High-throughput sequencing of human immunoglobulin variable regions with subtype identification

Area of Science:

• Immunology
• Genomics
• Biotechnology

Background:

• Antibody diversity is crucial for fighting infections, with both variable and constant regions influencing immune responses.
• Antibody isotypes (subtypes) have distinct effector functions, impacting natural infections and vaccine efficacy.
• High-throughput sequencing has advanced the study of antibody repertoire diversity.

Purpose of the Study:

• To develop a high-throughput sequencing protocol for simultaneously determining human IgG subtypes and full-length variable heavy chain regions.
• To enable linked analysis of antibody variable regions and their isotypes.
• To investigate the IgG variable region repertoire and subtype distribution in an HIV-1 infected individual.

Main Methods:

• Developed an Illumina MiSeq protocol using paired-end sequencing and customized identifier reads.
• Sequenced full-length antibody variable heavy chain regions and IgG subtypes from peripheral blood mononuclear cells (PBMC).
• Applied the method to analyze the IgG repertoire of an HIV-1 patient with MPER-specific antibodies.

Main Results:

• Successfully retrieved IgG transcripts with linked variable region and IgG subtype information.
• Observed increased frequencies of the IgG3 subtype in the memory B cell compartment of the HIV-1 patient after treatment interruption.
• Correlated the increase in IgG3 with heightened plasma antibody reactivity against the gp41 MPER domain.

Conclusions:

• The developed sequencing strategy effectively links antibody variable regions with their subtypes.
• IgG3 subtype expansion in memory B cells may be associated with specific immune responses in HIV-1 infection.
• The method is adaptable for broader Ig subtyping and phasing distant regions on amplicons.