Background
The HIV-1 envelope (Env) is the sole target of broadly neutralizing antibodies and, therefore, the context in which it is presented to the immune system is critical for efficiently engaging responder B cells. We previously designed and tested in pre-clinical models a virus-like particle (VLP)-forming env-gag mRNA vaccine that elicited tier-2 neutralizing antibodies and protection against heterologous SHIV infection in rhesus macaques. Here, we further optimize this platform by inclusion of the viral protease to process Gag and produce mature, rather than immature, VLPs. We hypothesized that protease supplementation could impart mobility to membrane-anchored Env spikes, resulting in Env clustering and subsequent B-cell receptor (BCR) crosslinking.
Methods
Appropriately dosed and timed expression of the protease was achieved using a full-length gag-pol mRNA transcript. Mice immunizations were performed to investigate the immunogenicity of the improved vaccine platform. To elucidate the mechanisms underlying the increased effectiveness of protease-containing vaccine platform, we used cryo-electron tomography (cryo-ET) to visualize the structure and organization of Env spikes on the surface of mature and immature virus like particles (VLPs).
Results
We found that mRNA formulations both with and without the protease produce VLPs with high density of Env trimers on their surface. Immunization of mice with HIV-1 env mRNA in combination with SIV gag and gag-pol mRNAs induced higher titers of trimer-binding and neutralizing antibodies than env alone or env + gag mRNAs. By cryo-ET, immature VLPs featured a well-organized, assembled Gag lattice underneath the viral membrane and displayed infrequent Env spikes on their surface, which did not show clustering. In contrast, in mature VLPs an organized Gag lattice was not observed, while some particles showed initial core condensation; mature VLPs displayed a higher density of Env spikes, with apparent clustering of 3-4 spikes visible on some particles.
Conclusions
Clustering of Env spikes on the surface of mature SHIV gag VLPs may have enhanced BCR crosslinking and, thereby, imparted superior immunogenicity to our gag-pol supplemented VLP-forming mRNA vaccine. These results suggest that vaccines capable of generating mature VLPs may have an enhanced effectiveness.
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