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Germinal Center Function in the Spleen during Simian HIV Infection in Rhesus Monkeys¹

David H. Margolin^2,*, Erika H. Saunders^3,*, Benjamin Bronfin^*, Nicole de Rosa^4,*, Michael K. Axthelm, Olga G. Goloubeva, Sara Eapen, Rebecca S. Gelman and Norman L. Letvin^5,*

^* Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR 97006; and Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with CT and GA replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.

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