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Origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing.

ABSTRACT: Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS coronavirus (SARS CoV), and Hendra and Nipah viruses (henipaviruses). Although broadly neutralizing antibodies (bnAbs) against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS CoV receptor-binding domain (RBD), and soluble G proteins (sG) of henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity, and a lower extent of somatic mutation. In this study, we identified antibody maturation intermediates that are related to bnAbs against the HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

SUBMITTER: Prabakaran P

PROVIDER: S-EPMC3410596 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

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Origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing.

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