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COVID-19 vaccines are playing a vital role in controlling the COVID-19 pandemic. As SARS-CoV-2 variants encoding mutations in the surface glycoprotein, Spike, continue to emerge, there is increased need to identify immunogens and vaccination regimens that provide the broadest and most durable immune responses. We compared the magnitude and breadth of the neutralizing antibody response, as well as levels of Spike-reactive memory B cells, in individuals receiving a second dose of BNT126b2 at a short (3–4 week) or extended interval (8–12 weeks) and following a third vaccination approximately 6–8 months later. We show that whilst an extended interval between the first two vaccinations can greatly increase the breadth of the immune response and generate a higher proportion of Spike reactive memory B cells, a third vaccination leads to similar levels between the two groups. Furthermore, we show that the third vaccine dose enhances neutralization activity against omicron lineage members BA.1, BA.2 and BA.4/BA.5 and this is further increased following breakthrough infection during the UK omicron wave. These findings are relevant for vaccination strategies in populations where COVID-19 vaccine coverage remains low. Author summary: COVID-19 vaccines have been vital in controlling the current pandemic. With the emergence of SARS-CoV-2 viral variants, it is important to understand factors that influence the neutralization breadth of vaccine responses. Here we study the impact of the interval between the 1st and 2nd BNT162b2 vaccine dose on neutralization breadth and how this is further affected by vaccine boosters and breakthrough infections. [ABSTRACT FROM AUTHOR]

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