During the ongoing COVID-19 pandemic, one of the many unknowns has been the quality of SARS-CoV-2 immune memory following infection. Horiuchi and colleagues took advantage of the golden hamster as a model system to address how SARS-CoV-2 immune memory develops and how protective it is against reinfection with variants of concern (VOC). They found that SARS-CoV-2 infection in the golden hamster induces strong adaptive immunity and generates memory T and B cells. Significantly, the induced immune memory effectively protected the animals from reinfection by VOC, such as the SARS-CoV-2β variant.
Small animal models are critical to help understanding adaptive immune responses against infectious agents. For example, golden hamsters are susceptible to any circulating SARS-CoV-2 strain infection and also demonstrate transmission features comparable to those observed in people. These features make them an ideal model to study SARS-CoV-2 immune memory (Sia et al. 2020). Unfortunately, there is a lack of reagents and methodologies to analyze the immune response in golden hamsters, making it difficult to address some of these questions.
Detecting SARS-CoV-2 Specific Memory T and B Cells
To analyze the impact of the memory response against SARS-CoV-2, Horiuchi and colleagues first established a method to identify immune cell populations by flow cytometry analysis. By using commercialized antibodies, they evaluated the phenotype of T and B cells after primary immune responses in various tissues (i.e., lung, lung draining mediastinal lymph node (mLN), blood, and/or spleen) over a prolonged period (effector phase and memory phase) following intranasal infection with the SARS-CoV-2 Washington strain (SARS-CoV-2WA).
Horiuchi and colleagues applied two methods to evaluate T and B cell responses during the memory phase. First, to identify CD4+ T cell populations responsive to SARS-CoV-2 antigens, the team relied on synthetic peptides, specifically a peptide mix containing Spike, Nucleocapsid, and Matrix sequences.
Mononuclear cells isolated from various tissues were rested overnight and subsequently cultured for 24 hours with the SARS-CoV-2 synthetic peptide mix. CD4+ T cells activated by these peptides were then identified by flow cytometry analysis.
Second, SARS-CoV-2 Spike protein specific B cells were assayed by incubating mononuclear cells with biotin-labeled SARS-CoV-2 Spike protein and following detection with streptavidin-BV786 and -BV650.
These SARS-CoV-2 specific T and B cells detected in the hamsters after more than 40 days post infection (dpi) were defined as memory T and B cells.
T cell activation assay with SARS-CoV-2 synthetic peptides. To develop an antigen-specific T cell detection assay, Horiuchi and colleagues relied on peptides synthesized by GenScript corresponding to sequences within three critical SARS-CoV-2 antigens, Spike, Nucleocapsid, and Matrix proteins. Mononuclear cells originating from various tissues were Ficoll separated, rested overnight, and subsequently incubated with the SARS-CoV-2 peptide mix. Following stimulation, cells were analyzed by flow cytometry to identify activated T cell populations. “Created with BioRender.com.”
First, to assess protection from SARS-CoV-2WA reinfection, Horiuchi and colleagues rechallenged a group of hamsters at four months post-primary infection. Nasal and lung viral titers were evaluated to determine the extent of clearance.
Next, the team designed a series of adoptive transfer studies to dissect the role of memory T and B cells in viral clearance. To this end, lymphoid cells were isolated from SARS-CoV-2WA recovered hamsters and transferred into naïve animals. Transfused animals were then challenged with SARS-CoV-2WA (i.e., one-day post lymphocyte transfer) and antibody responses evaluated at 3 and 8 dpi. Additionally, to distinguish between the roles of memory T and B cells in viral clearance, Horiuchi and colleagues performed adoptive transfer experiments with B cell-depleted lymphoid cells.
Lastly, to determine if immune memory to SARS-CoV-2WA would protect from infection by VOC, the team challenged recovered hamsters with the SARS-CoV-2β variant.
Having established the timing of the memory phase following SARS-CoV-2WA infection in the hamster model, investigators were able to detect a population of SARS-CoV-2 specific memory CD4+ T cells expressing high levels of the activation markers Ki67 and IRF4 in lung and spleen tissues. Similarly, after 40 dpi, SARS-CoV-2 specific memory B cells were detected in the lung, spleen, and blood.
Antigen-specific T and B cell numbers declined over time in the hamster following SARS-CoV-2 infection and recovery. However, Horiuchi and colleagues demonstrated that SARS-CoV-2 specific memory cells remain in recovered animals past 40 dpi and successfully enact viral clearance from the respiratory tract.
Adoptive transfer studies allowed investigators to test the contribution of memory cells to protective immunity. Transfer of total lymphocytes and B cell-depleted lymphocytes, isolated from SARS-CoV-2WA recovered hamsters (i.e., after 40 dpi), boosted anti-RBD antibodies and induced nasal viral clearance in newly infected animals.
Lastly, it has been shown that antibodies against SARS-CoV-2WA are less effective in neutralizing VOC in vitro. Nevertheless, Horiuchi and colleagues found that SARS-CoV-2WA specific memory B and T cells are protective against rechallenge with VOC such as SARS-CoV-2β and effectively support viral clearance, primarily by generating functional antibodies against SARS-CoV-2β.