class II tetramer-based assays will test directly the hypothesis that amino acid sequences encoded by F8 loci containing ns-SNPs comprise immunodominant epitopes that, when allogeneically mismatched, stimulate T cells of HA patients. Antigenicity will also be tested directly by surface plasmon resonance assays to (1) evaluate binding of patient-derived antibodies to recombinant FVIII proteins corresponding to different F8 haplotypes, and (2) determine the epitopes recognized by these antibodies. If the hypotheses are confirmed, this will motivate preclinical testing of novel, less immunogenic FVIII replacement proteins that could ultimately eradicate this long known race-based disparity in hemophilia A, and potentially extend in concept to other areas of transfusion medicine and protein therapeutics.

Public Health Relevance

In a collaboration of hemophilia treatment centers and expert laboratories, the mechanisms of immune response to factor VIII in hemophilia A and the risk factors for inhibitor development will be studied. The previously-suggested inhibitor risk of a possible mis-match between a patient's racially-determined background factor VIII structure and the factor VIII structure of exogenous therapeutic factor VIII products will be further studied. The possibility of a therapeutic factor VIII, better matched to minority recipients, will be explored.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
High Impact Research and Research Infrastructure Programs (RC2)
Project #
1RC2HL101851-01
Application #
7861345
Study Section
Special Emphasis Panel (ZHL1-CSR-R (O5))
Program Officer
Link, Rebecca P
Project Start
2009-09-30
Project End
2011-08-31
Budget Start
2009-09-30
Budget End
2010-08-31
Support Year
1
Fiscal Year
2009
Total Cost
$3,548,764
Indirect Cost
Name
Sepulveda Research Corporation
Department
Type
DUNS #
030380682
City
Sepulveda
State
CA
Country
United States
Zip Code
91343
Ettinger, Ruth A; Paz, Pedro; James, Eddie A et al. (2016) T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire. Blood 128:2043-2054
Dutta, Debargh; Gunasekera, Devi; Ragni, Margaret V et al. (2016) Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers. Blood Adv 1:231-239
Gunasekera, Devi; Ettinger, Ruth A; Nakaya Fletcher, Shelley et al. (2015) Factor VIII gene variants and inhibitor risk in African American hemophilia A patients. Blood 126:895-904
Sauna, Zuben E; Lozier, Jay N; Kasper, Carol K et al. (2015) The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics. Blood 125:223-8
Lin, Jasper C; Ettinger, Ruth A; Schuman, Jason T et al. (2015) Six amino acid residues in a 1200 Å2 interface mediate binding of factor VIII to an IgG4? inhibitory antibody. PLoS One 10:e0116577
Nguyen, Phuong-Cac T; Lewis, Kenneth B; Ettinger, Ruth A et al. (2014) High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance. Blood 123:2732-9
Kim, Benjamin; Hing, Zachary A; Wu, Andrew et al. (2014) Single-nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family. Br J Haematol 165:154-8
Pandey, Gouri Shankar; Yanover, Chen; Howard, Tom E et al. (2013) Polymorphisms in the F8 gene and MHC-II variants as risk factors for the development of inhibitory anti-factor VIII antibodies during the treatment of hemophilia a: a computational assessment. PLoS Comput Biol 9:e1003066
Pandey, Gouri Shankar; Yanover, Chen; Miller-Jenkins, Lisa M et al. (2013) Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A. Nat Med 19:1318-24
Lewis, Kenneth B; Hughes, Richard J; Epstein, Melinda S et al. (2013) Phenotypes of allo- and autoimmune antibody responses to FVIII characterized by surface plasmon resonance. PLoS One 8:e61120

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