R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 T Cells after Recombinant Influenza Hemagglutinin Vaccination

Abstract

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.

Flublok Quadrivalent Vaccine Adjuvanted with R-DOTAP Elicits a Robust and Multifunctional CD4 T Cell Response That Is of Greater Magnitude and Functional Diversity Than Conventional Adjuvant Systems

“…Previous studies of ours showed that in the B6 mouse model there were no significant H1-derived CD4+ T cell epitopes detectable [ 67 , 68 ], as shown in Supplementary Figure S1 . In our previous studies of influenza infection [ 67 ] and recombinant protein vaccination [ 61 ] in the B6 mouse strain, we defined three major HA-B epitopes (see Materials and Methods). Epitope discovery for the H3 protein in B6 mice was accomplished via infection and confirmed by infection and vaccination.…”

“…EliSpot assays used to quantify cytokine-secreting cells were performed as previously described [61]. Briefly, 96-well filter plates were coated with purified antibody at 2 µg/mL (IL-2: clone JES6-1A12, IFN-γ: clone AN-18, and IL-4/IL-5: clones 11B11/TRFK.5, respectively, BD Biosciences, San Diego, CA, USA) or granzyme B (1:60 dilution, 50 µL/well, R&D Systems, Minneapolis, MN, USA) in PBS overnight at 4 • C. Following incubation, the unbound antibody was removed by washing with media (complete DMEM media with 10% FBS (Fisher Scientific, Gibco, Waltham, MA, USA)).…”

“…We were particularly excited to explore the ability of a newly developed enantiospecific cationic lipid nanoparticle (R-DOTAP), promising as an antigen delivery system that has demonstrated safety in Phase I and Phase II clinical trials (NCT02065973, NCT04260126, NCT04580771, NCT05232851) [ 56 ]. In animal models, R-DOTAP has primarily been studied for CD8 T cell responses and tumor-specific immunity [ 57 , 58 , 59 , 60 ] and, more recently, for elicitation of other T cells and antibodies [ 61 , 62 ]. We hypothesized that the properties of R-DOTAP would also enhance CD4+ T cell responses and differentiation to recombinant protein antigens.…”

“…These arrays of features suggest that R-DOTAP is a promising candidate for inducing CD4+ T cell responses that could convey protective immunity to pathogens such as influenza. We recently studied the ability of R-DOTAP and other adjuvant systems in combination with a recombinant HA-B antigen to elicit the cytokines IL-2 and IFN-γ from elicited CD4+ T cells and found that the most robust response was conveyed by R-DOTAP [ 61 ].…”

Emerging Cationic Nanovaccines

“…The pressing challenge of developing a universal influenza vaccine lies in overcoming the limitations of current vaccines, which often fail to provide broad-spectrum protection due to the high antigenic variability of influenza viruses; in order to address this issue, it was developed an influenza universal vaccine based on the fused antigen NM2e; to this end, the adjuvant used was the cationic formulation composed of DODAB and poly(lactic acid) (PLA); the DODAB/PLA NPs were synthesized through a nanoprecipitation method, resulting in uniformly sized particles of 151 nm with a zeta potential of +30 mV; the NM2e@DODAB/PLA cationic nanovaccine exhibited a fourfold increase in antigen uptake by bone marrow-derived DC compared to the antigen alone accompanied by robust activation and maturation of DC, characterized by elevated expression of markers such as CD40, CD86, and MHC II, as well as increased production of cytokines including IFN-γ, TNF-α, and IL-6; in vivo studies demonstrated the prolonged presence of the antigen in lymph nodes for over 14 days post-immunization; the formulation induced high levels of specific IgG antibodies and strong cytotoxic T lymphocyte activity as indicated by increased secretion of granzyme B and IFN-γ; most notably, the NM2e@DDAB/PLA nanovaccine achieved over 90% cross-protection efficiency in mice challenged with both homosubtypic (H3N2) and heterosubtypic (H1N1) influenza viruses; NM2e@DDAB/PLA nanovaccine showed no significant toxicity from serum biochemical analyses and histopathological examinations [146]. This formulation for the influenza nanovaccine was based on the concept of combining a biocompatible polymer and a cationic compound as developed earlier in our group for PMMA/ DODAB hybrid NPs which yielded also similar results presenting the model antigen OVA [15,61,112,147] In another attempt to produce more effective vaccine strategies to induce broad and protective immunity against influenza, the cationic lipid DOTAP was used to prepare DOTAP NPs to carry the recombinant influenza hemagglutinin (HA) antigen; DOTAP NPs significantly enhanced the recruitment of CD4 T cells producing IL-2 and IFN-γ compared to the squalene-based adjuvant AddaVax, even when AddaVax was combined with the TLR9 agonist CpG; DOTAP-adjuvanted vaccine elicited high frequencies of cytokine-producing CD4 T cells in both draining lymph nodes and spleens, with an approximately 9 to 12-fold increase in IL-2-producing cells and a 5.6 to 6.5-fold increase in IFN-γ-producing cells; the response to HA was broadly distributed across three identified CD4 T cell epitopes, with DOTAP NPs inducing a balanced and robust response to each epitope suggesting that DOTAP NPs generated a comprehensive immune response that is less susceptible to viral antigenic variation [148].…”