Animals

Rhesus macaques were housed in pairs according to the “Guide for Care and Use of Laboratory Animals” as outlined by the American Association for Assessment and Accreditation of Laboratory Animal Care at the California National Primate Research Center (Davis, CA). All animal procedures were approved by the UC Davis Institutional Animal Care and Use Committee before study implementation. The study included 18 nursery-reared rhesus macaques (Macaca mulatta) that were between 3 and 10 days old at the time of the first immunization.

All procedures were performed under ketamine anesthesia (10 mg/kg body weight) administered IM.

Neonates were primed with two previously described SIV/adjuvant-coexpressing DNA plasmids, D_G and D_40L.^28–30 The amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted in vivo after immunization of macaques with D_G are not known, but transient transfection of 293T cells has been found to result in production of ∼200 ng GM-CSF per 10⁶ cells.²⁸ The D_40L plasmid coexpresses SIV_mac239 virus-like particles (VLP) and a membrane-bound form of macaque CD40L, resulting in secretion of VLP, which express CD40L on their surface.²⁹ For oral immunizations, the two DNA plasmids were formulated in cationic 1,2-dioleoyl-3-trimethylammonium liposomes by Encapsula Nanosciences (Brentwood, TN). The recombinant DR2 MVA-SIV_mac239 gag, pol, env, and empty MVA vectors used for boosting were provided by Dr. Bernie Moss (National Institute of Allergy and Infectious Diseases). Viral vectors were expanded and titrated for plaque forming units (PFUs) using chick embryo fibroblasts as previously described.³¹

The vaccination groups, vaccine dosages, and schedule are outlined in Table 1. Neonatal macaques in Groups B and C were immunized on weeks 0 and 3 with a 1:1 mixture of D_G + D_40L (hereafter referred to as DNA-SIV), and boosted on weeks 9 and 12 with MVA-SIV. Group B was IM immunized with a total of 3 mg DNA-SIV in 1 mL, and Group C received 1.5 mg DNA-SIV IM in 0.5 mL. The IM immunizations with DNA were done by injecting 250 μL (Group B) or 125 μL (Group C) into both the left and right quadriceps and biceps. For O immunizations (Group C), 0.7 mL of liposomal DNA-SIV was topically applied in each cheek pouch (1.4 mL total containing 1.5 mg DNA-SIV). All IM immunizations with MVA were done by injecting 0.5 × 10⁷ PFUs in 0.25 mL into both the left and right quadriceps (10⁸ PFU total). For SL administration of MVA-SIV, 10⁸ PFU in 30 μL was placed under the tongue. Group A mock control animals were administered saline both orally (1 mL) and IM (0.25 mL) on weeks 0 and 3, then boosted SL + IM on weeks 9 and 12 with empty MVA by applying 10⁸ PFU topically onto the SL mucosa and injecting 10⁸ PFU into the quadriceps. It should be noted that neonates in Groups A and B were immunized (and challenged) in parallel. Age-matched Group C neonates started the immunization regimen 1 month later.

Vaccination Groups and Schedule

SIV, simian immunodeficiency virus; O, oral; IM, intramuscular; SL, sublingual; MVA, modified vaccinia Ankara; PFU, plaque forming units.