Effective vaccination of eligible populations has proven to be one of the most successful strategies for preventing the spread of communicable diseases to humans, especially during pandemic and epidemic outbreaks. Indeed, the implementation of standardized childhood vaccination schedules for measles, polio, diphtheria, and other common infectious diseases has drastically reduced the incidence of infection worldwide, according to the WHO and CDC. In general, the types of vaccines administered include live, subunit, polysaccharide, viral vector, and messenger RNA (mRNA). Typically, vaccines using live or inactivated organisms induce longer-lived immunity than subunit vaccines containing viral or bacterial antigens, whereas plain polysaccharide vaccines do not generate memory (Petrovsky and Aguilar, 2004; Pollard and Bijker, 2021).

Serology is the conventional method for assessing vaccine efficacy, primarily by measuring vaccine-induced neutralizing antibodies in serum or plasma (Plotkin, 2010; Chen et al., 2022). In addition to serology, memory B and T cells present in circulating peripheral blood post-vaccination, which persist throughout an individual's lifetime, serve as valuable indicators of vaccine efficacy and longevity (Waltari et al., 2019; Davis, 2020). Several studies have investigated the frequency of memory B and T cells specific to vaccine antigens both before and after booster vaccination using ELISpot and flow cytometry. These studies have noted the necessity of booster vaccines to significantly enhance the detection of memory cells specific to vaccine antigens in peripheral blood, given the initially low levels of circulating memory cells (Perrett et al., 2014; Chen et al., 2018; Chapman et al., 2020; Hartley et al., 2020). Considering the decline in memory cells specific to childhood vaccine antigens over an individual's lifespan, the primary objective of this study was to standardize a methodology to monitor existing memory cells as an indicator of vaccine efficacy and longevity in vaccinated individuals without the need for recall booster vaccines.

Several studies have aimed to devise in vitro methodologies for detecting rare antigen-specific memory B cells in the peripheral blood of vaccinated individuals. For example, Bernasconi et al. were first to demonstrate that memory B cells isolated from peripheral blood mononuclear cells (PBMCs) proliferate and differentiate to antibody-secreting cells (ASCs) in response to CpG with interleukin (IL)-15 (Bernasconi et al., 2002). Subsequent research refined this method and found that stimulation of purified B cells with CpG along with IL-2, IL-10, and IL-15 led to the detection of antigen-specific memory and IgG production (Buisman et al., 2009). Further work by Cao et al. compared various polyclonal activators and confirmed the effectiveness of CpG or CpG with CD40L and IL-21 in inducing antigen-specific IgG-secreting B cells (Cao et al., 2010). Conversely, Pinna et al. tested R848, another toll-like receptor (TLR) agonist, in combination with IL-2 and showed that it efficiently stimulates memory B cells in total PBMCs (Pinna et al., 2009). Finally, Jahnmatz et al., discovered that treating PBMCs with R848, either alone or in combination with IL-2, proved to be more effective than using CpG with or without IL-2 and IL-10 in stimulating the expansion of memory B cells into ASCs within total PBMCs (Jahnmatz et al., 2013). In addition, studies have reported correlations between the frequency of measles-specific memory T cells in peripheral blood and antibody serum levels, as well as correlations between lymphoproliferation, serum measles-specific IgG levels, and IFN-γ production (Ovsyannikova et al., 2003; Dhiman et al., 2005).

In our study, we expanded upon these prior investigations, comprehensively evaluating the efficacy of the two aforementioned B cell polyclonal activators in transitioning preexisting vaccine-specific memory cells into ASCs. Knowing the role of T cells in the development of antigen-specific memory B cells post-vaccination, we concurrently assessed the transition of preexisting antigen-specific memory T cells into effector cells upon direct exposure to the vaccine antigen. Herein, we report on the effectiveness of our approach as a straightforward and reliable method of measuring the frequency and function of rare memory B and T cells specific to childhood vaccines.