mRNA-nanoparticles for therapeutic vaccination against HIV

In 2021, 38.4 million people were living with HIV globally. New HIV infections and AIDS-related deaths are steadily declining due to effective antiretroviral treatment. However, although this treatment in most cases normalizes the CD4+ T cell count in the blood, immune function is not always restored. Moreover, HIV is able to integrate its DNA into the genome of the host cell and when the infected cells enter a resting state, a viral reservoir is created. Therefore, when the treatment is stopped, viral rebound inevitably occurs. In this regard, a functional cure would be interesting, as the viral replication would be suppressed without necessarily eradicating the virus from the body. To induce a functional cure, a therapeutic vaccine should skew the HIV-specific immune response towards viral control and as a result, HIV replication would be suppressed without the need for daily treatment.

A small number of HIV-infected individuals, the so-called elite controllers, show the ability to control HIV to undetectable levels in the blood, without the need for antiretroviral treatment. Therefore, elite controllers are often taken as a model for HIV control. An important observation is that the HIV-specific CD8+ T cells in this population display polyfunctional characteristics, meaning that they produce many different cytokines, chemokines and other factors simultaneously. Moreover, these T cells are suggested to suppress rebounding virus from the prime location of the reservoir, the (gut-associated) lymphoid tissues. Therefore, it was hypothesized that eliciting a polyfunctional CD8+ T cell response is a crucial requirement for a therapeutic vaccine. Nevertheless, although many attempts have been made over the years, no efficient T cell-inducing therapeutic vaccines have been developed so far. Given the ease of manufacturing and its promise in therapeutic vaccination of cancer patients, our laboratory ventured in mRNA vaccine research as the basis for a therapeutic vaccine against HIV. An important feature of mRNA is that it results in intracellular presence of the antigen when taken up by the cell, thereby stimulating a CD8+ T cell-mediated immune response. In a first clinical trial, mRNA encoding HIV antigens was pulsed in dendritic cells from people living with HIV and used as a vaccine. In a second clinical trial, intranodal delivery of unformulated mRNA as a therapeutic vaccine against HIV was investigated. Although these approaches were safe and vaccine-induced immune responses were demonstrated in most patients, no clinical effects were observed. Therefore, the need for a more efficient delivery system led to packaging of the mRNA in nanoparticles to create an off-the-shelf universal therapeutic vaccine. In this thesis, we investigated two mRNA-nanoparticles for their ability to induce CD8+ T cell responses and their potential as a therapeutic vaccine against HIV.

Firstly, we investigated two cationic peptides, containing arginine (LAH4-L1R) or lysine (LAH4-L1) residues. The cationic peptides in combination with the mRNA spontaneously formed nanoparticles through electrostatic interactions and were able to protect the mRNA from degradation. Remarkably, mRNA in combination with LAH4-L1R formed slightly smaller and more positively charged nanoparticles in comparison to LAH4-L1. In the context of mRNA delivery, LAH4-L1 outperformed the arginine variant in transfecting dendritic cells. Furthermore, we demonstrated that both cationic peptides were able to induce maturation and assembly of the inflammasome in primary in vitro generated dendritic cells. Consequently, dendritic cells transfected with modified mRNA-peptide nanoparticles induced a polyfunctional CD8+ T cell response in vitro. In experiments performed in mice, we noticed that after intradermal immunization with mRNA-peptide nanoparticles, the roles tended to be reversed and LAH4-L1R-mRNA nanoparticles took the upper hand in stimulating CD8+ T cells. However, even though both cationic peptides showed very efficient transfection and T cell induction characteristics in vitro, in vivo the antigen-specific T cell stimulation was insufficient.

Secondly, we compared a lipid nanoparticle based on the ionizable lipid C12:200 with an αgalactosylceramide containing variant, the galsomes, in mice. The galsomes were designed to induce activation of invariant natural killer T (NKT) cells. Indeed, after intramuscular immunization, the mRNAgalsomes instructed an expansion of NKT cells in the draining lymph nodes. We used modified mRNA encoding ovalbumin and showed that after intramuscular immunization, the mRNA-galsomes gave rise to a stronger proliferative response of CD8+ T cells in lymphoid tissues compared to the conventional mRNA-lipid nanoparticles. Next, antigen presentation in the context of MHC I was observed in the migratory CD103+ dendritic cell subset after immunization with either formulation. Moreover, in in vitro generated dendritic cells, both formulations were able to induce secretion of IL-1β. By using modified HIV-gag mRNA, we noticed that even for doses substantially lower than those used in literature, we still observed an efficient immune response. Subsequently, immunization with the mRNA-galsomes and mRNA-lipid nanoparticles led to equally efficient polyfunctional CD8+ T cell responses. Moreover, the gag-specific CD8+ T cells were able to lyse target cells in (gut-associated) lymphoid tissues. In conclusion, because the mRNA-lipid nanoparticles and the mRNA-galsomes have the ability to induce polyfunctional antigen-specific CD8+ T cells, they are interesting candidates for therapeutic vaccination against HIV.