Briefly, protein expression was carried out in HeLa cells using the VTF7-3 transient expression system. of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy. Introduction It has been over 25 years since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS [1], [2]. However, the tremendous research effort has not yet yielded an effective AIDS vaccine strategy. Earlier clinical trials using HIV Env-based subunit vaccines elicited antibodies that reacted with gp120 but were not neutralizing antibodies (NAbs), and vaccination LDC000067 failed to show protection against HIV infection [3]C[8]. The failure of these trials promoted a shift to the development of HIV vaccines that focus on eliciting T cell responses [9]C[11]. However, the disappointing outcome from a recent clinical trial of a T-cell-based vaccine regimen, the STEP trial conducted by Merck and HIV Vaccine Trials Network (HVTN), dealt another setback to AIDS vaccine development [12]. The failure of the STEP trial further reinforced the notion that an effective AIDS vaccine will need to induce both strong CTLs and broadly neutralizing antibodies (bNAbs) against HIV infection [13]C[15]. Nevertheless, effort to engineer vaccines that can induce HIV bNAbs has encountered great difficulties. Rabbit Polyclonal to PEG3 Extensive sequence variation of concurrently circulating HIV strains poses a great challenge for inducing HIV bNAbs [16]. While conserved neutralizing epitopes have been identified in the HIV Env protein, induction of HIV LDC000067 bNAbs against such epitopes has been difficult largely due to camouflage of these cryptic sites by the highly variable sequences in the HIV Env surface subunit gp120 [17]. This is supported by structural studies of the HIV Env [18]C[20], which indicate that conserved neutralizing epitopes are either hidden behind variable loops or obscured by carbohydrates. Therefore, induction of HIV bNAbs will require the design and development of novel vaccine strategies that can overcome these obstacles. The HIV Env transmembrane subunit gp41 serves to anchor the Env protein to cellular and viral membranes and mediate membrane fusion during virus entry into the cell. Although most of gp41 appears to be occluded in the HIV Env, a number of studies indicate that the membrane proximal external region of gp41 (MPER) is accessible to several HIV bNAbs and can be a promising target for vaccine design [21]. Several monoclonal antibodies (MAbs), which neutralize a broad range of primary HIV-1 isolates, are known to bind to adjacent epitopes LDC000067 located in the MPER [22]C[24]. The MPER is highly conserved and plays important roles in HIV Env incorporation and virus entry into the cells [25], [26], and the identification of these conserved neutralizing epitopes in the MPER spurred great effort to design vaccines for inducing HIV bNAbs against this region [27]. However, little success has been achieved by various approaches [28]C[33]. We previously reported the construction of an HA/gp41 chimeric protein, in which the gp120 subunit of HIV Env is replaced by the HA1 subunit of the influenza virus A/Aichi/2/68 (H3N2) HA protein [34]. This chimeric protein is efficiently transported to the cell surface and exhibits.