Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID50) geometric mean titers of 501 in the 10-g dose group and 3481 in the 100-g dose group. Vaccination induced type 1 helper T-cell (Th1)Cbiased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-g dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. Conclusions Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (Covid-19), is responsible for the 2020 global pandemic.1,2 Developing a vaccine that is safe, effective, and rapidly deployable is an urgent global health priority. The majority of vaccine candidates have focused on inducing antibody responses against the trimeric SARS-CoV-2 spike (S) protein, a class I fusion protein that facilitates binding to the angiotensin-convertingCenzyme 2 (ACE2) receptor and triggers virusCcell-membrane fusion.3 A variety of vaccine approaches4 and formulations for targeting the SARS-CoV-2 S protein are being pursued, including nucleic acid PKC-theta inhibitor 1 vaccines (RNA and DNA),5-8 human and simian replication-defective adenoviral vaccines,9,10 whole-inactivated SARS-CoV-2,11,12 and subunit protein vaccines.13 In assessing the immunogenicity and protection of vaccines in preclinical animal models, nonhuman primates provide several advantages for clinical translation. They are outbred, have greater similarity to humans than rodents in innate immune responses and B-cell and T-cell repertoires, and allow for the use of clinically relevant vaccine doses. Recent studies have shown that SARS-CoV-2 targets similar replication sites and recapitulates some aspects of Covid-19Clike disease in nonhuman primates.7,14 After SARS-CoV-2 infection, nonhuman primates have transient viral replication in the upper and lower airways and mild inflammation in the lung that resolves within 14 days.7 Thus, nonhuman primates are a useful animal model for assessing vaccine-mediated protection against early viral replication.7,14 The use of messenger RNA (mRNA) is a promising approach for Covid-19 vaccination, since it combines rapid manufacturing and expeditious modification of the encoded immunogen, both of which accelerate vaccine development.14 RNA vaccines encoding viral antigens have been shown to be safe and immunogenic in several clinical trials,5,15 including in a recent phase 1 clinical trial of mRNA-1273, a SARS-CoV-2 vaccine candidate.16 Data from a mouse model showing that a low dose of mRNA-1273 induced a robust neutralizing antibody response and high-level protection against SARS-CoV-26 raised the possibility that vaccination with mRNA-1273 could prevent or limit both upper- and lower-airway infection in nonhuman primates. Methods Vaccine mRNA and Lipid Nanoparticle Production We synthesized a sequence-optimized mRNA encoding prefusion-stabilized SARS-CoV-2 S-2P protein in vitro. The mRNA was purified by oligo-dT affinity purification and encapsulated in a lipid nanoparticle through a modified ethanol-drop nanoprecipitation process, as described previously.17 Rhesus Macaque PKC-theta inhibitor 1 Model Experiments in animals were performed in compliance with National PKC-theta inhibitor 1 Institutes of Health (NIH) regulations and with approval from the Animal Care and Use Committee of the Vaccine Research Center and from Bioqual (Rockville, MD). Challenge studies were conducted at Bioqual. The authors vouch for the accuracy and completeness of the data in this report. Female and male Indian-origin rhesus macaques (12 of each sex; age range, 3 to 6 years) were sorted according to sex, age, and weight (see Supplementary Appendix 2, available with the full text of this article at and then stratified into groups of three. Within each stratum, one of the three animals was assigned to each study group arbitrarily. Animals were vaccinated intramuscularly at week 0 and at week 4 with either 10 or 100 g of mRNA-1273 in 1 ml of 1 1 phosphate-buffered saline (PBS) into the right hind leg. Unvaccinated control animals were administered an equal volume of 1 PBS. At week 8 (4 weeks after the second vaccination), all animals were challenged with a total dose of 7.6105 plaque-forming units (PFU). The stock of 1 1.9105 PFU per milliliter SARS-CoV-2 (USA-WA1/2020 strain) was administered in a volume of 3 ml by the intratracheal route and in a volume of 1 ml by the intranasal route (0.5 ml per nostril). Pre- and postchallenge specimen collection is detailed CXCL5 in Figure S1 in Supplementary Appendix 1 (note that all supplementary.