Immunoglobulin Gene Diversity in an African Population and Impact Antibody Function in HIV Infection
Antibodies are critical components of immune defense and are mediators of vaccine-elicited protection as well as autoimmunity. The heavy and light chains that comprise an antibody molecule are encoded by germline immunoglobulin (Ig) gene fragments that recombine and undergo somatic mutation thereby generating billions of specificities able to contend with a multitude of foreign antigens. The majority of the diversity lies within the antigen-binding (Fab) portion of antibodies although variation also exists within the conserved (Fc) region of the heavy chain that interacts with cellular receptors to mediate non-neutralizing functions. Diversity within both regions impacts on antibody function. Given the protective role of antibodies in HIV and other infections, we propose to mine the depth of the antibody repertoire and study how Fc diversity impacts antiviral activity. It is well known that Africans are more genetically diverse than other populations and yet there is a scarcity of data on African genomes including Ig genes. Our preliminary data using samples from an ethnic Zulu population residing in South Africa has revealed that approximately half of the heavy chain variable (IGHV) genes as well as a significant number of single nucleotide polymorphisms (SNPs) in the Fc are not recorded in public databases. Many of these novel IGHV alleles occur at high frequency and in some cases are highly expressed. Importantly, we have shown that they are used to make functional antibodies. We hypothesize that there is significant undiscovered variability within the African Ig gene repertoire some of which will impact on the functionality of anti-HIV antibodies. To explore this, we propose to sequence the entire rearranged, antigen-naïve heavy and light variable chain gene repertoires in 40 individuals of Zulu ethnicity using Illumina MiSeq. Data will be analyzed using a new bioinformatics tool called IgDiscover that will enable the identification of novel and known germline variable and joining region gene segments from both heavy and light chains that are expressed by functional antibodies. Variability in the Fc region of IgG and IgA will be analyzed using SNP analysis in 100 Zulu individuals. Furthermore, the Fc regions from individuals with novel IgG3 SNPs will be sequenced and used to make fully native antibodies for functional studies, including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis and neutralization. In this way the diversity within both the Fab and Fc regions will be analyzed and the impact on function assessed. This project will contribute new knowledge on African germline immunoglobulin genes, which is essential for a fully comprehensive immunogenetics database. These studies will be of great benefit to the HIV research community as they embark on major vaccine efficacy trials as well as to others studying the immune responses to infection, vaccination, cancer and auto-immunity in this population.
Antibodies are an essential component of the host immune response to infection and vaccination. In humans, the repertoire is vast with an estimated 1013 unique antibodies (Pieper, et al., 2013). These arise as a result of recombination from a limited number of germline genes followed by somatic hypermutation when B cells encounter antigen. Variability is mainly focused in the antigen-binding regions of the heavy and light chains that are responsible for specificity and neutralizing activity. There is also significant interest in characterizing the constant regions of the heavy chain that interacts with the Fc receptors on immune cells to mediate important effector functions. High-throughput approaches including next generation sequencing have made it possible to mine the depths of antibody diversity and examine the gene repertoire at a population level. Currently, antibody sequences from African populations are vastly understudied and underrepresented in public databases. Here we aim to study immunoglobulin gene repertoires from individuals living in KwaZulu-Natal, South Africa and define how gene variations impact on antibody effector function.
Stored blood samples from the well characterized CAPRISA cohort of young women at high risk of HIV infection established in 2004 will be used in this study. Infection rates in some communities in this region exceed 50% and so there is an urgent need for a preventative HIV vaccine. Through the use of this cohort we have made significant contributions to understanding how the neutralizing response to HIV develops providing vital information for HIV vaccine design (Moore, et al., 2012; Doria-Rose, et al., 2014; Bhiman, et al., 2015). More recently we have examined how antibody effector functions can enhance the antiviral activities of neutralizing antibodies (Richardson et al., unpublished). An understanding of the genetic variation that underpins these functions is needed to fully interpret the antibody response to HIV infection and vaccination.
This project will contribute new knowledge on African germline immunoglobulin genes, which is essential for a fully comprehensive immunogenetics database. Such information can inform relative usage in the overall expressed repertoires of different individuals as well as offer insight into which germline genes are preferentially expressed to produce functional antibodies. These studies will be of great benefit to the HIV research community as they embark on major vaccine efficacy trials as well as to others studying the immune responses to infection, vaccination, cancer and auto-immunity in this population.
Specific Aim 1: To characterize immunoglobulin variable germline genes in an African population.
We propose to sequence immunoglobulin germline genes and alleles of 40 women in the CAPRISA cohort. Rearranged, antigen-naïve IgM, Kappa and Lambda sequences will be amplified from cDNA, sequenced using Illumina MiSeq and analyzed using a novel bioinformatics tool called IgDiscover (Corcoran, et al., 2016). This will enable the identification of novel and known germline VH, VK and VL sequences, in addition to JH, JK and JL sequences expressed in functional antibodies. Based on our preliminary data of the germline IGHV genes (Scheepers, et al., 2015) we hypothesize that multiple novel V and J genes and alleles will be identified in Africans, some of which will be used in response to HIV-1.
Specific Aim 2: To sequence IgG and IgA germline constant regions
Our preliminary data has identified novel single nucleotide polymorphisms (SNPs) in the germline IgG constant regions. We propose to expand this analysis by sequencing IgG1, IgG2 and IgG3 as well as IgA1 and IgA2 germline genes from 100 CAPRISA participants to more accurately assess the frequency of these SNPs. The constant regions of selected participants will be cloned in order to link SNPs and resolve heterozygosity. The focus will be on IgG3 as this is the most genetically diverse subclass and has the most polyfunctionality. We anticipate identifying a number of novel SNPs and alleles in the antibody germline constant region.
Specific Aim 3: To examine the functional activity of IgG3 with natural and/or novel Fc alleles
We have shown that antibody effector functions can be improved when the biologically matched Fc region is used. Antibodies expressing novel and native SNPs will therefore be created and tested using a suite of assays including phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) and neutralization in order to determine their impact on anti-HIV activity. Our hypothesis is that antibodies expressing native and/or novel IgG3 alleles will show altered antiviral functions. This information may help to improve vaccine efficacy.