Experimental design

Model of study and experimental design

The ANRS recombinant MVA HIV B vaccine (MVATG17401; Transgene, Illkirch-Graffenstaden, France) was injected subcutaneously to five adult male cynomolgus macaques (macaque ID: BB078, BB231, BC641, BD619, and BD620), at 4x108 PFU, two months apart.

MVA HIV B contains the full-length codon-optimized sequence of gag (encoding amino acids [aa] 1 to 512) fused with fragments from pol (encoding aa 172 to 219, 325 to 383, and 461 to 519) and nef (encoding aa 66 to 147 and 182 to 206) from the Bru/Lai isolate (Los Alamos database accession number K02013).

Blood samples were collected longitudinally in EDTA to count leukocytes and in Lithium-Heparin to perform single-cell mass cytometry analyses. Fifteen samples per macaque were gathered. They were collected twice before any immunization, six times after the prime, and six times after the boost.

Mass cytometry antibody panel

Fixed leukocytes were stained with a 32 antibodies panel for mass cytometry targeting innate myeloid cells. Targeted markers included several FcR, migration, adherence, activation and co-stimulation molecules as well as cytokines. Cells were acquired using a CyTOF (Fluidigm).

The associated clone and metal are indicated for each marker. The right columns indicate whether the markers were extra- or intra-cellularly stained. The TET2 antibody clone recognizes four members of the CD66 family: CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), and CD66e (CEACAM5). The FLI8.26 antibody clone reacts with CD32a and CD32b. The LT27/295 antibody clone stains most IFNa subtypes, but not IFNa2b.