SARS-CoV-2: Repetitive boosters causes humoral and cellular immune tolerance

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A new study by researchers from Chongqing Medical University-China and the First Affiliated Hospital of Chongqing Medical University- China has found that repetitive COVID-19 booster shots cause humoral and cellular immune tolerance.

The study findings were published in the peer reviewed journal: iScience.
https://www.sciencedirect.com/science/article/pii/S2589004222017515

Currently, vaccination against COVID-19 has been promoted worldwide, although sustained protection against the newly emerged SARS-CoV-2 variant strains has been continuously challenged. Clinical evidence has proven that the inclusion of an additional booster vaccine can re-stimulate the protective immune response (Cheng et al., 2022; Gruell et al., 2022).

Whether such re-establishment of vaccine-induced immune response could be repeated by continued application of boosters is being questioned, yet largely unknown at present. Here, we compared the effects of repeated RBD vaccine boosters with a conventional immunization course to those with an extended vaccination strategy, in a Balb/c mice model.

We found that the protective effects from the humoral immunity and cellular immunity established by the conventional immunization were both profoundly impaired during the extended vaccination course. Specifically, extended vaccination not only fully impaired the amount and the neutralizing efficacy of serum RBD-specific antibodies, but also shortened the long-term humoral memory.

This is associated with immune tolerance in germinal center response, along with decreased numbers of spleen germinal center B and Tfh cells. Moreover, we demonstrated that extended immunization reduced the functional responses of CD4+ and CD8+T cells, restrained the population of memory T cells, and up-regulated the expression of PD-1 and LAG-3 in Te sub-type cells.

An increased percentile of Treg cells was also observed, accompanied by significant elevation of IL-10 production. Together, we provided crucial evidence that repetitive administration of RBD booster vaccines may negatively impact the immune response established by a conventional vaccination course and promote adaptive immune tolerance.

In our recent study, a three-dose course of RBD vaccines successfully yielded both humoral and cellular immune protection for 4 months in a Balb/c mice model (Gao et al., 2021). In the current study, we found that a subsequent fourth administration of the same vaccine continued to stimulate the production of RBD-specific neutralizing antibodies, whose serum levels were sustained for at least 6 weeks.

These findings were in accordance with the reported neutralizing effect of the fourth dose of the Pfizer vaccine on the SARS-CoV-2 mutants (Tanne, 2022). However, when we administrated additional doses of the same vaccine booster, with the attempt to induce a similarly enhanced or at least sustained immune response, we observed an overt reduction of the overall immune responses.

Both the titer of RBD-specific antibodies and the serum neutralizing potency against SARS-CoV-2 pseudo-viruses were severely impacted, with more than two folds decrease in the IC50 against the most recently emerged SARS-CoV-2 variants, including the Delta and Omicron mutants.

This suggests that repetitive administration of RBD booster vaccines may actively promote humoral immune tolerance, instead of functional humoral immunity. A recent independent report made similar observation that one additional booster with inactivated SARS-CoV-2 vaccine in human significantly reduced the titer of the RBD-specific antibodies, when administered at a time with already observed loss in protective efficacy (Perez-Then et al., 2022).

It suggested that for booster vaccines developed targeting wild-type SARS-CoV-2 RBD, the doses or the immunization course might be a key factor that could be negatively influenced by immune tolerance. It might be of importance to monitor the serum levels of antibodies prior to any extended vaccination.

The evidenced immune tolerance from repetitive dosing with homologous boosters in our study suggests that caution should be exercised when optimizing the extended plan for SARS-CoV-2 booster vaccination. Instead of continuous dosing with homologous prime vaccines, a mid-way switch to heterologous booster choices may offer a chance of improvement to the observed energy against Omicron mutants (Reynolds et al., 2022).

Such vaccination strategy may take advantage of the otherwise unsatisfying immune response consequential to the serum phenomenon termed antibody imprinting or original antigenic sin (OAS), which has been an emerging subject in SARS-CoV-2 vaccination, especially for children (Lavinder and Ippolito, 2022). Encountering heterologous boosters, the OAS-dominated immune memory response might generate a faster and stronger neutralizing protection from a preferential activation of existing B cell clones with antibodies recognizing epitopes of the wild-type strain.

This might provide a window of opportunity for sufficient time and accumulation of heterologous antigens that could induce proper recruitment of new naive B cells to generate another primary or secondary response to new epitopes presented. It is reasonable to speculate that such variant-specific immune adaptation may enhance the durability and/or efficacy for the evolving protective need. Within such framework, tailored mRNA vaccines may be a good choice to circumvent the loss of effective humoral and cellular immunity from conventional vaccines developed with the wild-type virus. Given the differences between human and mice in mechanism of OAS, further studies are definitely needed to strategically optimize the application of vaccine boosters for durable protection against SARS-CoV-2.

In the attempt to explain the mechanism of humoral immune tolerance associated with our extended immunization course, we analyzed the mechanisms involved in RBD-specific antibody production. With prolonged booster vaccination to mice, we observed significantly reduced number of elementary factors and assistant T cells that would be required for B cell maturation and activation, relative to the conventional course of immunization.

Insufficient availability of Tfh cells might hinder the conventional process of B cell functional differentiation, and the decreased amount of serum IL-4 might impede B cell activation. These assumptions were supported by the fact that a significantly lower number of active B cells was detected within the germinal center from mice of the extended immunization group as comparing to the animals received conventional course of vaccination.

Notably, we found that the proportion of memory B cell was markedly reduced in the extended immunization group, together with signs of B cell immune tolerance, indicating the repetitive vaccination of booster shots shared similar mechanisms as seen from humoral immune tolerance of repeated antigen exposure, as during chronic viral infections (Han et al., 2013).

In addition to the humoral immune responses, cellular immune tolerance was observed during the extended course of RBD booster vaccination. Limited levels of antigen-specific memory T cell activation and profoundly decreased IL-2 and IFN-γ secretion were found in the sera of the extended group, contrast to sustained cellular immune responses after 4 dosings of RBD vaccines.

It was reported that the chronic infection with HBV virus could result in antigen-specific cellular immune tolerance, which was manifested as a partial or complete inability to induce active immune response from antigen-specific CD8+T cells and significant increase in the surface expressions of inhibitory receptors, including PD-1, Tim-3 and CTLA-4.

Similarly, we found that prolonged administration of RBD booster vaccines overtly increased the levels of PD-1 and LAG-3, accompanied by significant reduction of the memory CD8+T cells (Han et al., 2013). This is of particular importance, because memory CD8+T cell response is shown to play a predominant role for effective response against newly emerged SARS-CoV-2 variants, which greatly challenged humoral immunity with collective neutralization escape mutations (Tarke et al., 2022; Naranbhai et al., 2022; Swadling et al., 2022).

Therefore, over-stimulation with the same booster vaccine or reinfection after vaccination may severely hamper the cellular immune response established by conventional vaccine course, which, together with challenged humoral immune responses, may lead to prolonged disease duration and/or aggravation of symptoms in recipients.

Moreover, over-vaccination may generate an immunosuppression micro-environment that is also an important facilitator of immune tolerance. We demonstrated that both the percentage of CD25+Foxp3+CD4+ Treg cells and the levels of immunosuppression cytokines IL-10 were up-regulated after extended RBD vaccine booster vaccination.

This may result in reduced activation and differentiation of B cells on antigen stimulation, as well as functional inhibition of antigen-presenting cells (APCs) and consequential decrease in CD8+T cell activation (Damo and Joshi, 2019; Field et al., 2020; Turner et al., 2020).

Indeed, we observed both humoral and cellular immune tolerance with the doses of extended booster administrations, which made it safe to speculate that over-vaccination might severely impact the immune protective efficacy established by conventional SARS-CoV-2 immunization, and probably enhance disease severity for new COVID-19 patients or re-infectants.

Although RBD subunit vaccines cannot entirely represent inactivated or mRNA vaccines, especially in antigen delivery way. A recent report in The New England Journal of Medicine demonstrated that a fourth mRNA vaccination of healthy young health care workers only shows marginal benefits (Regev-Yochay et al., 2022). Whether extended vaccination with other COVID-19 vaccines based on wild-type SARS-CoV-2 sequence will induce immune tolerance, further investigations are required.

In summary, we characterized the comprehensive effects of extended immunization with RBD booster vaccines in a balb/c mouse model. Our findings revealed that repeated dosing after the establishment of vaccine response might not further improve the antigen-specific reactivity; instead, it could cause systematic tolerance and inability to generate effective humoral and cellular immune responses to current SARS-CoV-2 variants. Our study provides timely information for the prevention of COVID-19. It puts an extended immunization course with two or more RBD-based vaccine boosters at debate, and warns for the future applications of vaccine enhancers without proper evaluation of serum antibody titers and T cell functions.

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