Scale bar, 30m. function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies. Subject terms:Immunological memory, RNA vaccines, Viral infection, Viral infection Here the authors use three different mouse models to show that prior infection or mRNA vaccination can protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) independently of antibodies, highlighting the importance of T cell-derived interferon- (IFN-) in host defense and the need to consider this measure of protection in vaccination. == Main == Neutralizing antibody responses have been viewed traditionally as the main bulwark against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) re-infection for vaccinated or previously infected individuals13. While the Rabbit Polyclonal to Tubulin beta wider effector functions of antibodies extend beyond neutralization4,5, there is an inherent vulnerability owing to the transient nature dBET57 of these neutralizing antibodies68. This vulnerability is further exacerbated by the emergence of viral variants that can evade these responses911. This has led to a prevailing notion that vaccines might necessitate periodic updates to counteract the evolving variants of concern (VOCs) and might require consistent boosting to maintain efficacy. Conversely, T cells are acknowledged for their role in averting severe manifestations of COVID-19, and their contribution to immunity has been echoed across multiple animal studies12,13. The lingering question, however, revolves around the capability of T cells to provide robust protection against a heterologous SARS-CoV-2 challenge in the absence of antibodies. This scenario is not only hypothetical; it could manifest in cases where VOCs dodge neutralizing antibodies, or in patients like those with agammaglobulinemias1416, or those undergoing treatments for cancer or multiple sclerosis who are treated with B cell-depleting agents1720. Evaluating the protective role of T cells independent of antibodies is fraught with challenges. For example, mice inherently deficient in B cells might not only be devoid of antibodies but may also have anomalies in their lymphoid tissue architecture2123, altered macrophage phenotype24,25and defective T cell responses26. Additionally, using adoptive transfer of T cells as a methodology poses its own set of challenges, potentially failing to station adequate numbers of T cells where and when they are most needed. To overcome these limitations, we took advantage of DHLMP2a mice, which possess B cells and retain normal lymphoid tissue architecture, yet are devoid of both surface and circulating immunoglobulins27. Using three independent mouse models, including one hybrid (hy) angiotensin-converting enzyme 2 (ACE2) knock-in mouse model generated ad hoc for this study, we show here that prior infection or mRNA vaccination can indeed offer protection against heterologous SARS-CoV-2 challenge, all while circumventing the need for antibodies. == Results == == Antibody-independent protection in K18-hACE2 transgenic mice == To determine if protection against heterologous SARS-CoV-2 challenge exists independently of antibodies, we initially made use of K18-hACE2 transgenic mice28. These mice, known for expressing human ACE2 (hACE2) predominantly in epithelial dBET57 cells under the control of the cytokeratin 18 dBET57 (KRT18) promoter, were crossbred with DHLMP2a mice, which maintain B cells and normal lymphoid tissue architecture yet lack both surface and circulating immunoglobulins27. Hereinafter, these are referred to as antibody-deficient (Ab) mice. Using a specialized inhalation tower system29, both antibody-sufficient (Ab+) and AbK18-hACE2 transgenic mice were exposed to aerosolized SARS-CoV-2 (D614G) under consistent pressure, temperature and dBET57 humidity (Extended Data Fig.1a). This exposure leads to robust viral replication in the respiratory tract29. However, unlike intranasal inoculation, this method averts fatal viral neuroinvasion29. Accordingly, all Ab+K18-hACE2 transgenic mice survived SARS-CoV-2 infection without detectable viral RNA in their brains (Extended Data Fig.1b,c). Conversely, ~70% of AbK18-hACE2 transgenic mice died between 9 and 12 days postinfection, revealing pronounced viral RNA in their brains (Extended Data Fig.1b,c). These results are consistent with the unusually elevated hACE2 expression in K18-hACE2 transgenic mice28,29, highlighting the protective role mucosal antibodies have in defending against mucosal infections30. == Extended Data Fig. 1. Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection in K18-hACE2 transgenic mice. == (a) Experimental setup. Antibody-sufficient (Ab+,n= 3-10) and antibody-deficient (Ab,n= 328) K18-hACE2 transgenic mice were infected with a target dose of 2 105TCID50of SARS-CoV-2 D614G through aerosol exposure. Brain was collected and analyzed 9 days postchallenge. (b) Survival curve of Ab+(n= 10, blue line) and Abmice upon infection (n= 28, red line).(c)Quantification of SARS-CoV-2 RNA in the brain of the indicated mice. RNA values are expressed as copy number per.