Differential peripheral blood mononuclear cell reactivity against SARS-CoV-2 proteins in naïve and previously infected subjects following COVID-19 vaccination

The decline in vaccine efficacy and the risk of reinfection by SARS-CoV-2 make new studies important to better characterize the immune response against the virus and its components. Here, we investigated the pattern of activation of T-cells and the expression of inflammatory factors by PBMCs obtained from naïve and previously infected subjects following COVID-19 vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins. PBMCs showed low levels of ACE2 and TMPRSS2 transcripts, which were not modulated by the exposure of these cells to SARS-CoV-2 proteins. Compared to S1 and RBD, N-RBD stimulation showed a greater ability to stimulate T-cell reactivity, according to CD25 and CD69 markers. Interestingly, T-cell reactivity was more pronounced in vaccinated subjects with prior SARS-CoV-2 infection than in vaccinated donors who never had been diagnosed with COVID-19. Finally, N-RBD stimulation promoted greater expression of IL-6 and IFN-γ in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. These data suggest that PBMCs from previously infected and vaccinated subjects are more reactive than those derived from just vaccinated donors. Moreover, the N-RBD together viral proteins showed a greater stimulatory capacity than S1 and RBD viral proteins.

The pathophysiology of COVID-19 is not fully understood at this time, but acute inflammation and disseminated intravascular coagulation appear as the main causes of mortality worldwide [2] . The immune response to viral infection involves both innate and adaptive immunity components participating in this process, and, depending on the viral In the present study, we investigated the pattern of T-cell activation and the expression of inflammatory factors by peripheral blood mononuclear cells (PBMCs) from naïve and previously infected subjects following vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins.

Sample collection and peripheral blood mononuclear cell isolation
Peripheral blood samples were obtained from 19 donors at the School of Health Sciences of the University of Brasilia (UnB), including convalescent COVID-19 donors and individuals unexposed to SARS-CoV-2. All donors followed the vaccination protocol established in Brazil. PBMCs were isolated from whole blood by density gradient centrifugation according to the manufacturer's instructions (Histopaque, Sigma-Aldrich, USA). The study protocols were approved by the Institutional Ethics Committee.

Data analysis and statistics
FlowJo software 10.0.7. was used for Flow Cytometry data analysis (FlowJo LLC, USA). Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis and plotting. Statistical significance was calculated using Student's t -test analyses. The value of p < 0.05 was considered statistically significant.

Volunteers' characterization
A total of 19 donors were recruited for this study (6 male and 13 female). All of them received at least 2 doses of vaccine against COVID-19. Among such individuals, 10 were convalescent from previously COVID-19 infection, and 9 were not infected by SARS-CoV-2. Convalescent patients had COVID-19 diagnosis confirmed by nasopharyngeal swab qRT-PCR. The average age of the volunteers is 27 years (16-60 years), and all convalescent COVID-19 donors had mild disease without hospitalization ( Table 1 ).

Activation marker in PBMCs stimulated with SARS-CoV-2 proteins
Stimulation of PBMCs with RBD protein did not change the expression of any evaluated lymphocyte activation markers ( Fig. 1 a-d). In contrast, PBMC stimulation with S1 protein induced an increase in CD25 ( p = 0.03) ( Fig. 1 e). Interestingly, stimulation of PBMCs with N-RBD protein also promoted an increase in the expression of CD25 ( p = 0.002). Interestingly, between two groups of vaccinated individuals, only the previously infected subjects showed a statistically significant increase in CD25 expression ( p = 0.002) ( Fig. 1 h). CD69 expression was also induced on T-cells after stimulation with N-RBD ( p = 0.005). Both samples from naive and previously infected individuals showed an increased expression of this receptor ( p = 0.03 and p = 0.04, respectively) ( Fig. 1 i).

Expression levels of SARS-CoV-2 receptors and inflammatory factors in PBMCs stimulated with SARS-CoV-2 proteins
PBMCs expressed very low levels of the ACE2 gene, with one sample showing no gene amplification. Furthermore, none of the SARS-CoV-2 proteins evaluated was able to stimulate the expression of ACE2 in PBMCs ( Fig. 2 a-c). We identified a certain variability in the expression of TMPRSS2 among the samples of PBMCs evaluated, but, once again, the antigenic stimuli tested using the different SARS-CoV-2 proteins failed to exert a significant modulation on the transcriptional levels of this gene ( Fig. 2 d-f).
Interestingly, the stimulation of PBMCs with RBD or S1 protein promoted a reduction in the transcriptional levels of IL-6 ( p = 0.01 and p = 0.006, respectively). In both cases, the reduction of IL-6 was shown to be statistically significant in samples from donors who did not have COVID-19 ( p = 0.03 and p = 0.02, respectively). In contrast, PBMCs stimulated with N-RBD protein had increased transcriptional levels of IL-6 ( p = 0.02) and IFN-( p = 0.02). Samples from donors who had COVID-19 sustained increased IFN-( p = 0.04), different from samples of partici- pants with no COVID-19 history. Stimulation of PBMCs with RBD led to a reduction in TNF-transcription only in samples from donors who did not have COVID-19 ( p = 0.04). Finally, the stimulation of PBMCs with S1 protein also promoted a reduction in TNF-( p = 0.03), which was only maintained in samples from donors who did not have COVID-19 ( p = 0.02) ( Fig. 2 g-o).

Discussion
Recently, countries with broader access to vaccination have made significant advances in immunizing the population through the use of booster doses, given the reported decline in vaccine efficacy with time. However, this decline in vaccine efficacy and the risks associated with the emergence of new variants of SARS-CoV-2, keep the world on alert regarding the risks that still exist due to the COVID-19 pandemic [9] . In this context, a better understanding of the immune response of immunized patients becomes essential. In this study, after stimulating PBMCs of vaccinated patients with specific proteins of SARS-CoV-2, we demonstrated that the expression of ACE2 and TMPRSS2 is not modulated by the exposure of these cells to proteins S1 and RBD, and that the N-RBD protein stimulates greater T-cell reactivity, mainly in patients who were vaccinated and had previous SARS-CoV-2 infection.
Remarkably, it has been demonstrated that the incubation of epithelial cells with S protein or RBD induces ACE2 expression, which can worsen the infection by providing more receptors to the virus [10] .
Our results demonstrated a reduced expression of ACE2 mRNA levels in PBMCs, some of which did not even show gene amplification. Furthermore, incubating these cells with S1, RBD, or N-RBD did not modulate their ACE2 and TMPRSS2 mRNA levels. In accordance, little to no expression of ACE2 has been observed in most human PBMC samples, as assessed at the protein level [11] . Surprisingly, even in patients with COVID-19 there seems to be lower ACE2 levels in circulating blood cells compared to healthy individuals [12] . Despite this, even at low levels, the amount of ACE2 on the T-cell membrane is sufficient to mediate virus binding and entry into these cells [13] .
The cellular immune response mediated by T-cells is essential for the control of the infection caused by a coronavirus. Individuals who recovered from the infection have been shown to have SARS-CoV-specific memory T-cells [14] . In this study, the stimulation of T-cells with S1 protein increased CD25 expression on T-cells. On the other hand, the stimulation of T-cells with the N-RBD protein stimulated the expression of CD25 and CD69 on T-cells from vaccinated patients, which suggests that the N-RBD segment showed a greater ability to stimulate the T-cell reactivity in our samples, compared to the RBD and S1. In agreement with our observations, it has been shown that convalescent patients from previously COVID-19 infection have specific T-cells against N and S proteins [15] and that N protein has an outstanding immunogenic potential in these patients [16] . It is also important to note that the increased expression of CD25 was observed in subjects who had previously COVID-19 infection, but not in vaccinated individuals who were not exposed to the virus. This finding indicates that possibly the vaccinated convalescent individuals have greater immunologic memory and reactivity against the N-RBD viral protein, compared with subjects vaccinated but not infected by SARS-CoV-2.
Intriguingly, PBMC stimulation with either S1 or RBD did not stimulate IL-6 and TNF-transcription in such cells. We actually found a reduction in the levels of these transcripts, which was maintained in vaccinated patients who did not have SARS-CoV-2 infection. We also did not observe changes in INF-levels after stimulating PBCMs with S1 and RBD proteins. On the other hand, stimulation with N-RBD promoted higher expression of IL-6 and IFN-in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. More importantly, the increase in the expression of activation markers, associated with the greater production of IFN-, indicates a greater capacity for viral protein recognition and protection against the virus in the vaccinated donors [17] .
Our study has as main limitation the small number of samples. However, the results obtained consistently show that PBMC exposure to S1, RBD, and N-RBD proteins does not stimulate ACE2 and TMPRSS2 expression in these cells. Furthermore, N-RBD protein has greater immunogenic potential in vaccinated donors, and T-cells from vaccinated patients who had SARS-CoV-2 infection show greater reactivity when exposed to virus antigens, especially to N-RBD protein. In addition to contributing to the investigation of T-cell reactivity, in vitro studies with models of human immune cells and viral proteins are important for a better understanding of the immunogenic potential of SARS-CoV-2 in vaccinated patients and for the establishment of platforms that allow a better understanding of the inflammatory process involved in COVID-19.

Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) and Fundação de Apoio à Pesquisa do Distrito Federal.

Ethics approval
The present study was approved by the local Ethical Committee. Written informed consent was obtained from all subjects included in the study.