Large-scale expansion of spike-reactive T cells feasible after COVID-19 vaccination

The rollout of several different vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19), has significantly reduced the number of new SARS-CoV-2 infections worldwide.  

Many of these vaccines provide the body with information on the spike (S) glycoprotein of SARS-CoV-2, which mitigates the entry of the virus into host cells. With this information, the vaccinated individual's immune system can recognize the protein and generate specific antibodies that will attack this virus if the host is infected in the future.

Since antibody production is impaired in people with innate and/or acquired immunodeficient health conditions, better immunization results might be obtained by expanding the generation of S protein-reactive T cells; namely, CD4+ and CD8+ cells ex vivo.

A new bioRxiv* preprint research paper reports on the successful use of a protocol to expand SARS-CoV-2-reactive T-cells a thousand-fold, thus providing a basis for T-cell-based adoptive cell immunotherapy for this illness.

The critical role of cellular immunity

The newer variants of SARS-CoV-2 evade detection by the immune system, thereby making it more difficult to achieve antibody-mediated neutralization. More specifically, mutations in the S glycoprotein often change the conformation of this antigen.

This conformational change in the S protein makes it more difficult for specific antibodies to bind efficiently to neutralize the virus. As a result, anti-S protein antibodies react less effectively to the antigen. This becomes particularly concerning when considering the current vaccines that are being administered throughout the world today, as they predominantly target the S protein almost exclusively. This immunization mechanism can ultimately render the current vaccines quite vulnerable to immune evasion by mutations.

The adaptive cellular response is a critical part of antiviral immunity, as it makes use of cytotoxic CD8+ T-cells that attach to infected cells expressing viral proteins. However, this attachment is based on viral peptides that are presented in a background of major histocompatibility complexes (MHC) and T-cell receptors (TCRs).

T-cell immunity is not seriously affected by the SARS-CoV-2 mutations that have emerged so far; therefore, enhancing this immune response presents a promising complement to the role of humoral immunity on viral neutralization. One challenge that researchers encounter when looking to improve this aspect of the immune response is that patients with weakened immune systems typically cannot activate cellular immunity, which depends on recruitment by B cells.

Study aim

The current study conducted in Prague, Czech Republic describes the decision of the researchers to explore alternatives to the in vivo expansion of cellular immune responses to the virus. To this end, the researchers were looking to develop a novel COVID-19 immunization technology that would benefit patients who lack the humoral immune capacity.

Healthy antibody response

The researchers began this study by first identifying healthy participants with no history or serological evidence of SARS-CoV-2 infection. The study participants were then administered two doses of the Pfizer mRNA SARS-CoV-2 spike-based COVID-19 vaccine, BNT162b2, at an interval of 3-4 weeks, to induce the production of antibodies to the viral spike.

No antibodies were detected in the test subjects against the nucleocapsid, envelope, or papain-like protease, which rules out any prior infection in these individuals. All vaccine recipients showed receptor-binding domain (RBD)-specific immunoglobulin G (IgG) antibodies after one dose, with this response further enhanced after the subjects received the second dose.

One and two recipients in the study also showed IgG responses to the S2 subunit of the S glycoprotein after the first and second doses, respectively, indicating that the RBD is immunodominant. IgA antibodies were also found to be even more specific for the RBD. IgM antibodies were found to arise in only one donor after receiving the first dose.

Thus, the COVID-19 mRNA vaccines produced a precise antibody response that was not reactive to other coronaviruses.

Cellular immunity after vaccination

Cellular immunity is the important layer of the immune protection against viruses, as it prevents virus amplification after infection.” In fact, cellular immunity against viruses is primarily mediated by cytotoxic CD8+ T-cells.

Isolated peripheral blood mononuclear cells (PBMCs) from vaccine recipients failed to show the presence of S protein-reactive CD8+ T-cells before or after vaccination.

Upon this discovery, both the pre-and post-vaccination samples were enriched by culturing with interleukin-2 (IL-2) for 12 days. Samples were then stimulated with a pool of peptides from the SARS-CoV-2 S glycoprotein.

Pre-vaccination samples did not enrich the reactivity of CD8+ T-cells to the S protein; however, after two doses of the vaccine, cell samples were found to contain higher numbers of T-cells that produce tumor necrosis factor α (TNF-α), interferon γ (IFNγ), and TNF-α/IFNγ.

After the first dose, the researchers found reactive CD4+ cell populations producing TNF-α. After the second dose, the samples were still richer in reactive CD4+ T-cells producing TNF-α. No effect was observed on CD4+ IFNγ- or TNF-α /IFNγ-producing T-cells in any of the samples, including those that underwent 12-day-enriched cell cultures.

Image Credit: Orpheus FX /

Thus, vaccination against COVID-19 allowed donor PBMCs in culture to become enriched with the SARS-CoV-2 S glycoprotein-reactive CD4+ and CD8+ T-cells. The vaccination-induced antibody response correlated closely with the cellular responses to the S protein antigen.

These results mirror earlier cancer studies, where patient samples showed higher frequencies of T-cells that were reactive to peptides associated with tumor antigens, following vaccination with tumor cell-bearing dendritic cells obtained from culture.  

Large-scale expansion of spike-reactive T cells in culture

The researchers expanded the number of reactive T-cells by a mean of 744-fold using the rapid expansion protocol (REP). The expanded cultures showed higher proportions of viable cells, as well as S protein-specific CD4+ and CD8+ T-cells.

What are the implications?

The findings showed that the combination of COVID-19 vaccination, peptide-mediated enrichment, and REP could lead to the production of therapeutically relevant numbers of SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T-cells.”

Such antigen-specific T-cell-based therapeutic approaches have already been applied for adoptive cellular immunotherapy (ACI) in some cancers and after stem cell transplants in bone marrow disorders. In the latter case, the aim is to restore antiviral immunity by transferring expanded populations of virus-specific CD8+ T-cells from the stem cell donor.

The type of approach discussed here has the potential to help patients with impaired cellular immunity to mount an effective response to SARS-CoV-2 by expanding cytotoxic spike-specific T-cells enriched and expanded on a large scale.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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