The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA ...sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.
Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The ...application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.
The dynamics of emerging nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI) resistance in hepatitis B virus (HBV) are not well understood because standard dideoxynucleotide direct ...polymerase chain reaction (PCR) sequencing assays detect drug-resistance mutations only after they have become dominant. To obtain insight into NRTI resistance, we used a new sequencing technology to characterize the spectrum of low-prevalence NRTI-resistance mutations in HBV obtained from 20 plasma samples from 11 NRTI-treated patients and 17 plasma samples from 17 NRTI-naive patients, by using standard direct PCR sequencing and ultra-deep pyrosequencing (UDPS). UDPS detected drug-resistance mutations that were not detected by PCR in 10 samples from 5 NRTI-treated patients, including the lamivudine-resistance mutation V173L (in 5 samples), the entecavir-resistance mutations T184S (in 2 samples) and S202G (in 1 sample), the adefovir-resistance mutation N236T (in 1 sample), and the lamivudine and adefovir–resistance mutations V173L, L180M, A181T, and M204V (in 1 sample). G-to-A hypermutation mediated by the apolipoprotein B mRNA editing enzyme, catalytic polypeptide–like family of cytidine deaminases was estimated to be present in 0.6% of reverse-transcriptase genes. Genotype A coinfection was detected by UDPS in each of 3 patients in whom genotype G virus was detected by direct PCR sequencing. UDPS detected low-prevalence HBV variants with NRTI-resistance mutations, G-to-A hypermutation, and low-level dual genotype infection with a sensitivity not previously possible
It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in ...antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown.
Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016).
Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available.
Major histocompatibility complex (MHC) class I alleles of nonhuman primates have been associated with disease susceptibility, resistance, and resolution. Here, using high-resolution pyrosequencing, ...we characterized MHC class I transcripts expressed in Mauritian cynomolgus macaques (MCM), a nonhuman primate population with restricted MHC diversity. Using this approach, we identified 67 distinct MHC class I transcripts encoded by the seven most frequent MCM MHC class I haplotypes, 40 (60%) of which span the complete open reading frames. These results double the number of MHC class I sequences previously defined by cloning and Sanger sequencing of cDNA-PCR products and provide a rapid, high-throughput, and economical method for MHC characterization. Overall, this approach significantly expanded our knowledge of MCM haplotypes and will facilitate future studies on disease pathogenesis and protective cellular immunity.
Interference with the innate apoptotic activity is a hallmark of neoplastic transformation and tumor formation. In this study we characterize the cytotoxic effect of phenoxodiol, a synthetic ...anticancer drug analog of genestein, and demonstrate the mechanism of action by which phenoxodiol affects the components of the Fas apoptotic pathway on ovarian cancer cells. Primary ovarian cancer cells, isolated from ascitic fluids of ovarian cancer patients, resistant to conventional chemotherapy, undergo apoptosis following phenoxodiol treatment. This effect is dependent upon the activation of the caspase system, inhibiting XIAP, an inhibitor of apoptosis, and disrupting FLICE inhibitory protein (FLIP) expression through the Akt signal transduction pathway. We suggest that phenoxodiol is an efficient inducer of cell death in ovarian cancer cells and sensitizes the cancer cells to Fas-mediated apoptosis. We identified FLIP and XIAP signalling pathways as key factors regulating the survival of ovarian cancer cells. These findings demonstrate a novel nontoxic drug that controls FLIP/XIAP function and has the potential to eliminate tumor cells through Fas-mediated apoptosis.
B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To ...assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
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•Human antibody gene repertoire sequencing identifies vaccine-specific B cell clones•Quantified B cell clonal expansions correlate with serological vaccine responses•Human pandemic H1N1 2009 influenza vaccine responses show convergent antibodies
Infection stimulates B cells expressing antibodies specific to the pathogen. By sequencing human antibody gene repertoires, Jackson et al. determined that influenza vaccination or infection induces B cell clonal expansions, whose magnitude correlates with individual secreted antibody responses, and that different individuals use convergent antibody rearrangements specific to influenza antigens.
Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that ...could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.
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•White blood cell DNA sequences reveal clonal B cell expansion in acute dengue patients•Multiple dengue cases exhibit convergent dengue-specific antibody (CDR3) signatures•Convergent CDR3s have diverse underlying nucleotide sequences•Dengue-specific CDR3s have unique amino acid physicochemical profiles
The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding ...antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.
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