The levels of neutralizing antibody titers against the same pseudoviruses increased significantly after Env/MF59 boosting to the same levels as those in animals that received Env/MF59 5 times (Figure ?(Figure5).5). evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 g) of formulated self-amplifying mRNA is safe and immunogenic. Keywords: self-amplifying mRNA, HIV, vaccine, antibodies, T cells Vaccination is one AZD4573 of the most important tools to combat infectious diseases such as human immunodeficiency virus (HIV) infection. Nucleic acid vaccines (eg, plasmid DNA, viral vector, and messenger RNA [mRNA] vaccines) have been evaluated in a variety of experimental clinical settings against cancer, malaria parasites, hepatitis B virus, and HIV type 1 [1C3]. RNA approaches in particular have been studied for cancer [4], allergy [5], and influenza vaccines [6], as well as for gene therapies [7, 8]. AZD4573 So far, DNA vaccines have shown low potency in humans during clinical trials, in contrast to the robust cellular and humoral immunogenicity elicited by standard intramuscularly injected vaccines in small animals [9]. As a consequence, the focus of many DNA vaccine strategies has shifted to their ability to prime the immune response in heterologous prime-boost regimens, using various viral vectors, such as adenovirus or modified vaccinia Ankara [10C12], or recombinant subunit proteins [13] as boosts. Recombinant viral vector technologies have the advantage of efficient delivery of the nucleic acid payload, but their usefulness is often hampered by preexisting antivector immunity, production limitations, and safety concerns, as in the case with adenovirus serotype 5Cbased vaccines [14, 15]. Also, antivector immunity develops rapidly after vaccination with recombinant viral vectors, thereby interfering with subsequent immunizations [16]. DNA/RNA vaccines are not limited by such constraints and can be safely and effectively administered repeatedly to humans [17, 18]. We have previously described the AZD4573 SAM vaccine platform [8, 19C21]. This platform, now in preclinical development, is based on a synthetic, self-amplifying mRNA delivered by a synthetic lipid nanoparticle [19]. We used a self-amplifying RNA based on an alphavirus genome [22], which contains the genes encoding the alphavirus RNA replication machinery LATS1 but lacks the genes encoding the viral structural proteins required to make an infectious alphavirus particle. The structural protein genes are replaced with genes encoding protein antigens, which are abundantly expressed from a subgenomic mRNA in the cytoplasm of cells transfected with these self-amplifying RNAs [19]. It was shown that nonviral delivery of AZD4573 a 9-kb self-amplifying RNA encapsulated within a lipid nanoparticle substantially increased immunogenicity, compared with delivery of unformulated RNA, and that this novel vaccine technology was able to elicit broad, potent, and protective immune responses in rodents, comparable to a viral delivery technology and to a 200-fold higher dose of pDNA delivered using electroporation. In the current study, we evaluated the usefulness of an alternative nonviral delivery system. This newer formulation is based on a cationic nanoemulsion (CNE) that binds to the self-amplifying mRNA or SAM vector and enhances the delivery and potency of the vaccine [23]. CNE is based on the oil-in-water emulsion adjuvant MF59, which has been extensively tested in clinical trials, is licensed in 30 countries, and has an established safety profile in children, adults, and elderly individuals [24, 25]. Here we evaluated CNE delivery of an HIV SAM vaccine in rhesus macaques and compared its immunogenicity to that of 2 vaccine modalities known AZD4573 to be immunogenic in humans: viral delivery of self-amplifying RNA using viral replicon particles (VRPs) [26] and recombinant envelope protein formulated with the potent adjuvant MF59 [27]. METHODS Preparation of CNE CNE was prepared as described elsewhere [23, 28]. Briefly, squalene, DOTAP, and sorbitan trioleate were combined and heated to 37C. The resulting oil phase was then combined with an aqueous phase consisting of polysorbate 80 in 10 mM citrate buffer at pH 6.5. The final weight by weight percentages of squalene, DOTAP, sorbitan trioleate, and polysorbate 80 were 4.3%, 0.4%, 0.5%, and 0.5%, respectively. This mixture was homogenized using a T25 homogenizer with a 13.4 mm diameter rotor (IKA, Wilmington, North Carolina) at 24 000 RPM to produce a primary emulsion. This was then passed through a M-110P Microfluidizer (Microfluidics, Newton, Massachusetts) with an ice bath cooling coil at a homogenization pressure of 137 Mpa approximately 8 times. The formulation was stored at 4C before use. The 100-nm CNE had a positive surface charge, which was used to adsorb the RNA to the surface of the oil droplet through an electrostatic interaction with the negatively charge phosphate backbone. RNA was diluted to 200 g/mL and was added to an equal volume of CNE, mixed, and allowed to equilibrate on ice for 30 minutes to 2 hours. Endotoxin levels were measured by the gel clot LAL assay per the manufacturer’s instructions (Cape Cod.