The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function.

Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.

Inclusion of an Optimized Plasmodium falciparum Merozoite Surface Protein 2-Based Antigen in a Trivalent, Multistage Malaria Vaccine.

Plasmodium falciparum merozoite surface protein (PfMSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant PfMSP2 (rPfMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rPfMSP8 as a carrier to optimize a rPfMSP2-based subunit vaccine. rPfMSP2 and chimeric rPfMSP2/8 vaccines produced in Escherichia coli were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high-titer anti-PfMSP2 Abs that recognized the major allelic variants of PfMSP2. Vaccine-induced T cells recognized epitopes present in both PfMSP2 and the PfMSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rPfMSP2 vaccines with glucopyranosyl lipid adjuvant-stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rPfMSP2/8 + glucopyranosyl lipid adjuvant-stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8, and the P. falciparum 25 kDa sexual stage antigen fused to PfMSP8 (Pfs25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rPfMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.

Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody.

Vaccine trials and cohort studies in Plasmodium falciparum endemic areas indicate that naturally-acquired and vaccine-induced antibodies to merozoite surface protein 2 (MSP2) are associated with resistance to malaria. These data indicate that PfMSP2 has significant potential as a component of a multi-antigen malaria vaccine. To overcome challenges encountered with subunit malaria vaccines, we established that the use of highly immunogenic rPfMSP8 as a carrier protein for leading vaccine candidates rPfMSP119 and rPfs25 facilitated antigen production, minimized antigenic competition and enhanced induction of functional antibodies. We applied this strategy to optimize a rPfMSP2 (3D7)-based subunit vaccine by producing unfused rPfMSP2 or chimeric rPfMSP2/8 in Escherichia coli. rPfMSP2 formed fibrils, which induced splenocyte proliferation in an antigen receptor-independent, TLR2-dependent manner. However, fusion to rPfMSP8 prevented rPfMSP2 amyloid-like fibril formation. Immunization of rabbits elicited high-titer anti-PfMSP2 antibodies that recognized rPfMSP2 of the 3D7 and FC27 alleles, as well as native PfMSP2. Competition assays revealed a difference in the specificity of antibodies induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. Rabbit anti-PfMSP2/8 was superior to rPfMSP2-elicited antibody at opsonizing P. falciparum merozoites for phagocytosis. These data establish rPfMSP8 as an effective carrier for a PfMSP2-based subunit malaria vaccine.