CLEC5A Activation in Inflammatory Monocytes: A Mechanism for Enhanced Adaptive Immunity Following COVID-19 mRNA Vaccination in a Preclinical Study

THP-1 cell line is a human monocytic leukemia cell line (THP-1—TIB-202, ATCC) used in the in vitro assays to verify any differentiation caused by the bivalent commercial COVID-19 mRNA vaccine (Pfizer BioNTech^®, Cambridge, MA, USA). The THP-1 cells were transfected with the vaccine, assisted by Opti-MEM I reduced-serum medium without antibiotics (Opti-MEM™ I Reduced Serum Medium—CAT: 31985062—Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 1, 2, and 3 days. This was based on the activation peak reported in the literature [12].

After establishing the peak of spike expression, the THP-1 cells were incubated with mRNA vaccine and lipofectamine, as vehicle-only control (mock) (Lipofectamine 2000, ThermoFisher, Waltham, MA, USA, Cat#11668019) to investigate whether the cells were able to express CD11b or CD38 as markers of transitioning to macrophage-M1 phenotype, as well as to antigen-presenting cells with CD86+ expression in the same time points (1, 2, and 3 days) [13,14,15].

As soon as the best time points were chosen, the transfected THP-1 was co-cultured with peripheral blood mononuclear cells (PBMC) in the ratio 1:10 (PBMC:THP-1) from vaccinated subjects without previous SARS-CoV-2 infection (30 days after second dose) to verify whether CD4+ and CD8+ T lymphocytes would be activated through the antigen presenting cell differentiation induced by the mRNA vaccine in the THP-1 [16]. The variety of T-cell phenotypes was assessed through flow cytometry, and the list of antibodies used is available in Supplementary Table S3 [17].

Five human PBMC samples from vaccinated subjects were from our previous study, which the samples were frozen in liquid nitrogen and thawed for this assay. All procedures for obtaining samples were approved by the Ethics Committee of Fiocruz (Protocol# 34728920.4.0000.5262) in accordance with the Helsinki Declaration as revised in 2013 [14]. Informed consent was obtained from all participants, and all methods were carried out in accordance with relevant regulations and guidelines [18].

Inflammatory cytokines MIP1a, MCP1, IL-8, IL-6, and IL-1b were measured from supernatant of transfected THP-1 cells at the optimal time point before incubation with human PBMC samples. An in-house multiplex liquid microarray test was performed using 106 xMAP microspheres (Luminex Corporation, Austin, TX, USA) which were coupled with anti-human purified mouse monoclonal antibodies at the following concentrations: anti–IL-6 at 100 μg/mL, anti-MIP1a at 500 μg/mL, anti-IL-8 at 50 μg/mL, anti-MCP1 at 50 µg/mL and anti-IL-1b at 50 µg/mL. Coupling reactions were performed using the Amine Coupling Kit (Bio-Rad) following the manufacturer's instructions [11,19].

Blood collected from five mice were treated with ACK lyse solution, as recommended by manufacturer (ThermoFisher, Framingham, MA, USA, cat#A1049201) and the cells were transiently transfected with the COVID-19 mRNA bivalent vaccine from Pfizer BioNTech^® (5 µg mRNA/1000 µL, Lot. PAA194703) using a transfection medium. After 1, 2 and 3 days of mRNA vaccine incubation, the cells were washed, collected, and resuspended in fluorescence-activated cell sorting (FACS) buffer (1 × PBS, 2% FBS, 5% horse serum, 0.05% sodium azide). The cells were stained with Live Dead Blue dye for 10 min, and then washed with FACS buffer and centrifuged (500× g, 5 min). After that, the cells were stained with anti-mouse antibodies purchased from BD Biosciences (Bedford, MA, USA) and R&D Systems as described by Supplementary Table S3. All antibodies were incubated for 25 min (2–8 °C). The cells were washed and then fixed with 1% PFA. Data acquisition was performed on a BD LSR Fortessa instrument (BD Biosciences) and analyzed using FlowJo software v.10.

To confirm the surface expression of the spike protein, THP-1 cells were incubated with the mRNA vaccine in a 5% CO₂ incubator at 37 °C. After incubation, the cells were fixed with 4% paraformaldehyde, washed with blocking buffer (PBS supplemented with 5% FBS and 2% horse serum), and stained with anti-CD11a to identify the cell surface (Supplementary Table S3). The cells were then treated with 0.1% Triton-X permeabilization solution. Following permeabilization, the samples were stained with anti-spike antibody to evaluate the cellular localization of the spike protein. Images were acquired at 400× magnification using the EVOS M7000 Imaging System.

Adult mice (Mus musculus) K18-hACE2 lineage weighing 20–30 g and 8–10 weeks old were used as an animal model for this study. Of the 60 mice, 40 were female. The study protocol was also approved by Fundação Oswaldo Cruz animal ethics committee (LW-17/20). All procedures involving animals were conducted in Animal Biosafety Level 3 at the Preclinical Testing Laboratory (LAEPC) of the Department of Experimental Development and Preclinical Research at Bio-Manguinhos/Fiocruz and we confirm that all experiments were performed in accordance with relevant guidelines and regulations.

The mice were divided into four groups (n = 15 mice/group): 1- control group receiving saline buffer (Tris HCl pH 7.4, 100 µL), 2- animals who received two doses of COVID-19 vaccine, 3- animals who were virally challenged and not vaccinated, 4- animals who were vaccinated with two doses and were virally challenged. The animals were followed through 14 days after viral challenge until euthanasia, and every day the weight was measured, and clinical signs were monitored until severity for humane endpoint (e.g., piloerection, arched back, respiratory distress, ocular discharge and lethargy) or euthanasia day (14th day after SARS-CoV-2 challenge). The SARS-CoV-2 RT-qPCR was performed after challenged and immunization using oropharyngeal swabs from third- and fifth-days post-infection (DPI), and from lung and brain sections extracted after euthanasia on days 5, 6, and 14 post infection.

A COVID-19 mRNA bivalent vaccine from Pfizer BioNTech^® (30 µg mRNA/300 µL, Lot. PAA194703) was used for immunization. The animals were contained for intramuscular immunization at 1/6 of the human adult dose (50 µL per hind limb). Two doses were administered, with an interval of 28 days between the first and second dose. For in vitro study, five animals (non-immunized; male: 3) were used.

The animals were anesthetized via inhalation using up to 1.5–3% isoflurane (dose-dependent) in an acrylic anesthesia chamber specifically designed for laboratory animals (INHALATION ANESTHESIA EQUIPMENT BY INFUSION—Bonther^®, Ribeirão Preto, SP, Brazil). After complete relaxation, the animals were kept in an upright position for intranasal inoculation. The Gamma variant (P.1) of SARS-CoV-2 was intranasally inoculated with 1 × 10⁵ PFU in 10 µL (5 µL per nostril) using a 2–20 µL automatic pipette (Rainin^®, Sigma-Aldrich, St. Louis, MO, USA) at the 14th day after the second dose of COVID-19 vaccine. The same anesthesia procedure was applied on the 3rd and 5th DPI for swab sample collection of oropharyngeal cavities.

Euthanasia was performed one day before viral challenge (at day 13 post 2nd dose of vaccination) and in specific euthanasia days (5th, 6th and 14th day after SARS-CoV-2 challenge) through anesthesia with doses of 200 mg/kg of ketamine hydrochloride and 20 mg/kg of xylazine hydrochloride administered intramuscularly. After several minutes of anesthetic administration and the absence of palpebral and interdigital reflexes (tested by touch), blood sample collection by cardiac puncture was performed. Next, sodium thiopental was administered intraperitoneally at a dose of 150 mg/kg for spleen, lung, and brain sample collection.

The adult mice (Mus musculus) K18-hACE2 lineage were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), maintained by the Institute of Science and Technology in Biomodels (ICTB)/Fiocruz and housed at a density of five animals per cage, with weights varied to ensure heterogeneity within each group. The airtight cages measured 36 cm in length, 17 cm in width, and 15 cm in height, and were set up in a ventilated microisolator rack system (ALESCO^®, Fort Myers, FL, USA) using high negative pressure system (ALN 2) with autoclavable wood shavings as substrate. Water and food were changed twice a week under unrestricted access conditions. The mice were maintained under a 12-h light/12-h dark cycle in a controlled environment with temperatures ranging from 18 to 24 °C, relative humidity between 40 and 70%, and a constant air exchange rate of 20 changes per hour.

The experimental laboratory has an Environmental Enrichment Program that includes a polysulfone igloo as a shelter, with cotton rolls, shredded paper, and cardboard being interchanges monthly as forms of environmental enrichment. Clinical evaluations of the animals were conducted by a veterinarian based on the ARRIVE guidelines, and humane endpoints were established based on the severity of clinical signs.

The health monitoring of the colony providing the animals was conducted quarterly based on a specific pathogen list, as recommended by the Federation for Laboratory Animal Science Associations (FELASA), with all animals included in this study considered free of specific pathogens (SPF).

To evaluate the gene expression after the vaccination and the viral challenge, total RNA was obtained from blood samples from 60 animals using the TRIzol extraction method (Invitrogen, Carlsbad, CAM, USA). RNA mass was quantified using a spectrophotometer (260 nm; Nanodrop Technologies, Wilmington, DE, USA), followed by a complementary DNA (cDNA) synthesis from 250 ng of total RNA using the High-Capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific, Framingham, MA, USA), with both procedures carried out according to the manufacturer's instructions. The experimental design for analyzing gene expression is illustrated in Figure 1, with the timing of sample collection.

For the quantitative RT-qPCR reaction, Fast Sybr Green master mix (Applied Biosystems, Foster City, CA, USA) with 200 nM of each primer () and 10 ng of each cDNA were used in a final reaction volume of 10 µL. QuantStudio 7 PRO (Applied Biosystems, Foster City, CA, USA) instrument was used to calculate the Ct values during the RT-qPCR assay and the cycling conditions involved: polymerase activation at 95 °C for 20 s; followed by up to 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Supplementary Table S1

The data obtained were normalized to the average cycling threshold of the reference genes PPIA and GAPDH and expressed as fold change (2-ΔΔCt) using the control group receiving saline buffer as a reference for calibration.

A sub-analysis using total RNA from blood samples of 15 animals was conducted to characterize the transcriptomic profile of a panel of innate-immunity-related genes. To test this panel, we used 96.96 gene expression Integrated Fluidic Circuit for Biomark HD platform (99.96 GE IFC, Fluidigm, #BMK M-96.96).

The cDNA from each of the 96 samples was synthesized from 250 ng of RNA using Reverse Transcription Master Mix (Fluidigm, South San Francisco, CA, USA, #100-6297). Afterward, the cDNAs were simultaneously preamplified with a pool of all primer pairs in a conventional thermocycler. To this end, we used Preamp Master mix (Fluidigm #1005581) with 500 nM of each primer (forward and reverse) and 1.25 µL of each cDNA in a final reaction volume of 5 µL for 14 cycles. Preamplified cDNA was treated with Exonuclease I (New England BioLabs, MA, USA, # M0293S), and diluted 1:5 in TE Buffer pH 8.0 (10 mM Tris-HCl, 0.1 mM EDTA). The preamplified cDNAs mixed with 2x SsoEvaGreen Supermix with low ROX (Bio-Rad, Hercules, CA, USA, #64461016) as well as 20 nM of individual primer pairs were loaded in the 99.96 GE IFC using the Juno system (Fluidigm). Real-time PCR reactions were conducted in the Biomark HD microfluidic system (Fluidigm). The melting curve of each primer pair and sample quality were analyzed on the company's software and unexpected values were considered for primer or sample exclusion.

After quality control exclusions, a panel of 78 genes was analyzed including genes related to innate immunity and the references B2M, GAPDH, and ACTB (). The data obtained were normalized to the average cycling threshold of the reference genes and expressed as fold change (2-ΔΔCt) using the control group receiving saline buffer as a reference for normalization. Supplementary Table S2

Microtiter ELISA plates (Maxisorp NUNC—Thermo Fisher Scientific) were coated with 2.5 μg/mL of Spike trimer XBB Omicron (cat.101677) in carbonate/bicarbonate buffer pH 9.6 overnight at 4 °C. Coated plates were washed with PBST (PBS and Tween 20 at 0.05%) and blocked for 1 h at 37 °C with blocking and diluent solution—BDS (PBS, Tween 20% at 0.05%, bovine serum albumin at 0.05%, and skim milk at 5%). The plates were washed again with PBST, then 3-fold serially diluted mice serum samples were added to each well and incubated for 2 h at 37 °C. Serial 3-fold dilutions ranging from 0.007 to 5 µg/mL of mouse anti-spike monoclonal antibodies against SARS-CoV-2 (lot 201112S134SPIKP) were used as standard curve. The plates were washed again before the addition of anti-mouse IgG-peroxidase (cat. A9044—Sigma-Aldrich, Burlington, MA, USA) diluted 1:20,000 in BDS and incubated for 1 h at 37 °C. Finally, the plates were washed before the addition of TMB plus (Kementec Solutions/Bio-Connect Diagnosis BV, Huissen, The Netherlands) for 15 min in the dark at room temperature. The colorimetric reaction was stopped with H₂SO₄ 2 M and measured at 450 nm in a plate reader VersaMax™ (Molecular Devices, San Jose, CA, USA). The optical density (OD) of the sera dilutions was plotted on the standard curve, and the antibody titers were calculated by a four-parameter logistic regression using the software SoftMax^® (version 7.1.0) (Molecular Devices, San Jose, CA, USA) and expressed as µg/mL.

The spleens of the mice were collected and placed in RPMI medium containing 10% FBS and 1% penicillin-streptomycin solution. Splenocytes were isolated into a single-cell suspension by passing the spleens through a 40-μm filter. Erythrocytes were removed using a red blood cell lysis buffer, and the resulting splenocytes were washed, counted, adjusted to 1 × 106 cell/well and exposed to spike peptides at 1 µg/mL (PepMix^TM SARS-CoV-2, Spike BA.4/5) and purified αCD28/αCD49d mouse antibodies using a commercial kit for IFN-gamma ELISPOT (Mabtech #3321-4APT-2) as well as in 96-well plates for antibody staining for FACS analysis. The splenocytes with peptides were incubated for 48 h, and then the ELISPOT assay was performed following the commercial kit instructions. The splenocytes for phenotyping assay were washed with FACS buffer and stained with specific antibodies for flow cytometry analysis. The negative control was set up using only cultivation media (RPMI1640 supplemented as described previously). The positive control was concanavalin A at 1 µg/mL. The antibodies were purchased from BD Biosciences, and the list is provided in Supplementary Table S3. After staining assay, the cells were fixed with paraformaldehyde 1%, acquired using LSR Fortessa (BD Biosciences), and analyzed through FlowJo v.10.8 software (BD Biosciences). The gating strategy to identify B and T cells upon spike stimulation is displayed in Supplementary Figure S1.

In silico analysis was performed to investigate the peptide epitopes for CLEC5A interaction as well as lymphocyte T and B cell activation through the spike glycoprotein used in the in vitro stimulation assay (Pepmix JPT peptides, PM-WCPV-S-2). IEDB at http://tools.iedb.org/bcell/↗ (accessed on 13 March 2025) was used to predict linear T-cell and B-cell epitopes in the protein sequence. ClusPro 2.0 server (https://cluspro.bu.edu/login.php↗ (accessed on 15 March 2025)) was used to dock the CLEC5A receptor with spike glycoprotein (PDB: 6VXX). After the docking prediction by Cluspro, the interactions between the CLEC5A receptor and spike protein were predicted through PDBsum server (http://www.ebi.ac.uk/pdbsum↗ (accessed on 5 May 2025)) to obtain residue interactions and bounding information.

Statistical analyses were conducted using GraphPad Prism (version 8.4.2 for Windows). Data normality was assessed with the Kolmogorov–Smirnov or Shapiro–Wilk tests. Group differences were analyzed using Kruskal–Wallis test, and results are presented as mean ± standard error in bar graphs. Statistical significance was set at p < 0.05. Final data were obtained from two independent assays using a mouse model and triplicates in the in vitro experiments. In vitro tests were repeated at least one more time to confirm the data. Linear correlation was performed to evaluate the kinetics of protein expression through in vitro assays.

The kinetics of the THP-1 cells incubated with the mRNA vaccine, showed that the highest level of spike and CLEC5A expression was observed at day 2 post-incubation compared with control samples (Kruskal–Wallis test, p = 0.013) (Figure 2). A positive linear correlation was found between spike protein and CLEC5A expression (R² = 0.75, p = 0.0003).

Flow cytometry demonstrated that the mRNA vaccine induced THP-1 activation toward an M1-like macrophage phenotype, with ~60% higher activation than mock (lipofectamine [LPF]) after two days. While vehicle-only (LPF) also promoted activation over time, its effect remained consistently lower than that of the mRNA vaccine at the same time points (Figure 3).

To assess T cell activation induced by antigen-presenting cells during THP-1 differentiation, a 2-day incubation with the mRNA vaccine was performed. PBMCs were then co-cultured with THP-1 cells, and after 24 h the co-culture was resuspended and processed for phenotypic staining by flow cytometry. A significant activation of both CD4+ and CD8+ T cells was observed (Figure 4). In addition, the chemokines MCP-1 (CCL2) and IL-8 (CXCL8) showed 2.07-fold and 4.98-fold increases, respectively, compared with the control at day 2 of THP-1 culture with the mRNA vaccine alone (Kruskal–Wallis test, p = 0.03). No significant changes were observed for the other cytokines.

The cells from murine blood incubated with mRNA vaccine at day 2 showed a significant increase of inflammatory monocytes (Ly6C+CCR2+CX3CR1low) (Figure 5) and a significant increase of CLEC5A co-expressed with CD11c (dendritic like) by those cells (Figure 5) compared with control cells. The gating strategy is displayed in Figure 5.

As the peak of spike expression was detected at 2 days in flow cytometry assays, fluorescence microscopy was performed at this time point to determine the cellular localization of spike in THP-1 cells. To facilitate membrane identification, an anti-CD11a antibody, commonly expressed on the surface of this cell line, was used. The images demonstrate spike protein expression in THP-1 cells, along with CD11a staining at the cell surface (Figure 6).

The vaccinated group exhibited a maximum weight loss of 4.64% on Day 7 over the 14-day period, while the control group showed a significantly higher weight loss of 19.98% on the same day (Figure 7A). Between Days 7 and 11, a slight difference in weight loss was observed between the control and vaccinated groups, though both groups demonstrated a reduction in weight loss toward the end of the observation period (Figure 7A). The survival curve revealed that the vaccinated group was effectively protected against the viral challenge (Figure 7B). All animals were humanely euthanized after 14 days. The viral load significantly decreased three days after the SARS-CoV-2 challenge with the Gamma strain in the vaccinated group, as determined by oropharyngeal swabs (Figure 7C). In lung (Figure 7D) and brain tissue samples (Figure 7E), a significant reduction in viral load was observed in the vaccinated mice group on days 5–6 post-challenge. By the 8th day post-infection, >50% animals in the control group had died, eliminating the possibility of statistical analysis. The standard curve for viral load quantification was generated using a minigene construct containing the SARS-CoV-2 target sequences N1 and N2, which were cloned into a plasmid.

The gene expression profile of mice after immunization and after immunization + challenge was evaluated using a panel of innate-immune-response related genes, where only significant analysis was plotted (p < 0.05) (Figure 8). The genes related to inflammatory profile (IL1B, TNF and CLEC5A) were upregulated in mice infected with SARS-CoV-2, as expected. Meanwhile, the genes related to innate antiviral activity were downregulated (NFKB, IFNA, IFNB) (Figure 8A). The immunization with bivalent mRNA vaccine was characterized by upregulation of genes expressing proinflammatory cytokines (CCL2, and IL1B), complement activation (CFH), inflammasome (NLRP3), and genes involved with receptors of innate immunity that trigger inflammation (TLR2, TYK2, JAK1) (Figure 8B). After the viral challenge, as expected, mice presented upregulation of genes related to the innate antiviral response (STAT2 and RNASEL). Interestingly, the expression of TLR2 and NLRP3 after challenge remained upregulated (1.03-fold-change ± 0.40 and 0.74-fold-change ± 0.25 respectively), in levels not statistically different from the ones triggered by immunization (1.18-fold-change ± 0.37 and 1.14-fold-change ± 0.40) (Figure 8B). Quantification of CLEC5A gene expression in animals revealed a slight increase following immunization compared with controls, which was also observed—at a higher level—in non-immunized animals challenged with SARS-CoV-2. However, in animals immunized with two doses of the vaccine and subsequently challenged with the virus, CLEC5A expression was significantly reduced compared with both vaccinated-only and non-immunized infected animals (Figure 8A–C).

Immunogenicity was assessed through ELISA, ELISPOT and FACs analysis to evaluate whether any change in the adaptive immune response could be affected. The spike IgG antibodies were significantly elevated after immunization and after viral challenge (Figure 9). The splenocytes stimulated with spike from Omicron XBB.1.5 variant showed a significant increase in cells secreting IFN-gamma in ELISPOT data after vaccination (Figure 10A), and the percentage of follicular memory B cells was also increased after immunization (Figure 10B). Regarding memory T lymphocytes, it was observed that the percentage of total T cells was higher in vaccinated mice compared with non-vaccinated mice (control group) (Figure 10C–E). On the other hand, central memory CD8+ T cells were also elevated in immunized mice compared with the control group (Figure 10F).

It was possible to observe that bioinformatics findings showed that T lymphocyte epitopes as well as B cell epitopes are not located in the same region of CLEC5A in the linear map of the spike glycoprotein amino acids sequence (Figure 11A,B). It was clearly demonstrated through molecular docking that the interaction with CLEC5A is between 330–390 residues of spike glycoprotein, while ACE-2 binds in 437–508 (Figure 11A–E). In addition, the local receptor binding modif (ACE-2) interaction with spike glycoprotein are distant from lymphocytes and CLEC5A predicted epitopes (Figure 11D,E).