VERMONT COBRE: PROJECT 2: INNATE IMMUNE RESPONSES TO CRYPTOSPORIDIUM PARVUM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Aim 4: Genetic Susceptibility to Cryptosporidium infection: In year 3, we extended our observations of the role of human genetics in susceptibility to Cryptosporidium infection by studying human mannose binding lectin (MBL). MBL is an evolutionarily conserved protein which functions in human innate immunity by binding microbial surfaces and promoting opsonophagocytosis. MBL has been shown to bind Cryptosporidium sporozoites and prior work has suggested that the protective role of MBL may be most important in childhood. We evaluated the association of polymorphisms in the MBL gene (mbl2) and serum MBL deficiency with childhood infection due to Cryptosporidium, Entamoeba histolytica, and Giardia intestinalis. We used anonymous specimens from a large, prospectively-followed cohort of Bangladeshi preschool children which followed for over three years. Clinical outcomes, MBL serum levels, and mbl2 polymorphisms and haplotypes were determined. Genotype determination of the MBL2 gene polymorphisms were performed by polymerase chain reaction (PCR) and direct sequencing. Analysis of polymorphisms was started with a 14-primer set, but modified to the methods below due to inability to interpret indistinct bands. Four single nucleotide polymorphisms (SNPs) of interest were determined, three located in the structural region on exon 1, and one located in the promoter region, Table 1. The three structural polymorphisms occur at codon 54 (glycine [GGC] to aspartic acid [GAC] substitution), codon 57 (lycine [GGA] to glutamic acid (GAA) substitution) and at codon 52 (arginine [CGT] to cysteine [TGT] substitution). The wild type allele is termed A at all three positions and polymorphisms are referred to as B, C, and D respectively.4 Any structural polymorphism (B, C, D) are collectively referred to as an "O" polymorphism. The promoter polymorphism studied is located at position -221 (X/Y variant) where there is a (G¿C) nucleotide substitution. In analysis of data, significant associations with E. histolytica and G. intestinalis were not found. However, serum MBL deficiency, polymorphisms in the -221 promoter region and the XA/YO mbl2 haplotype were strongly associated with Cryptosporidium infections, particularly recurrent infection. Children with multiple infections with Cryptosporidium were more likely to be MBL deficient (OR 10.45), carry the -221 promoter variant (OR 4.02), and the YO/XA haplotype (OR 4.91). Please see Table 1 and Figure 1. We have identified a potentially important component of the human innate immune response to Cryptosporidum infection. Further work is needed to evaluate the mechanism of protection of MBL in Cryptosporidium infections. Aim 5: Future work on Cryptosporidium will be focused on exploring the functional significant of the MBL mutations in control of human cryptosporidiosis. Using western blotting and chemoluminesent detection, we will examine the isoforms of MBL in the sera of children with known MBL genetic variants. Once confirmed, we will then clone the wild-type and variant MBL genes into CHO cells using established methods. Using antibody-based techniques, we will then evaluate the differing abilities of the MBL protein variants to bind both Cryptosporidium oocysts and sporozoites and correlate binding with clinical outcomes. Concurrently, we are also looking forward to studying Cryptosporidium in a birth-cohort of 700 children in Dhaka, Banglaesh in order to examine pre-and post-infection cytokine production and lymphocyte phenotypes. Working with collaborators at the University of Virginia, where genome-wide associations will be performed on these 700 children, we hope to confirm and add to our findings of MBL and HLA as genetic components changing susceptibility to infection. ***Please see paper copy for figures and tables (would not reproduce here)*** Mentoring Summaries: Elizabeth Bonney Beth Kirkpatrick, a COBRE Junior Faculty and I share an interest in human mucosal immunology and infectious disease, and I have met with her and provided consultation on her projects. She has greatly expanded her work in clinical and translation research around the development of vaccines to several important infectious agents. My plan is to meet with her monthly to further define career goals and encourage the publication of results. She has already obtained significant funding to support her work. Ralph Budd Dr. Kirkpatrick has been productive during the past year in terms of publications and success with the vaccine center evolution. She now has received a subcontract from Johns Hopkins for a dengue virus vaccine trial. In addition, she has been successful in obtaining funding through the Navy for the Campylobacter work, and biotech support for a typhoid vaccine trial. In addition, Dr. Kirkpatrick has taken on the task of developing a human immunology core laboratory, which is needed to grow the vaccine center, but will also be useful for the COBRE group in general. This will be submitted in May as a T1 supplement request. The main issue for the coming year will be defining which project/organism can be developed into an R21 or R01 application. Dr. Kirkpatrick's K08 award ends this year and there is a need to now focus on the one or two areas that we allow her to compete for NIH funding. This was also mentioned in the EAC report.