demonstrates that non-replicating mRNA is able to induce antibody titers in NHPs. mRNA vaccines exhibited a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, PSI-7977 our data PSI-7977 demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated computer virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that this mRNA technology is usually a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases. Introduction The introduction of prophylactic vaccination has been one of the most effective medical interventions to fight and eradicate infectious diseases. Despite its great successes, the continuous threat of infectious brokers for which no vaccine exists and the introduction of new pathogens into the human population emphasize the need for the development of novel safe vaccines and even vaccine platforms capable of rapidly responding to those needs. Ideally, PSI-7977 these vaccine platforms should be highly versatile at minimal development and production costs. Vaccines based on mRNA could meet these requirements because they offer multifaceted advantages including flexible antigen design, a cost-effective manufacturing process allowing for parallel production of multiple mRNA vaccines1C5 and rapid manufacturing, which could be of crucial importance during pandemics.6 Using exclusively unmodified nucleosides, we have demonstrated that non-replicating mRNA vaccines are immunogenic and capable of inducing protection against lethal rabies and influenza virus infections after intradermal vaccination (i.d.) in mice, rats, ferrets, and pigs.7,8 These vaccines contained free and protamine-complexed mRNA to support both strong antigen expression and immunostimulation. 9 This approach was PSI-7977 specifically optimized for i.d. administration and showed lower efficacy when given by the intramuscular route (i.m.) as preferred route for prophylactic vaccination, highlighting the important role of formulation in vaccine CCR2 design. Studies with self-amplifying mRNA vaccines investigated the effect of different formulations such as lipid-nanoparticles (LNP)10 or cationic nanoemulsion (CNE)11 on the immunogenicity of mRNA vaccines. Self-amplifying vaccines benefited from formulation with LNP or CNE and were able to induce protective antibody titers. In contrast, non-replicating mRNA did not induce any detectable antibody titers even when formulated with CNE. These data reveal a gap between self-amplifying and non-replicating mRNA, which cannot be closed only by formulation. This demonstrates a clear need to optimize the mRNA itself to PSI-7977 obtain sufficient expression levels. To this end, different approaches of optimization are pursued. In recent publications chemically modified nucleosides were used to reach sufficient antigen expression,12C15 which is in contrast to our proprietary mRNA technology, which employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.16 In these studies, mice were vaccinated i.m. with LNP-formulated mRNA vaccines inducing protection against Zika virus challenge infections,12 and protection against placental damage and fetal demise in challenged pregnant mice.13 Importantly, protective efficacy against Zika virus challenge infections was also demonstrated in non-human primates (NHPs) after i.d. vaccination.14 Immunogenicity data in NHPs are absolutely desirable taking into account the experiences with early DNA vaccines, for which efficacy could not be translated from mice to NHPs. The study by Pardi et al. demonstrates that non-replicating mRNA is able to induce antibody titers in NHPs. To this end, they applied ten separate i.d. injections distributed on the back of the animals. For prophylactic vaccination single injections are highly desirable with i.m. application being the routinely used route. Notably, previous studies have demonstrated lower vaccine immunogenicity after i.m. compared to i.d. vaccination, which could be due to the lower.