The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) consists of a trimer of noncovalently associated gp120/gp41 heterodimers, which form the functional viral spike and mediate entry into CD4 and CCR5 receptor-positive host target cells. The exterior envelope glycoprotein, gp120, and the transmembrane glycoprotein, gp41, are the sole virally encoded targets for neutralizing antibodies on the surface of the virus and likely represent a critical immunogenic component for an effective prophylactic vaccine against HIV-1 (7
). The HIV-1 Envs are also potential targets for cell-mediated immune responses, and as such, their inclusion in future HIV-1 vaccine candidates may contribute to the induction of both protective antibody and cellular immune responses (25
). In attempts to elicit antibodies that recognize the functional Env spike, soluble trimeric molecules containing full-length gp120 covalently linked to the gp41 ectodomain have been designed (5
). An incremental advance in neutralizing antibody elicitation using soluble trimeric Env spike mimetics compared to the use of monomeric gp120 was observed (28
), but further improvements in trimer immunogen design are still needed both to mimic better the functional viral spike and to elicit broadly neutralizing antibodies (reviewed in reference 7
The immunogenicity of cleavage-defective Env trimers derived from the primary R5 isolate YU2, possessing heterologous trimerization motifs derived either from T4 bacteriophage (foldon) or from the transcription factor GCN4, were examined in several small animals studies (6
). However, to date these trimeric Env immunogens were not analyzed for their ability to elicit neutralizing antibodies and Env-specific T-cell responses in nonhuman primates. Other oligomeric Env proteins, such as the SF162 gp140 proteins with or without a deletion of the second major variable region (ΔV2), were evaluated with nonhuman primates (1
). For example, a recent study demonstrated that gp140SF162ΔV2 administered in the MF59 adjuvant mediated protection against mucosal challenge with the SHIV-162P4 virus (2
), implicating Env-directed immune responses in mediating protection against this homologous virus challenge.
The capacity of different Env immunogens to stimulate humoral and cellular responses was also evaluated using genetic means of expression, such as plasmid DNA or recombinant viral vectors, followed by immunization of purified Env protein in an adjuvant to boost antibody responses (15
). While such heterologous immunization regimens may enhance Env-directed cellular immune responses, little is known about the quality of neutralizing antibody responses induced by viral vector priming followed by a protein boost or about the relative responses elicited by regimens consisting of purified Env protein in an adjuvant using homologous or heterologous protein priming/boosting. One potential concern when Env is expressed in vivo from DNA or viral vectors is that the actual dose and the antigenic integrity of the immunogen are not easily assessed. For example, incorrectly folded but immunogenic Env protein released from dying cells in vivo may adversely affect the quality of the elicited antibody response. Since many candidate vaccines which are currently moving into clinical trials rely on in vivo genetic expression (9
), analysis of the quality of antibody responses elicited by genetic platforms is warranted and is an aim of our present study.
Previously we performed a head-to-head study using rabbits to characterize neutralizing antibody responses stimulated by sequential administrations of YU2 Env protein trimers emulsified in the Ribi adjuvant compared to two immunizations of single-round infectious recombinant Semliki Forest virus (rSFV) particles expressing YU2 trimers followed by a boost with YU2 trimeric proteins in an adjuvant (15
). SFV is an alphavirus, genetically related to Sindbis virus and Venezuelan equine encephalitis virus (VEE), two other viruses for which single-round replicon systems have been developed (8
). SFV has been used extensively in preclinical immunogenicity experiments (3
), human vaccine candidates based on VEE are already in clinical trials (11
), and currently the chimeric VEErep/SINenv vector is one of the most frequently used alphavirus-based vector systems for preclinical HIV-1 vaccine studies (35
). In our previous study, we demonstrated that rSFV infection of BHK-21 cells in vitro resulted in the secretion of homogeneous and stable gp140 trimers into the culture medium (15
). Here, to evaluate recombinant alphavirus priming in greater detail, we performed a comprehensive study with nonhuman primates to examine both antibody and cellular responses induced by gp140 trimers with and without rSFV priming. We inoculated cynomolgus macaques with Env trimers administered as purified protein in the AS01B adjuvant system from GlaxoSmithKline Biologicals (GSK) or expressed in vivo from rSFV particles. We used cleavage-defective (−) gp140 trimers possessing the heterologous foldon (F) trimerization motif (gp140-F) (54
), hereafter referred to as gp140-F trimers. As an additional arm of the study, we used a modified gp120 core protein in AS01B as a prime, followed by boosts with Env trimers, both formulated in AS01B. We sought to test the concept of “immunofocusing” by first inoculating with the stable gp120 core, followed by the gp140-F proteins in sequence to focus responses on the shared and conserved core elements of the two forms of Env. The modified gp120 core proteins, hereafter referred to as “stable core,” contain pocket-filling mutations and were stabilized by disulfide-linked cysteine pairs spanning the inner and outer domains of gp120, as previously described (56
). These modifications were designed to reduce the flexibility of gp120 and to improve presentation of conserved but discontinuous cross-domain antigenic surfaces of Env, such as the CD4-binding site. We hypothesized that priming with a stable core protein and boosting with gp140-F trimers would favor the expansion of Env-specific B cells recognizing common determinants between the two immunogens and that this might translate into an increased breadth of neutralization. We also wished to determine if priming with stable cores and boosting with trimers would alter the Env-specific T-cell response compared to immunization with gp140-F trimers alone, since this may represent a strategy to focus the cellular response on conserved sequences common to the two immunogens. Our results show that the properties of the Env antigen used to prime the response are more important for shaping the overall Env-specific immune response than the platform by which Env is delivered. Priming with the stable core protein and boosting with trimers strikingly focused the T-cell response on core sequences of Env even as measured after the trimer boost. However, this regimen did not improve the neutralizing antibody response, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved structural determinants on the functional viral spike to stimulate antibodies possessing an increased breadth of neutralization.