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An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

A promising strategy for the enhancement of vaccine-mediated immune responses is by directly targeting protein antigens to immune cells. Targeting of antigens to the dendritic cell (DC) molecule Clec9A has been shown to enhance antibody affinity and titers for model antigens, and influenza and enterovirus antigens, and may be advantageous for immunogens that otherwise fail to elicit antibodies with sufficient titers and breadth for broad protection, such as the envelope protein (Env) of HIV. Previously employed targeting strategies often utilize receptor-specific antibodies, however it is impractical to conjugate a bivalent IgG antibody to oligomeric antigens, including HIV Env trimers. Here we designed single chain variable fragment (scFv) and single chain Fab (scFab) constructs of a Clec9A-targeting antibody, expressed as genetically fused conjugates with the soluble ectodomain of Env, gp140. This conjugation did not affect the presentation of Env neutralising antibody epitopes. The scFab moiety was shown to be more stable than scFv, and in the context of gp140 fusions, was able to mediate better binding to recombinant and cell surface-expressed Clec9A, although the level of binding to cell-surface Clec9A was lower than that of the anti-Clec9A IgG. However, binding to Clec9A on the surface of DCs was not detected. Mouse immunization experiments suggested that the Clec9A-binding activity of the scFab-gp140 conjugate was insufficient to enhance Env-specific antibody responses. This is an important first proof of principle study demonstrating the conjugation of a scFab to an oligomeric protein antigen, and that an scFab displays better antigen binding than the corresponding scFv. Future developments of this technique that increase the scFab affinity will provide a valuable means to target oligomeric proteins to cell surface antigens of interest, improving vaccine-generated immune responses.