Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice

Abstract

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

A Single Dose of Inactivated Influenza Virus Vaccine Expressing COBRA Hemagglutinin Elicits Broadly-Reactive and Long-Lasting Protection

“…R-DOTAP is designed to attach to the negatively charged cell membrane and deliver the peptide or protein antigens into the host cells, which stimulates cellular immunity and cross-presentation (40). R-DOTAP adjuvanted COBRA rHA vaccines elicit antibodies with wider HAI breadth and stimulated more multifunctional T cells (CD8+ and CD4+ T cells) than unadjuvanted COBRA rHA vaccines (24). The addition of R-DOTAP to COBRA-SIV improved the antibody activity spectrum (Figure 2F) with only one vaccination and induced more stem-binding antibodies than unadjuvanted COBRA-SIV or AddaVax adjuvanted COBRA-SIV (Figure 4B).…”

“…AddaVax and these T cell receptors (TCRs) also recognized by more distinct influenza peptides (24). Future studies will evaluate the cellular immunity elicited by COBRA-IIV vaccines and the map TCR usage in order to optimize the vaccine formulation.…”

“…AddaVax, as an equivalent of the commercial influenza vaccine adjuvant MF59, can stimulate a balanced Th1/Th2 activation (22). The novel cationic nanoparticle R-DOTAP can stimulate cellular immunity, especially CD8T cells, to eliminate intracellular pathogens (23,24). In the previous study, the primary vaccination of COBRA-IIVs successfully recalled memory B cells in the pre-immune mice (H. Shi, X. Zhang, P. Ge, V. Meliopoulos, P. Freiden, B.…”

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

“…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.…”

“…Published studies indicate that R-DOTAP induces Type I IFN responses via MyD88 and endosomal TLR-7 and TLR-9 [ 59 ]. Moreover, broadly reactive IgG antibody responses to SARS-CoV-2 and HA recombinant proteins were elicited in response to R-DOTAP vaccination in prime boost strategies [ 62 ]. 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.…”