Human immunodeficiency virus (HIV) is responsible for nearly two million deaths annually, and although the overall incidence appears to have stabilized, the epidemic continues to spread (WHO, 2011). A vaccine represents the best possibility for eradication of the virus, but despite unprecedented efforts, an effective vaccine has not yet been developed.
Two recent vaccine efficacy trials provide clues to potential components that can contribute to protective immunity against HIV. Specifically, the Step study used a mixture of recombinant adenovirus serotype 5 (Ad5) vectors expressing HIV-1 proteins 
. That study terminated early after interim analyses demonstrated that the vaccine neither prevented infection nor lowered viral load, and perhaps had the adverse effect of increasing HIV acquisition in subjects with preexisting Ad5 neutralizing antibodies. Though the reasons for this remain unclear, follow up investigations indicated that adenovirus-specific CD4+ T cells might have impacted the availability of potential HIV target cells 
. In contrast, the RV144 trial was the first ever to demonstrate modest protection from HIV infection 
. That trial used an ALVAC-HIV prime in combination with a VaxGen AIDSVAX bivalent gp120 clade B/E protein boost. The regimen induced mostly humoral and low-level CD4+ T-cell responses, supporting the hypothesis that balancing both arms of the immune response will induce improved protection.
New York vaccinia virus (NYVAC) vector is a highly attenuated Copenhagen virus strain capable of inducing humoral and T-cell responses 
. NYVAC-CN54 encodes cell-released HIV-1 Env gp120 and Gag/Pol/Nef, an intracellular polyprotein harboring cytotoxic T lymphocyte epitopes 
. Preclinical studies in mice demonstrated that this vector, when used as a boost after a DNA prime, induces HIV-specific CD8+ T-cell responses and IgG production 
. In monkeys, a similar NYVAC vector expressing HIV gp120 and SIV Gag/Pol/Nef induced CD4+ and CD8+ T cells and antibodies to Env, with protection following SHIV89.6p challenge 
. In a phase I clinical trial NYVAC-CN54 induced a robust immune response, in particular Env-specific IFN-γ production by CD4+ and CD8+ T cells 
. Additionally, vaccinia viruses are advantageous vaccine vectors because pre-existing immunity at the population level is restricted to aged groups, since smallpox vaccination was terminated in the mid-1970s. Even in individuals with pre-existing immunity, smallpox-specific T cells are less frequent in the mucosal tissues of healthy volunteers than adenovirus-specific T cells 
, suggesting that use of this vector may avoid problems encountered in the Step study. Finally, vaccinia viruses are capable of activating innate immune responses through TLR-dependent pathways 
. TLR activation is recognized as a key component in several vaccines, including the yellow fever vaccine, the activity of which seems largely to be due to the generation of Th1-inducing mature DCs 
. Such findings have opened a new field of research on novel adjuvants, many of which are TLR ligands, to mimic the pathogen-associated molecular patterns recognized during an encounter with a natural pathogen.
Thus far, eleven TLRs have been identified in mice. Among them, TLR3, TLR7, TLR8 and TLR9 recognize nucleic acids; in particular, TLR9 recognizes CpG motif-containing DNA sequences. Several CpG oligodeoxynucleotide (CpG ODN) formulations have already been used as adjuvants in vaccine studies for infectious diseases, including HIV 
. In this regard, CpG ODN was shown to improve the humoral and cellular responses in a vaccination regimen consisting of Gag protein prime and adenovirus boost, both in mice 
and in a primate model 
. The recently developed compound IC31 consists of a combination of ODN1a, a TLR9 ligand, and the antimicrobial peptide KLKL5
KLK, which contributes to the stabilization of ODN1a and to depot formation 
. This combination was shown to induce a strong cellular and humoral immune response through activation of DCs and antigen-specific T-cell proliferation. Furthermore, IC31 increases the activation and cytotoxic activity of CD8+ T cells 
In this study we compared different vaccination regimens using NYVAC-CN54 and recombinant Env-CN54 protein, in combination with alum alone, with IC31 alone, or with IC31 and alum together. We evaluated induction of Env-specific antibodies, CD4+ and CD8+ T-cell effector function, and the formation of mucosal central and effector memory T-cell subsets. Our findings indicate that IC31 greatly improves vaccination immunogenicity in terms of the development of the Env-specific response.