FIG 1.
Plasma viral RNA load and antibody-based immune responses following repeated low-dose SIVmac251 mucosal challenge. (A) Viral loads of four rhesus macaques infected by repeated low-dose challenge with SIVmac251 were measured 1, 2, 3, 4, 6, 8, 10, 14, 18, 23, 25, and 29 weeks from initial challenge by real-time RT-PCR of plasma viral RNA and reported as SIV RNA copy equivalents/ml of plasma. All four male rhesus macaques were positively infected, showing peak of viral replication 2 weeks after the last SIV-negative assay. Viral loads were synchronized to the last week in which vRNA was not detectable in each infected animal. (B) Env-specific antibody responses were measured by ELISA using recombinant soluble SIVmac239 gp140. The level of binding in plasma from each animal was tested by serial 4-fold dilution in duplicate. Antibody titer was calculated as the inverse of the lowest dilution with a value at least 2-fold above background. (C) Neutralizing antibody titers in plasma were measured against laboratory-adapted SIVmac251. Plasma was tested over eight serial 2-fold dilutions in duplicate to determine the concentration of plasma which reduced infection by 50%, termed the IC50. The IC50 was reported for every time point tested for each animal in the cohort. (D) Two million mononuclear cells were stained with anti-human monoclonal antibodies CD3-V450, CD20-allophycocyanin (APC)-H7, CD4-APC (BD Bioscience, San Jose, CA), and Dead/Live Aqua dead cell stain (Invitrogen, Carlsbad, CA). Data were acquired using an Aria I flow cytometer (BD Bioscience, San Jose, CA) and analyzed using FlowJo software (TreeStar, Cupertino, CA). (E and F) Mononuclear cells from peripheral blood (PBMC), bone marrow (BM), spleen (SP), axillary lymph node (AxLN), and inguinal lymph node (InLN) were stimulated with a pool of overlapping peptides corresponding to the complete amino acid sequence of SIVmac239 Env (E) and Gag (F). The average number of spot-forming cells (SFC) is reported for duplicate wells seeded at 200,000 cells per well. Results correspond to PBMC at 3 weeks p.i. and BM, SP, AxLN, and InLN at necropsy.