Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are rarely elicited during natural infection and to an even lesser extent during vaccination with Env-based immunogens. The primary challenge in the development of a vaccine capable of inducing broadly neutralizing antibodies against HIV-1 lies in the design of the immunogen (6
). However, a successful vaccine against HIV-1 will likely need to induce both effective cell-mediated immune responses and broadly neutralizing antibodies. Viral systems are therefore of interest, since they are capable of stimulating both cellular and humoral immune responses. In addition, they can be used to express optimized envelope glycoprotein immunogens in vivo. To obtain potent antibody responses, virus-based vaccines often require subsequent immunizations with recombinant protein. Thus, the characterization of prime-boost regimens, which combine virus-based expression with recombinant protein-based immunogens, is an important area of investigation. The efficacy of neutralizing antibodies in protection against HIV-1 has been demonstrated in passive transfer studies (3
). It has also been shown that the in vitro neutralizing capacity of well-characterized antibodies against HIV-1 correlates with their ability to protect against virus challenge in vivo (27
). In vitro neutralization assays are therefore important for the screening of new envelope glycoprotein immunogens as well as to evaluate antibody responses elicited by different vaccine vector systems used for immunogen delivery.
Multiple lines of evidence suggest that gp120 and gp41, the HIV-1 envelope glycoproteins, are organized into trimeric spike complexes on the surface of infected cells and infectious virus particles (7
). The functional spike is labile, and monomeric gp120 dissociates readily from gp41, resulting in the exposure of nonneutralizing gp120 and gp41 protein surfaces to the immune system (15
). Monomeric gp120 has been shown to be a highly flexible molecule (36
) which is likely to present many different conformations to the immune system, thereby diverting the immune response away from epitopes found on the functional spike. Other immune-evasion strategies inherent in the spike include a high density of glycans on the accessible outer domain of gp120 and V1/2 loops as well as orientation of the immunodominant gp120 variable loops to shield the conserved, receptor-binding regions of the functional spike (48
). Significant efforts have therefore been made to design and construct immunogens that better resemble the functional trimeric envelope glycoprotein complexes, thereby preferentially exposing relevant neutralizing determinants to the immune system (1
). Several approaches have been taken to generate soluble stable HIV-1 envelope glycoprotein trimers, a majority of which is based upon gp140 molecules that possess both gp120 and the ectodomain of gp41. Frequently, the natural cleavage site between gp120 and gp41 has been rendered defective by site-directed mutagenesis or deletion of sequences to maintain gp120-gp41 association in a covalent manner (11
). Soluble gp140 molecules with an intact cleavage site have also been generated (4
). In these molecules, the heterodimeric monomeric subunits are stabilized by the introduction of disulfides between gp120 and gp41. A series of reports have shown that the addition of heterologous trimerization domains at the C terminus of the gp41 ectodomain increases the stability and homogeneity of soluble HIV-1 envelope trimers (51
). Molecules have been constructed by fusing either a trimerization domain derived from the yeast transcription factor (GCN4) or the trimeric motif from the T4 bacteriophage fibritin (FT) to cleavage-defective gp140 molecules to generate YU2gp140(-/GCN4) and YU2gp140(-/FT) constructs, respectively. The trimeric gp140s have been shown to stimulate neutralizing antibodies with greater breadth than YU2gp120, suggesting an advance over the homologous monomeric immunogens (16
The potential of viral systems to express oligomeric gp140 envelope immunogens and to stimulate neutralizing antibodies against HIV-1 has been investigated in previous studies, most frequently by using systems based on adenoviruses, poxviruses, or alphaviruses (8
). However, much remains to be understood about the qualitative and quantitative aspects of the proteins that are expressed in such viral systems as well as the immune responses stimulated by these systems. For example, recombinant virus-based vaccines typically induce a Th1-biased response and it is unclear whether this also allows a sufficient stimulation of humoral immunity. Protein-based regimens are generally the most effective in stimulating antibody responses, since high doses of antigen can be administered. In addition, depending on the adjuvant, protein-based vaccines generally stimulate a Th2-biased immune response, which drives humoral immunity efficiently (17
). The nature of the antibody response stimulated by different virus prime-protein boost compared to protein-only regimens has not been thoroughly investigated. To address some of the issues raised above, head-to-head comparisons between selected immunization regimens utilizing matched envelope glycoprotein immunogens are required.
In this study, we used the rSFV vector system to express trimeric envelope glycoproteins derived from the primary isolate YU2. This system allows the packaging of suicidal, single-round infectious viral particles (23
) that have been shown to stimulate protective immunity in a number of different infection models (2
). The potential of rSFV as a valuable component of a vaccine against HIV-1 has also been demonstrated, but these studies have primarily focused on the ability of rSFV to stimulate CD8+
T-cell responses (21
). Here, we have characterized the ability of recombinant Semliki Forest virus (rSFV) particles to express soluble YU2gp140(-/GCN4) trimers in vitro and compared their expression to monomeric YU2gp120 proteins expressed by rSFV. Further, we investigated the potential of rSFV particles expressing YU2gp140(-/GCN4) to stimulate humoral responses against HIV-1. The availability of a soluble envelope immunogen allowed us to directly compare immunization regimens based on repeated inoculation of purified protein in adjuvant to regimens based on rSFV priming followed by a single boost with purified YU2gp140(-/GCN4) protein in adjuvant.
The results presented in this report show that rSFV expression supports the production of conformationally intact YU2gp140(-/GCN4) molecules that are secreted as homogenous soluble trimers. Immunogenicity experiments were performed with mice and rabbits to study quantitative and qualitative aspects of the YU2gp140(-/GCN4)-directed immune responses (antibody isotypes, CD4+ T-cell responses, and neutralizing antibodies) stimulated by the different immunization regimens. We demonstrate that rSFV-based vaccination stimulated a Th1-biased response, which remained Th1 biased after a boost with recombinant protein in adjuvant. In contrast, and as expected, the immune response was Th2 biased in animals immunized with repeated inoculations of recombinant YU2gp140(-/GCN4) protein in Ribi adjuvant. Interestingly, these qualitative differences did not affect the neutralizing antibody responses, which were comparable using both immunization regimens.