Moreover, the E2 system is naturally designed to present up to 60 copies of the E1 (~150 kDa) or E3 (~100 kDa) enzymes noncovalently attached on its surface. CTL activity even in the absence of IFN-producing CD4+ T cells. Keywords:Virus-like particles, HIV, vaccine, E2 scaffold, Gag(p17) == Introduction == After twenty-five years of HIV research and several failed vaccine trials (Buchbinder et al., 2008), despite some evidence for modest vaccine protection in humans (Rerks-Ngarm et al., 2009), new vaccine modalities are still needed to elicit the high-titer and durable immune responses. These responses include, but may not be limited to, cytotoxic T cells (CTL) directed to multiple viral proteins, strong T helper responses, and neutralizing antibodies (NAbs) that are effective against a broad range of primary HIV-1 isolates. Proof-of-principle experiments in nonhuman primate challenge models have been useful in understanding the role of antibodies in blocking infection, when they are present at sufficient titers in advance of exposure (Baba et al., 2000;Hessell et al., 2009; Hessell et al.;Mascola et al., 2000;Shibata et al., 1999). Similarly, the presence of T cell responses is correlated with viral control, as shown by T cell depletion studies in nonhuman primates (Lifson et al., 2001;Schmitz et al., 2005) and protection from disease when strong T cell responses are induced by vaccination with recombinant viral vectors (Hansen et al., 2009;Santra et al., 2005). Efforts have thus been focused on designing vaccines and delivery systems that can elicit strong, durable immunity that is directed against multiple viral antigens, to prevent ready escape. Current methods for eliciting T-cell responses for HIV, in particular, have presented limitations due to poor immunogenicity that is limited in seropositive individuals (OBrien et al., 2009). However, in the case of some vectors, such as adenovirus, strategies have been proposed to circumvent anti vector immunity (Roberts et al., 2006). Moreover, combining two or more vaccine modalities in prime-boost regimens can elicit stronger immune responses than either vaccine alone (Kibuuka et al., 2010;Koup et al., 2010;Rerks-Ngarm et al., 2009). The inactivated HIV virion itself has long been considered as a vaccine candidate and this approach, especially for therapeutic vaccines, was championed by polio vaccine luminary Jonas Salk (Salk, 1987). Protective efficacy with formalin-inactivated SIV in nonhuman primates was demonstrated (Johnson et al., 1992;Murphey-Corb et al., 1990) but the vaccine was complicated by the presence of host HLA, which was shown to be the mechanism for protection-unrelated to the SIV antigens (Arthur et al., 1995;Stott, 1991). Recent advances in understanding of virus assembly have led to the development of methods that chemically and irreversibly inactivate whole virions and that maintain the receptor binding activity of Envelope (Arthur et al., 1998;Lifson et al., 2004). The attractiveness of VLPs that can mimic the natural HIV virion without any risk of HIV infection led to the early development of recombinant Gag-Env particles produced in mammalian cells by recombinant vaccinia virus (Haffar et al., 1990;Haffar et al., 1992;Haffar et al., 1991;Luo, Li, and Yong Kang, 2003), or rhinovirus chimeras (Arnold et al., 1994;Lapelosa et al., 2010), yeast (Tsunetsugu-Yokota et al., 2003), or in other systems, examined in (Deml et al., 2005). While clearly immunogenic in eliciting Gag-specific CTL (Paliard et al., 2000) and antibodies, including some neutralizing antibodies (Ding et al., 2002), none Betulin of these methods has elicited strong protective immunity. Explanations for low level immunogenicity may include dose, stochiometry of important immunogens such as Envelope (when present), or delivery or adjuvant systems used to day. We have previously designed and investigated a new delivery vehicle in which antigenic determinants are put on the surface of an icosahedral scaffold created from the acyltransferase component (E2 protein) of the multienzyme pyruvate dehydrogenase complex (PDH) fromGeobacillus stearothermophilus, and reported the ability of this scaffold to display peptides in a high immunogenic form (De Berardinis et al., 2003;Domingo et al., 2003). The core C-terminal catalytic website of E2 self-assembles into trimers, which in turn aggregate to generate a 60-chain core with icosahedral symmetry (Domingo, Orru, and Perham, 2001;Henderson, Perham, DC42 and Finch, 1979;Perham, 2000). Moreover this 60-meric icosahderal structure can be regenerated with high effectiveness from denaturing conditionsin vitro, without the need of chaperonins (Allen and Perham, 1997;Lessard et al., 1998). The robustness of this peptide centered virus-like particle offers rendered it a good macromolecular scaffold for demonstration of exogenous Betulin molecules on its surface (De Berardinis et al., 2003;Domingo et al., 2003;Domingo, Orru, and Perham, 2001) and for molecular encapsulation in its cavity (Dalmau et al., 2008;Dalmau, Lim, and Wang, 2009a;Dalmau, Lim, and Wang, 2009b). Efficient refolding Betulin of E2 to the 60-mer is also possible with foreign peptides replacing the natural peripheral domains,.