Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan (China) in December 2019, the virus has spread rapidly, causing the coronavirus disease 2019 (COVID-19) pandemic (Meo et al., 2021). Several vaccines have been developed, successfully protecting against symptomatic COVID-19 cases and deaths. Among the vaccines available in Spain, two of themBNT162b2 (Pfizer—BioNTech) (PZ) and mRNA-1273 (Moderna—NIAID) (MD), are messenger RNA (mRNA) vaccines. Other vaccines, such as ChAdOx1-S (University of Oxford—AstraZeneca) (AZ), is based on the “Chimpanzee” adenovirus vector (Gobierno de España, 2024).

In response to SARS-CoV-2 infection, humans produce specific antibodies, CD4⁺ and CD8⁺ T cells. SARS-CoV-2-specific antibodies are directed against the spike protein (S) and nucleocapsid (N). Special roles are played by neutralizing antibodies against the S1 subunit on the receptor-binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2) sites, thereby facilitating endocytosis, viral entry into host cells (Tretyn et al., 2021). In case of the T cells (including CD4⁺ T and CD8⁺ T cells) recognize, in addition to the nucleocapsid and spike protein, membrane proteins (M) of the virus and are present in most COVID-19 patients (Sette and Crotty, 2021). It is known that while antibodies, produced by B cells in response to viral infection, provide a first line of defense against subsequent exposures, SARS-CoV-2 -specific-T cells (including CD4⁺ and CD8⁺ T cells) could limit disease severity, reduce its duration and drive rapid recovery (Kedzierska and Thomas, 2022). During natural infection, CD8⁺ T cells play an important complementary role to contain the infection through their ability to eliminate already infected cells, while CD4⁺ helper T cells, amongst other functions, provide signals that support the development of antibody responses (Castro Dopico et al., 2022). Therefore, a combined humoral and cell-mediated response are required for an optimal immunity to SARS-CoV-2 infection (Goletti et al., 2021).

In line with natural infection, SARS-CoV-2 vaccination has also been shown to induce robust humoral and T-cell responses (Goletti et al., 2021; Goel et al., 2021; Ben Ahmed et al., 2022; Zhang Z. et al., 2022). Vaccine-induced immunity remains effective at preventing severe disease, hospitalization, and death, even at later time points when antibody levels may have declined (Griffin et al., 2021; Thomas et al., 2021).

Hence, our purpose was to evaluate the humoral and cellular immunity after SARS-CoV-2 infection and/or vaccination according to the type of vaccine, number of doses and combination of vaccines.

In the present study, we evaluated post-COVID-19 and/or post-vaccination immunization according to the type of vaccine, number of doses and combination of vaccines. The IgG antibodies against the S protein and IFN-γ- producing CD4⁺ and CD8⁺ T lymphocytes are a reliable read-out of humoral and cellular immunity to SARS-CoV-2 following natural infection and vaccination.

Our results revealed that previous exposure to SARS-CoV-2 antigens by infection strongly affected subsequent cellular and humoral immunity. In fact, among the unvaccinated participants, infected people managed to develop IFN- γ levels up to 10 times higher than those without prior infection. The data from vaccinated individuals demonstrated that humoral and cellular immune responses were significantly higher in SARS-CoV-2 experienced compared to naive participants, regardless of the type, number of doses and combination of vaccines. Our observations are consistent with previous studies showing that history of SARS-CoV-2 infection influences the increase in immunity of vaccinated people (Angyal et al., 2021; Tretyn et al., 2021; Desmecht et al., 2022).

Among healthy people, the level of protection against COVID-19 could be enhanced with vaccination, reaching antibodies and IFN- γ values similar or even higher than those detected in COVID-19 patients (Grigoryan and Pulendran, 2020; Kedzierska and Thomas, 2022). Based on our results, high antibody titer was observed in all vaccinated individuals compared to titer from unvaccinated participants. In addition to these effects on the antibody response, CD4⁺ and CD8⁺ T cell responses were also induced following vaccination. As described Grigoryan et al., the mRNA and viral-vector-based platforms may be beneficial for the induction of T cell responses (Grigoryan and Pulendran, 2020). Given that Ag1 stimulates production of IFN- γ by CD4⁺ T cells and Ag2 stimulates both CD4⁺ and CD8⁺ T cells, mainly median concentration of IFN- γ by both cell subpopulations was greater than that of CD4⁺ alone. Nevertheless, several authors have shown that IFN-γ by CD4⁺ T cells numerically dominated over IFN- γ by CD8⁺ T cells after COVID-19 immunization (Murugesan et al., 2021; Barreiro et al., 2022), similar to what was found in our study for individuals vaccinated with 2 doses of PZ and previous infection. Moreover, Matew et al (Mathew et al., 2020) observed that CD8⁺ T cells are not present in all infected patients and ~20% of patients did not have substantial CD8⁺ T-cell activation. Hence, it could be possible that in the present study, those individuals with a prior infection before receiving 2 doses of PZ would not have had a CD8⁺ T-cell activation.

Conversely, patients with acquired or inherited immune disorders may show variable immune responses to vaccination against SARS-CoV-2 (Oyaert et al., 2022; Fiorcari et al., 2023). In the present study, the two immunocompromised participants developed a negative humoral and cellular response after 8 months and 1 month after administration of the second and third dose, respectively. These results are in line with Oyaert et al. (2022) who evaluated the humoral and cellular immune response in a large cohort of patients diagnosed with immune deficiency disorders and revealed incomplete and/or delayed humoral and cellular immune responses up to 3 months after administration of the second dose. However, the participant who had been infected between the first and second dose, had positive IgG anti-S antibodies at 2 months after administration of the third dose, confirming that prior SARS-CoV-2 infection improves immune response.

One vaccine dose could be sufficient to increase both cellular and humoral immune response in COVID-19–recovered subjects (Angyal et al., 2021; Ebinger et al., 2021; Mazzoni et al., 2021; Tretyn et al., 2021; Varghese et al., 2022), even surpassing the titer achieved by the second dose of vaccine in SARS CoV-2 naive recipients (Varghese et al., 2022). These findings were obtained in the present study, the immune response after the first dose of the AZ vaccine in previously infected people was higher than after two doses of AZ in those who had never had the disease. A prior SARS-CoV-2 infection triggered the immune system to a very strong response to a single dose of the vaccine. The first dose, given in to people whose immune systems had already been stimulated by the natural infection, had a similar effect when given as a second “booster” dose (Tretyn et al., 2021).

Our data is part of two projects in which the immune response to several SARS-CoV-2 vaccines was evaluated. The results of these studies demonstrated that after vaccine inmunization, those people without previous infection developed a level of anti-N IgG and IgM antibodies values of 0.00142-0.0071 and 0.00142-0.0142 BAU/ml, respectively. These antibody titers were lower than those found in the three patients vaccinated with two doses of AZ and without knowledge of previous infection, whose immune response (anti-S antibodies and IFN- γ produced by T-cell) values were outside the ranges of the rest of the group. Therefore, despite anti-N antibody titers of < 0.142 BAU/ml, probably these three patients could have had asymptomatic SARS-CoV-2 infection at some point prior to analysis. Our observations are in line with previous studies showing a higher concentration of IFN- γ produced by T cells in asymptomatic than in symptomatic patients (Le Bert et al., 2021; Zhao et al., 2021).

Regarding a two-dose vaccination regimen, the highest humoral and cellular immunity detected was for AZ/PZ combination. Several reports have published that the mixing of the AZ and PZ vaccine induce a more robust immune response compared to that induced by the two doses of either of these vaccines (Borobia et al., 2021; Schmidt et al., 2021). Besides, our results demonstrated that the cellular immune response to the heterologous mix was even significantly greater than that to AZ/AZ/AZ and PZ/PZ/PZ combinations.

On the other hand, a third boosting vaccine dose not only reduces the risk of symptomatic breakthrough infections by new SARS-CoV-2 variants (Zhang B. et al., 2022) but also the incidence of severe cases and mortality related to COVID-19 (Muhsen et al., 2021), particularly in uninfected individuals. Moreover, both humoral and cellular immunity waned within 6 months after the first two vaccine doses, highlighting the urgency of administering a booster dose to induce stronger immunity against SARS-CoV-2 (Tartof et al., 2021; Uwamino et al., 2022). In addition, the benefit of the booster dose is greater in native individuals who have lower persisting levels of humoral or cellular immunity before the booster dose (Desmecht et al., 2022). Based on both our results and previous study published by Demecht et al, the booster dose induced an increase in humoral and cellular immune responses (Desmecht et al., 2022). Based on the concentration of anti-Spike antibodies, we found that individuals after receiving a third boosting vaccine dose (MD) had significantly higher antibody titers than participants who had received two doses of homologous vaccination (PZ/PZ or AZ/AZ). These findings are consistent with previous study reporting that mRNA Booster Vaccination (PZ or MD) Enhanced Antibody Response against SARS-CoV-2 in individuals vaccinated with either two prior doses of mRNA (PZ) or inactivated virus vaccine (AZ) (Zhang B. et al., 2022).

One of the limitations of our study was the heterogeneity in terms of the number of participants in each group studied, since the sample size was not calculated at the beginning of the study. This is due to the random inclusion of subjects, the participants were recruited as they voluntarily provided consent and as the reagents became available to perform the assays.

In summary, the present study demonstrate that heterologous vaccination (AZ/PZ) induced the strongest immunity among the two-dose vaccination regimens. The immunity offered by the third-booster dose of SARS-CoV-2 vaccine depends not only on the type of vaccine administered but also on previous doses and prior infection. Previous exposure to SARS-CoV-2 antigens by infection strongly affect immunity of vaccinated individuals.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study involving humans was approved by Ethics Committee of Hospital Clınico San Carlos, Madrid (Spain) (References: 21/071-E and 21/ ́193-E). All research was conducted according to the Declaration of Helsinki principles. The participants provided their written informed consent to participate in this study.

EC: Supervision, Conceptualization, Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Data curation. MM: Writing – review & editing, Writing – original draft, Methodology, Investigation. CN: Methodology, Investigation, Writing – review & editing, Writing – original draft. JL: Writing – review & editing, Writing – original draft, Methodology, Investigation. EM: Writing – review & editing, Writing – original draft, Methodology, Investigation. AD-I: Validation, Supervision, Conceptualization, Writing – review & editing, Writing – original draft. ER: Visualization, Formal analysis, Data curation, Writing – review & editing, Writing – original draft, Validation.