Of interest to us, the production of all cytokines was not prominent in the SE36/AHG with D35 ODN group at both time points despite the clear antibody titer induction in this group. Open in a separate window Physique?2. a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers sera into and squirrel monkeys.8-11 Mouse and rat antibodies against the SE47, likewise, inhibited parasite growth in vitro.12-14 However, SE47 is highly hydrophobic, making large-scale manufacturing under good manufacturing practice (GMP) conditions a major challenge. Therefore, we constructed a new recombinant antigen, SE36, lacking the serine repeats. SE36 assimilated to the adjuvant aluminum hydroxide gel (SE36/AHG) was prepared under GMP conditions. SE36/AHG was highly immunogenic and anti-SE36 IgG titers lasted more than 1 y in chimpanzees.15 Squirrel monkeys vaccinated with SE36/AHG were guarded against high parasitemia, and serum anti-SE36 IgG titers were boosted after malaria parasite challenge. A human phase 1a Entrectinib clinical trial in Japan exhibited Entrectinib that SE36/AHG (100 g/1,000 g) was safe, well-tolerated and immunogenic.15 However, the mean titer of induced anti-SE36 antibody in Entrectinib the phase 1a trial was lower than that in African high responders. Synthetic oligodeoxyribonucleotides (ODNs) made up of immunostimulatory Entrectinib unmethylated cytosine-guanosine dinucleotides (CpG motifs) are potentially useful adjuvants and have been evaluated for veterinary and human vaccines.16 Rabbit polyclonal to PLAC1 These so-called CpG ODNs are categorized into two major classes, K- and D-type. K-type ODNs trigger the maturation of dendritic cells and stimulate the production of IgM and interleukin (IL)-6.17,18 The D-type ODNs trigger antigen-presenting cell (APC) maturation and preferentially induce interferon (IFN)- and – secretion.18,19 It has been reported that K3 ODN effectively induces IL-6 production and cell proliferation, and that D35 ODN effectively secretes IFN- in rhesus macaques.20 Another TLR9 ligand, synthetic hemozoin (sHZ, also known as -hematin), is also a potent adjuvant for malarial antigens.21 Hemozoin, a malaria pigment, is a detoxified product of heme molecules found in food vacuoles of the malaria parasites.22,23 In previous studies, it was shown that purified HZ activates macrophages, thereby producing pro-inflammatory cytokines, chemokines and nitric oxide. HZ has also been shown to enhance human myeloid dendritic cell maturation.21,24 Furthermore, the adjuvant function of sHZ was validated in a canine antiallergenic vaccine model.25 Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development. We recently decided that a formulation of K3 and D35 ODNs, or sHZ with SE36/AHG was effective for the induction of anti-SE36 IgG in a rodent malaria model (Tougan et al., unpublished data). The purpose of this study was to increase the levels of induced antibody using a vaccine formulation made up of TLR9 ligands as adjuvants. Here, we report the safety, immunogenicity and protective efficacy of the SE36/AHG formulation made up of either K3 ODN, D35 Entrectinib ODN or sHZ as an adjuvant in non-human primate models. Results Adjuvant efficacy of TLR9 ligands to SE36/AHG The adjuvant efficacy of K3 and D35 ODNs, and sHZ with SE36/AHG was examined. Twelve cynomolgus monkeys were randomly assigned to four groups. SE36/AHG, with or without each adjuvant, was administered four occasions and SE36-specific IgG titer was measured. Two weeks after the second immunization (Day 36), mean anti-SE36 antibody titers were 54.5, 432.9 (p < 0.05), 68.8 and 270.1 in the SE36/AHG, SE36/AHG with K3 ODN, SE36/AHG with D35 ODN and SE36/AHG with sHZ groups, respectively (Fig.?1A and B). Two weeks after the third immunization (Day 112), mean antibody titers were 182.2, 2258.9 (p < 0.05), 704.3 and 1276.1, respectively (Fig.?1A and B). Two weeks after the fourth immunization (Day 379), mean antibody titers were 237.8, 1960.8 (p < 0.05), 548.2 and 791.3 in the respective groups (Fig.?1A and B). At the three time points post-administration (Fig.?1A and B), the formulations including K3 ODN and sHZ elicited significantly higher anti-SE36 antibody titers. In particular, K3 ODN remarkably enhanced the antibody response after each administration. Furthermore, titers in individual monkeys from each group were compared immediately before each immunization to observe sustainability of the antibody titers. After the second immunization, mean antibody titers were 13.9, 176.0, 17.3 and 43.0 in the SE36/AHG, SE36/AHG with K3 ODN, SE36/AHG with D35 ODN, and SE36/AHG with sHZ groups, respectively (Fig.?1A and B). After the third immunization, mean antibody titers were 17.6, 157.7, 26.6 and 21.3, respectively (Fig.?1A.