Control of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal.The epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions, we determined potential sites of escape by comparing antibody binding of peptides containing wild-type residues versus peptides containing a mutant residue.Individuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination.The finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests that protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level.This work was supported by NIH grants AI138709 (PI JMO) and AI146028 (PI FAM). JMO received support as the Endowed Chair for Graduate Education (FHCRC). The research of FAM was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.When SARS-CoV-2 – the virus that causes COVID-19 – infects our bodies, our immune system reacts by producing small molecules called antibodies that stick to a part of the virus called the spike protein. Vaccines are thought to work by triggering the production of similar antibodies without causing disease. Some of the most effective antibodies against SARS-CoV-2 bind a specific area of the spike protein called the ‘receptor binding domain’ or RBD. When SARS-CoV-2 evolves it creates a challenge for our immune system: mutations, which are changes in the virus’s genetic code, can alter the shape of its spike protein, meaning that existing antibodies may no longer bind to it as effectively. This lowers the protection offered by past infection or vaccination, which makes it harder to tackle the pandemic. As it stands, it is not clear which mutations to the virus’s genetic code can affect antibody binding, especially to portions outside the RBD. To complicate things further, the antibodies people produce in response to mild infection, severe infection, and vaccination, while somewhat overlapping, exhibit some differences. Studying these differences could help minimize emergence of mutations that allow the virus to ‘escape’ the antibody response. A phage display library is a laboratory technique in which phages (viruses that infect bacteria) are used as a ‘repository’ for DNA fragments that code for a specific protein. The phages can then produce the protein (or fragments of it), and if the protein fragments bind to a target, it can be easily detected. Garrett, Galloway et al. exploited this technique to study how different portions of the SARS-CoV-2 spike protein were bound by antibodies. They made a phage library in which each phage encoded a portion of the spike protein with different mutations, and then exposed the different versions of the protein to antibodies from people who had experienced prior infection, vaccination, or both. The experiment showed that antibodies produced during severe infection or after vaccination bound to similar parts of the spike protein, while antibodies from people who had experienced mild infection targeted fewer areas. Garrett, Galloway et al. also found that mutations that affected the binding of antibodies produced after vaccination were more consistent than mutations that interfered with antibodies produced during infection. While these results show which mutations are most likely to help the virus escape existing antibodies, this does not mean that the virus will necessarily evolve in that direction. Indeed, some of the mutations may be impossible for the virus to acquire because they interfere with the virus’s ability to spread. Further studies could focus on revealing which of the mutations detected by Garrett, Galloway et al. are most likely to occur, to guide vaccine development in that direction. To help with this, Garrett, Galloway et al. have made the data accessible to other scientists and the public using a web tool.