An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice
Fulya Erendor, Fatih Uzer, Salih Sanlioglu
TL;DR
This study shows that an adenovirus-based vaccine encoding the SARS-CoV-2 Spike protein induces both antibody and immune cell responses in mice, supporting its potential as a vaccine platform.
Contribution
The study introduces and evaluates a new adenovirus-based vaccine candidate for SARS-CoV-2 that elicits measurable immune responses in mice.
Findings
Ad5Spike vaccination induced anti-Spike IgG antibodies that persisted for 90 days.
The vaccine triggered cytokine secretion (IFN-γ, TNF-α, IL-2) and neutralizing antibody activity in a dose-dependent manner.
Immune responses were detectable at both 30 and 90 days post-immunization, indicating sustained immunity.
Abstract
The global SARS‐CoV‐2 pandemic has underlined the urgent need for effective vaccine platforms. Adenoviral vectors have gained attention due to their high transgene capacity, broad tissue tropism, and innate immunostimulatory properties. This study aimed to develop and evaluate a recombinant adenoviral vaccine, Ad5Spike, encoding the full‐length SARS‐CoV‐2 Spike glycoprotein. The Ad5Spike vector was generated using Gateway Cloning Technology and produced by transient calcium phosphate‐mediated transfection of 293A cells. Viral particles (VP) were purified via CsCl density gradient ultracentrifugation. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with 108, 1010, or 101 2 viral particles. Humoral and cellular immune responses were evaluated at 30‐ and 90‐days post‐immunization using ELISA, ELISpot, and pseudovirus neutralization assays.…
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FIGURE 5- —KOSGEB R&D, Innovation and Support Program
- —Akdeniz University Scientific Research Projects Coordination Unit
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Taxonomy
TopicsSARS-CoV-2 and COVID-19 Research · Virus-based gene therapy research · Immune responses and vaccinations
Introduction
1
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in December 2019 rapidly escalated into a global pandemic. This crisis posed an unprecedented threat to public health and caused immense socio‐economic disruption worldwide [1, 2]. SARS‐CoV‐2, the causative agent of coronavirus disease 2019 (COVID‐19), is characterized by high transmissibility and a wide spectrum of clinical manifestations. These symptoms range from mild respiratory issues to severe pneumonia, acute respiratory distress syndrome (ARDS), and death. Patients with severe COVID‐19 often develop a “cytokine storm” marked by an excessive production of inflammatory cytokines, such as interleukin (IL)‐1β, IL‐6, IL‐12, interferon (IFN)‐γ, and tumor necrosis factor (TNF)‐α. This hyper‐inflammatory state can lead to ARDS, systemic inflammatory response, and multi‐organ failure [3, 4]. The devastating global impact of SARS‐CoV‐2 underlined an urgent and critical need for the rapid development of safe and effective prophylactic vaccines.
Vaccination remains the most effective strategy for preventing and controlling infectious diseases [5]. The COVID‐19 pandemic serves as a reminder that such large‐scale outbreaks are likely to recur, necessitating the development of robust and adaptable vaccine platforms for future pandemic preparedness. An ideal vaccine must possess an excellent safety profile, induce rapid protective immunity, and provide long‐lasting protection through both humoral and cellular immune responses [6]. Most COVID‐19 vaccines target the SARS‐CoV‐2 Spike (S) glycoprotein, as this protein mediates viral entry via the ACE2 receptor and contains the primary epitopes for neutralizing antibodies. However, the continuous emergence of SARS‐CoV‐2 variants—including the early Alpha and Delta strains, followed by the highly divergent Omicron subvariants (e.g., B.1.1.529, XBB, and beyond)—challenges the long‐term efficacy of first‐generation vaccines utilizing the ancestral Wuhan‐1 sequence [7]. These mutations, particularly within the receptor‐binding domain (RBD) of the S protein, can enhance transmissibility and facilitate immune evasion, highlighting the urgent need for novel vaccine strategies that can be rapidly updated.
Coronaviruses are enveloped, positive‐sense single‐stranded RNA viruses whose infectivity is primarily mediated by the surface‐expressed Spike (S) glycoprotein [8]. The SARS‐CoV‐2 S protein is a trimeric class I fusion protein responsible for receptor recognition and membrane fusion [9]. The S protein ectodomain comprises S1 and S2 subunits, generated by furin‐mediated cleavage at a polybasic site. The S1 subunit contains the RBD, which interacts with the angiotensin‐converting enzyme 2 (ACE2) receptor. Simultaneously, the S2 subunit facilitates membrane fusion [8, 10]. Due to highly immunogenic nature of the S protein and the S protein's critical role in viral entry, this glycoprotein serves as the primary target for inducing both antigen‐specific antibody responses and protective T‐cell immunity [11]. While antigen selection is a key determinant of vaccine efficacy, the choice of an appropriate delivery platform is equally critical for shaping the magnitude and quality of the immune response.
Among the various gene delivery platforms, adenoviral vectors (Ad vectors) stand out as effective vehicles for introducing foreign antigens into host cells [12, 13]. Furthermore, Ad vectors act as a self‐adjuvant by stimulating multiple innate immune pathways, thereby enhancing the immunogenicity of the encoded antigen [14, 15]. Importantly, the natural tropism of Ad vectors for respiratory epithelial cells makes this platform well‐suited for mucosal immunization strategies [16, 17].
By early 2026, the global COVID‐19 vaccine landscape has evolved into a stabilized, seasonal model, predominantly utilizing updated monovalent formulations that target dominant Omicron lineages, such as JN.1 and its sub‐variants [18]. While the current vaccination cycle is primarily led by mRNA‐based platforms and protein subunit alternatives, these technologies continue to face challenges regarding transmission blockade and duration of protection [19]. Consequently, the research frontier has shifted toward the development of ‘pan‐coronavirus’ vaccines and novel delivery systems—particularly mucosal (intranasal [20]) and viral vector‐based platforms—designed to provide broader cross‐reactivity and a more localized immune response [21]. Within this context, modular adenoviral vector systems remain a critical area of investigation due to their established ability to elicit measurable cellular immunity, which may offer more resilient protection against the rapid antigenic drift observed in evolving SARS‐CoV‐2 variants.
In this study, we designed a recombinant, replication‐deficient Ad5 vector encoding the full‐length SARS‐CoV‐2 Spike protein (Ad5Spike) and evaluated its immunogenicity in female BALB/c mice. Our work provides an initial proof‐of‐concept assessment of the Ad5Spike candidate, characterizing the induction of measurable Spike‐specific antibody and cytokine responses. While further studies are required to evaluate in vivo protective efficacy, cross‐variant neutralization, and detailed immune phenotyping of memory subsets, these results contribute to the broader understanding of adenoviral vector‐based vaccine platforms and their potential application for future infectious disease threats.
Methods
2
Cell Culture
2.1
HEK293A cells (human embryonic kidney, female, embryonic kidney tissue; official name: HEK293A, RRID:CVCL_6910) were obtained from Thermo Fisher Scientific (Cat. no. R70507) in 2020. HEK293T cells (human embryonic kidney expressing SV40 large T antigen, female, embryonic kidney tissue; official name: HEK293T, RRID:CVCL_0063) were purchased from ATCC (CRL‐3216) in 2019. HT1080 cells (human fibrosarcoma, male, fibrosarcoma tissue; official name: HT1080, RRID:CVCL_0317) were obtained from ATCC (CCL‐121) in 2020. RAW 264.7 cells (murine macrophage‐like, male, Abelson murine leukemia virus–induced tumor ascites; official name: RAW 264.7, RRID:CVCL_0493) were purchased from ATCC (TIB‐71) in 2021. THP‐1 cells (human monocytic leukemia, male, peripheral blood; official name: THP‐1, RRID:CVCL_0006) were obtained from ATCC (TIB‐202) in 2021.
All cell lines were authenticated by the suppliers using short tandem repeat (STR) profiling and have been verified against the International Cell Line Authentication Committee (ICLAC) database to ensure that they are not misidentified or contaminated. Cultures were routinely screened and confirmed to be negative for mycoplasma contamination throughout the experimental period.
Construction of Adenoviral Expression Plasmids
2.2
Codon‐optimized cDNAs encoding the full‐length SARS‐CoV‐2 Spike glycoprotein (3822 bp; Addgene #149329) or GFP were initially obtained in the Gateway entry vector pDONR223, flanked by attL1 and attL2 recombination sites. LR recombination reactions were performed using Gateway LR Clonase enzyme mix (Thermo Fisher Scientific) to transfer the Spike or GFP gene into the pAd/CMV/V5‐DEST destination vector, which contains attR1 and attR2 sites and a CMV promoter for high‐level transgene expression. These reactions resulted in the formation of recombinant adenoviral plasmids: pAd5Spike and pAdGFP. Plasmid maps and simulated digestion patterns were generated using SnapGene software (version 4.3.6). Following bacterial transformation into chemically competent E. coli cells, positive clones were selected via antibiotic resistance, cultured, and plasmid DNA was isolated using a high‐purity miniprep kit. The identity and orientation of the recombinant clones were validated by restriction enzyme digestion (with EcoRI) and analyzed by 1% agarose gel electrophoresis, confirming that the actual banding patterns matched the predicted in silico patterns.
Adenoviral Vector Production and Purification
2.3
HEK 293A cells were transfected with either pAd5Spike or pAdGFP using polyethylenimine (PEI) at a PEI:DNA ratio of 3:1 (w/w). Initial virus stocks were amplified in 100 mm dishes and scaled up to 150 mm dishes to achieve high‐titer production. Crude viral lysates were harvested following three consecutive freeze–thaw cycles (–80°C/37°C), clarified by centrifugation to remove cellular debris, and purified through two rounds of cesium chloride (CsCl) density gradient ultracentrifugation (20,000 × g, 16 h, 4°C). Distinct bands representing intact (full) and empty viral capsids were observed and collected. VP concentrations were determined by optical density at 260 nm (OD260) using the formula 1 OD260 = 1.1 × 10^1^ ^2^ particles/mL. Final viral stocks were desalted using Sephadex G‐50 columns into a storage buffer, plaque‐purified to ensure clonal stability, and stored in aliquots with 10% glycerol at –80°C.
Confirmation of Protein Expression
2.4
For validation of Spike protein expression, HEK293T cells were transfected with pAd5Spike and assessed by immunocytochemistry (IHC) 72 h post‐transfection using a rabbit polyclonal anti‐SARS‐CoV‐2 Spike antibody (Abcam, ab272504) and HRP‐conjugated secondary antibody, followed by DAB detection. For transgene expression from the viral vector, HT1080 cells were transduced with increasing multiplicities of infection (MOI: 10^2^–10^5^ for Ad5Spike, 10^1^–10^6^ for AdGFP). Spike expression was visualized via immunofluorescence using the same primary antibody and an Alexa Fluor 488–conjugated secondary antibody. GFP expression was directly monitored by fluorescence microscopy. Nuclei were counterstained with DAPI for localization. Images were captured using a fluorescence microscope (Olympus IX81).
Adenoviral Transduction Efficiency in Macrophage and Monocytic Cell Lines
2.5
Murine RAW 264.7 macrophages and human THP‐1 monocytic cells were seeded in 24‐well plates at 5 × 10^5^ cells/well. After 24 h of incubation, cells were transduced with AdGFP at varying doses to evaluate transduction efficiency. RAW 264.7 cells received 10^1^–10^6^ viral particles (vp) per well, while THP‐1 cells were transduced with 10^3^ and 10^4^ vp per well. At 72 h post‐transduction, GFP expression was visualized using an Olympus IX‐81 fluorescence microscope under both bright field (BF) and GFP channels. Representative images were captured at 20× magnification for RAW 264.7 cells and at 10×, 20×, and 40× for THP‐1 cells. Image acquisition settings, including exposure time and gain, were maintained constant across groups to allow for qualitative comparison of fluorescence intensity.
Animals and Immunization
2.6
For this pilot study, 6‐week‐old BALB/c female mice were obtained from the Animal Care Unit of Akdeniz University Hospitals. Female mice were selected to maintain cohort consistency for this initial immunogenicity evaluation. These mice were maintained in a humidity‐ and temperature‐controlled environment with a 12 h light/dark cycle, and they had ad libitum access to standard chow and water. Female BALB/c mice (6 weeks old) were used to evaluate the immunogenicity of the adenovirus‐based Spike vaccine. Mice were housed under specific pathogen‐free conditions in individually ventilated cages throughout the study. Animals were randomly assigned into five groups (n = 10 per group). Three groups received a single intraperitoneal injection of Ad5Spike at doses of 10^8^, 10^10^, or 10^1^ ^2^ vp. Control groups received either 10^1^ ^2^ viral particles of AdGFP (vector control) or 1X PBS (negative control). To assess both early and persistent immune responses, five mice from each group (n = 5 per timepoint) were humanely sacrificed on days 30 and 90 post‐immunization. Blood and spleen samples were collected for humoral and cellular immune response analyses. All animal procedures were conducted under the strict supervision and approval of the Institutional Animal Care and Use Committee of Akdeniz University School of Medicine (Approval No:1129/2020.04.48).
Spleen Cell Isolation
2.7
Spleen tissues were harvested from sacrificed animals and placed in a petri dish containing 5 mL of ice‐cold RPMI cell culture medium supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin‐streptomycin. The spleen tissues were mechanically dissociated into small pieces using a sterile scalpel. The spleen fragments were passed through a 70 µm cell strainer into 50 mL conical tubes to obtain a single‐cell suspension and centrifuged at 600 xg for 10 min at 4°C. The supernatant was discarded, and to remove red blood cells, the pellet was treated with an erythrocyte lysis buffer (ACK lysis buffer) before being resuspended in 500 µL of cold 1X PBS containing 2% FBS to a concentration of 1×10^8^ cells/mL. Cell viability was assessed using the trypan blue exclusion method. For long‐term storage, the cell suspensions were cryopreserved in a freezing medium containing 10% DMSO and stored in liquid nitrogen tanks.
ELISA
2.8
Ninety‐six‐well high‐binding plates (Corning, Thermo Fisher Scientific) were coated overnight at 4°C with 100 µL/well of recombinant SARS‐CoV‐2 Spike protein (Abcam, ab273063, 1 µg/mL) in carbonate–bicarbonate buffer (pH 9.6). The next day, plates were blocked with 1% BSA in PBS for 2 h at 37°C. Following three washes with PBS, containing 0.05% Tween‐20 (PBS‐T), serum samples from immunized mice were serially diluted in PBS‐T with 1% BSA and added in duplicate to the wells, then incubated for 2 h at 37°C. After washing, 1:1000 diluted HRP‐conjugated goat anti‐mouse IgG (Abcam, ab205718) was added and incubated for 1 h at 37°C. Plates were developed with 100 µL/well of TMB substrate, and the reaction was stopped after 20 min with 50 µL of 1 M H_2_SO_4_. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Multiskan spectrophotometer). Endpoint titers were defined as the highest dilution with OD values exceeding twice the background obtained from PBS‐control samples.
For IFN‐γ cytokine quantification, culture supernatants from antigen‐stimulated splenic T cells were collected after 48 h and analyzed using a sandwich ELISA kit specific for mouse IFN‐γ (Abcam, ab100689) according to the manufacturer's instructions. Absorbance was read at 450 nm, and cytokine concentrations were calculated based on standard curves. All cytokine concentrations were reported as net values after subtracting the background levels of unstimulated (media‐only) control wells.
ELISpot
2.9
Mouse T cell ELISpot assays were performed using pre‐coated 96‐well PVDF plates (R&D system EL485 for IFN‐γ, EL410 for TNF‐α; and EL402 for IL‐2) according to the manufacturers' protocols. Briefly, splenocytes were isolated from vaccinated mice and enriched for CD3^+^ T cells, using magnetic beads (Dynabeads, Thermo Fisher Scientific). Purified T cells (2 × 10^5^ cells/well) were stimulated in vitro for 24–48 h with a full‐length SARS‐CoV‐2 Spike peptide pool (comprising 15‐mer peptides overlapping by 11 amino acids; Abcam, ab273063) at a final concentration of 2 ug/mL per peptide in RPMI‐1640 supplemented with 10% FBS. After incubation, plates were washed thoroughly with PBS, and detection antibodies for IFN‐γ, TNF‐α, or IL‐2 were added. Following a second wash step, plates were developed using streptavidin–HRP and a chromogenic AEC substrate. Spot‐forming units (SFUs) were visualized and quantified using stereomicroscopy. Data were expressed as SFUs per 10^6^ cells after subtracting background spots from unstimulated (media‐only) control wells for each individual sample.
Neutralizing Antibody Assay
2.10
Neutralizing antibody responses were assessed using a pseudovirus neutralization assay based on third‐generation lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (Wuhan‐1 sequence; Addgene, #155130), encoding an RFP reporter gene. Pseudoviruses were generated in HEK293T cells by co‐transfection with plasmids encoding the Spike envelope (Addgene, #155130), lentiviral packaging mix (gag‐pol, rev), and an RFP reporter (Addgene, #26001) construct, followed by ultracentrifugation‐based concentration. For the assay, heat‐inactivated mouse sera (56°C, 30 min) were serially diluted in culture medium and incubated with LentiRFPSpike particles (MOI: 5) for 1 h at 37°C. Virus‐serum mixtures were then added to 293T cell monolayers in 96‐well plates. After 72 h incubation at 37°C with 5% CO_2_, RFP fluorescence intensity was imaged using an IX‐81 fluorescence microscope (Olympus). Neutralizing activity was expressed as percent inhibition relative to virus‐only controls, and ID_50_ values were calculated using nonlinear regression (four‐parameter logistic curve) in GraphPad Prism 10.
Statistical Analysis
2.11
Statistical analyses were performed using GraphPad Prism software. Data are presented as mean ± SD or SEM, as indicated. Normality of the data distribution was assessed using the Shapiro–Wilk test. For normally distributed data, one‐way ANOVA followed by Tukey's multiple comparison test was used. For data that did not follow a normal distribution, the non‐parametric Kruskal–Wallis test followed by Dunn's post‐hoc test was applied. A p‐value of < 0.05 was considered statistically significant. Exact p‐values and specific pairwise comparisons are indicated in the respective figure legends.
Results
3
Ad5Spike Vector Construction Enables Efficient Expression of the SARS‐CoV‐2 Spike Protein
3.1
We constructed a replication‐defective adenoviral expression vector, pAd5Spike, containing the SARS‐CoV‐2 Spike gene. Gateway LR recombination technology was employed for its high efficiency and site‐specific recombination. The Spike gene, flanked by attL1 and attL2 sites in the pDONR223‐Spike entry vector (Addgene plasmid #149329), was transferred to the pAd/CMV/V5‐DEST destination vector using LR Clonase enzyme mix. This reaction yielded pAd5Spike, which includes the full‐length Spike gene integrated into the adenoviral backbone. The recombination event was simulated using SnapGene software (Figure 1A). Successful cloning was confirmed by EcoRI restriction enzyme digestion, where the observed banding patterns on 1% agarose gel electrophoresis matched the in silico simulation precisely (Figure 1B). These results confirm the successful and directional cloning of the SARS‐CoV‐2 Spike gene into the adenoviral backbone. To evaluate the functional expression of the pAd5Spike plasmid, HEK 293T cells were transiently transfected. Immunohistochemical staining performed 72 h post‐transfection demonstrated measurable expression of the SARS‐CoV‐2 Spike glycoprotein, indicated by distinct brown cytoplasmic staining, thereby confirming protein expression from the construct (Figure 1C).
Construction, expression, and characterization of recombinant adenoviral vectors encoding the SARS‐CoV‐2 Spike protein. (A) Schematic representation of the adenoviral expression plasmid construction using Gateway LR recombination. The entry plasmid (pDONR223 SARS‐CoV‐2 S) containing attL1/attL2 recombination sites was recombined with the destination vector (pAd/CMV/V5‐DEST) containing attR1/attR2 sites using LR clonase enzyme mix. The resulting transfer plasmid (pAd5Spike) includes the full‐length Spike gene under a CMV promoter along with adenoviral vector backbone sequences. Simulation images were generated using SnapGene. (B) Agarose gel electrophoresis analysis of pAd5Spike plasmids. The left panel shows the simulated gel image, and the right panel displays actual gel electrophoresis of plasmids isolated from three independent bacterial colonies (lanes 1–3). MW: 1 kb Plus DNA Ladder (Thermo, SM0313). (C) Immunocytochemical analysis of SARS‐CoV‐2 Spike protein expression in HEK293T cells transfected with pAd5Spike. Cells transfected with pAd5Spike show strong brown immunostaining, indicating Spike expression, while control cells (transfected with PEI alone) show no signal. (D) CsCl density gradient ultracentrifugation of recombinant adenoviral particles. Two distinct bands are visible: the lower band represents genome‐containing Ad5Spike particles, and the upper band corresponds to empty viral capsids, indicating successful viral purification. (E) Immunofluorescence analysis of spike protein expression in HT1080 cells transduced with Ad5Spike at increasing multiplicities of infection (MOI: 102–105). Cells were stained 72 h post‐transduction using an anti‐Spike antibody (green, Alexa Fluor 488). Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.
For adenoviral vector production, pAd5Spike was transfected into HEK 293A cells, which provide the necessary E1 and E3 functions for adenoviral replication in trans. Initial VPs were harvested and amplified through subsequent infections in large‐scale 293A cultures. Viral vectors were then purified by cesium chloride density gradient ultracentrifugation, yielding purified Ad5Spike viral stocks (Figure 1D). The final VP concentration was quantified by spectrophotometric measurement at OD260, yielding approximately 1.65 × 10^12 ^particles/mL. To assess the functional expression of the Ad5Spike viral vector, HT1080 cells were transduced with purified Ad5Spike at various multiplicities of infection (MOIs ranging from 10^2^ to 10^5^). Immunofluorescence analysis at 72 h post‐transduction showed clear and dose‐dependent expression of the SARS‐CoV‐2 Spike glycoprotein, indicated by strong green fluorescence (Alexa Fluor 488), after anti‐Spike antibody staining (Figure 1E). Spike expression intensity increased in a dose‐dependent manner with higher MOIs, confirming efficient protein expression by the viral vector.
AdGFP Vector Construction Facilitates Functional Validation of the Adenoviral Platform
3.2
To validate the general functionality and transduction capability of the adenoviral vector system, a GFP‐expressing adenoviral plasmid (pAdGFP) was generated. This was achieved through Gateway LR recombination between a pDONR223‐GFP entry vector and the pAd/CMV/V5‐DEST destination vector, resulting in the pAdGFP construct (Figure 2A). Following the production of AdGFP VPs, their functionality was assessed by transducing HT1080 cells with increasing doses of AdGFP (ranging from 101 to 106 viral particles per well). Fluorescence microscopy performed at 72 h post‐transduction revealed clear and dose‐dependent GFP expression in HT1080 cells, indicated by increasing green fluorescence intensity correlated with higher VP concentrations. These observations confirmed the successful generation of functional AdGFP viral vectors and their ability to efficiently transduce target cells (Figure 2B).
Construction of the adenoviral expression plasmid and GFP expression in HT1080 cells. (A) Schematic representation of the Gateway LR recombination reaction for the generation of a GFP‐expressing adenoviral plasmid. The entry vector (pDONR223‐GFP) containing the attL1 and attL2 recombination sites and the GFP coding sequence was recombined with the destination vector (pAd/CMV/V5‐DEST), which contains attR1 and attR2 sites, using LR Clonase enzyme mix. This reaction resulted in the formation of the final expression construct (pAdGFP), in which the GFP gene is positioned under the control of the CMV promoter within an adenoviral backbone. Plasmid maps were generated using SnapGene software (version 4.3.6). (B) GFP expression in HT1080 cells following transduction with an adenoviral vector serotype 5 encoding GFP. Cells were transduced with increasing amounts of viral particles per well (0, 101, 102, 103, 104, 105, and 106 vp). Fluorescence images were captured at 10X and 20X magnifications. GFP expression is indicated by green fluorescence. Untransduced cells (0 vp) served as a negative control.
Ad5 Vectors Demonstrate Dose‐Dependent Transduction Efficiency in Macrophage and Monocytic Cell Lines
3.3
To assess the transduction capability of the adenoviral system in representative innate immune cell lines, murine RAW 264.7 macrophages and human THP‐1 monocytic cells were transduced with control AdGFP vectors. Fluorescence microscopy confirmed measurable transduction in both cell types, indicated by clear GFP expression (Figure 3A for RAW 264.7; Figure 3B for THP‐1). These findings demonstrate that both RAW 264.7 macrophages and THP‐1 monocytes are permissive to adenoviral entry and subsequent transgene expression. This permissiveness is an important characteristic for a vaccine vector aiming to engage the innate immune system and potentially elicit immune responses.
In vitro transduction efficiency of AdGFP in macrophage and monocytic cell lines. (A) Transduction of murine RAW 264.7 macrophages with increasing doses of AdGFP viral particles ranging from 0 to 105 viral particles per well. Cells were imaged 48 h post‐transduction using bright field (BF) and GFP fluorescence microscopy at 20X magnification. Green fluorescence indicates successful transgene expression of green fluorescent protein (GFP). (B) Transduction of human monocytic THP‐1 cells with AdGFP at viral doses of 0, 103, and 104 viral particles per well. Cells were imaged using BF and GFP channels at 10X, 20X, and 40X magnifications. Enhanced GFP expression was observed in a dose‐dependent manner.
Ad5Spike Vaccination Elicits Measurable and Persistent Cellular Immune Responses
3.4
To evaluate the cellular immune responses elicited by the Ad5Spike vaccine, early (day 30) and long‐term (day 90) post‐immunization responses were assessed in female BALB/c mice. The secretion of Th1‐type cytokines—IFN‐γ, IL‐2, and TNF‐α—was focused on using ELISpot and ELISA assays on T cell–enriched splenocyte populations following stimulation with a Spike peptide pool.
IFN‐γ production was quantified using both ELISA in culture supernatants and ELISpot at the single‐cell level. ELISA‐based quantification of IFN‐γ in splenocyte culture supernatants confirmed a highly significant increase in cytokine secretion across all Ad5Spike‐vaccinated groups at both day 30 and day 90 compared to controls (Figure 4A). In these assays, background values from unstimulated wells were subtracted to determine antigen‐specific responses. On day 30, mice immunized with all Ad5Spike doses exhibited a significantly higher number of IFN‐γ–secreting splenocytes compared to the AdGFP control group (Figure 4B). However, by day 90, ELISpot analysis showed that only the 10^1^ ^2^ dose group maintained a statistically significant increase in IFN‐γ–secreting splenocytes compared to the AdGFP control. No significant differences were observed in the lower dose groups at this time point (Figure 4B), indicating that the peak early cellular IFN‐γ response had waned by day 90 except at the highest vaccine dose.
*Evaluation of antigen‐specific IFN‐γ, IL‐2, and TNF‐α secretion in BALB/c mice immunized with the Ad5Spike vaccine vector. Female BALB/c mice (6–8 weeks old; n = 5 per group per timepoint) were immunized intraperitoneally with varying doses of the Ad5Spike recombinant adenoviral vector containing 108, 1010, or 101 2 viral particles at two time points: 30 days (left panels) and 90 days (right panels) post‐immunization. (A) IFN‐γ levels in mouse sera were measured using ELISA. (B) IFN‐γ–secreting splenocytes were quantified by ELISpot assay. (C) IL‐2–secreting splenocytes and (D) TNF‐α–secreting splenocytes were measured by ELISpot assays. Data are presented as mean ± SEM, with each data point representing an individual mouse. Normality was assessed using the Shapiro–Wilk test. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. The AdGFP‐immunized group served as the reference control. *p < 0.05, ***p < 0.0001.
IL‐2 secretion, a critical indicator of T cell proliferation and activation, was quantified by ELISpot analysis. Mice immunized with all tested doses of Ad5Spike consistently exhibited significantly elevated IL‐2‐secreting splenocytes compared to PBS and AdGFP control groups at day 30, while this remained significant only in the 10^12^ group at day 90 (Figure 4C).
TNF‐α production, indicative of proinflammatory potential, was also evaluated by ELISpot. On day 30 post‐immunization, all Ad5Spike‐vaccinated groups showed significantly increased numbers of TNF‐α–secreting splenocytes compared to controls (Figure 4D). However, by day 90, TNF‐α secretion levels had declined, and no statistically significant differences were observed between vaccinated and control groups.
Ad5Spike Vaccination Induces Measurable and Sustained Spike‐Specific Humoral Responses
3.5
To assess the vaccine's ability to stimulate the humoral immune system and induce Spike‐specific antibodies, IgG ELISA was performed on serum samples from immunized female BALB/c mice (n = 5 per group/timepoint) at days 30 and 90 post‐immunization. Mice received intraperitoneal immunization with 10^8^, 10^10^, or 10^12^ viral particles of Ad5Spike, while control groups received PBS or AdGFP. At day 30, mice immunized with all doses of Ad5Spike exhibited significantly elevated anti‐Spike IgG levels compared to the AdGFP control (Figure 5A). At day 90, anti‐Spike IgG levels remained significantly elevated in the 10^10^ and 10^12^ vp dose groups, compared to the AdGFP control. While the 10^8^ vp dose group also showed an increase in anti‐Spike IgG levels, this elevation was not statistically significant compared to the AdGFP control (Figure 5A). These results demonstrate that intraperitoneal administration of the Ad5Spike vector induces measurable production of anti‐Spike IgG antibodies in female BALB/c mice, contributing to the activation of the humoral arm of the immune response.
*Humoral immune responses induced by Ad5Spike vaccination in BALB/c mice. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with the Ad5Spike vaccine vector at doses of 108, 1010, or 101 2 viral particles. Control groups received PBS or an AdGFP vector. (A) Serum anti‐Spike IgG antibody titers were quantified by ELISA at days 30 and 90 post‐immunization. Data are presented as mean ± SD, with each data point representing an individual mouse. (B) SARS‐CoV‐2 Wuhan‐1 variant–specific neutralizing antibody titers were assessed in sera collected at days 30 and 90 using a pseudovirus‐based neutralization assay. Titers are expressed as ID50 values (reciprocal serum dilution). Each dot represents an individual animal; box plots indicate the interquartile range with whiskers representing the 95% confidence interval. Normality was assessed using the Shapiro–Wilk test. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test, with comparisons made against the AdGFP vector–immunized control group. *p < 0.05, ***p < 0.001, ***p < 0.0001.
Ad5Spike‐Induced Antibodies Demonstrate Dose‐Dependent Neutralizing Activity against SARS‐CoV‐2 Pseudovirus
3.6
Following the evaluation of cytokine and antibody responses, a pseudovirus‐based neutralization assay was performed to assess the functional capacity of the induced antibodies by measuring neutralizing antibody (NAb) titers in serum samples. This assay utilized lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (LentiRFPSpike) to safely evaluate neutralizing activity under BSL‐2 conditions.
Neutralization assays performed on serum samples collected at day 30 post‐immunization revealed significantly elevated NAb titers in all Ad5Spike‐vaccinated groups relative to the AdGFP control groups (Figure 5B). At day 90, NAb titers remained elevated across all vaccinated groups. The 10^10^ and 10^12^ dose groups showed significantly higher neutralizing activity compared to the AdGFP control. While the 10^8^ group also exhibited higher neutralization compared to the AdGFP control, no significant difference was observed between these two groups at day 90. These findings indicate that intraperitoneal immunization with Ad5Spike effectively induces measurable and persistent neutralizing antibody levels in female BALB/c mice across all tested doses, with the 10^12^ viral particle dose particularly showing sustained and statistically superior neutralizing activity at later time points, providing a baseline for future assessment of protective efficacy.
Discussion
4
In this study, we designed, constructed, and evaluated a replication‐defective adenoviral vector, Ad5Spike, encoding the full‐length SARS‐CoV‐2 Spike glycoprotein. Our results demonstrate that Ad5Spike elicits measurable and persistent cellular and humoral immune responses in BALB/c mice, highlighting its potential as a proof‐of‐concept for an Ad5‐based vaccine candidate against SARS‐CoV‐2.
The successful generation of the pAd5Spike expression plasmid via Gateway LR recombination was confirmed through restriction enzyme digestion and gel electrophoresis, demonstrating the integrity and correct insertion of the codon‐optimized Spike gene. In particular, the in silico simulation using SnapGene and confirmation by EcoRI digestion provided reliable structural validation of the construct, ensuring directional cloning and vector integrity, which is crucial for subsequent expression fidelity. Functional Spike expression from the plasmid in HEK293T cells and from the purified Ad5Spike vector in HT1080 cells was validated via immunohistochemistry and immunofluorescence, respectively. The dose‐dependent increase in Spike expression in HT1080 cells confirmed efficient transduction, while the successful gene delivery to murine RAW 264.7 and human THP‐1 monocytes using a control AdGFP vector further emphasized the broad tropism and functionality of the adenoviral system in innate immune cells, aligning with previous studies demonstrating the permissiveness of such cell lines to Ad5 vectors [22, 23].
Immunization with Ad5Spike induced a cellular immune response marked by elevated IFN‐γ, IL‐2, and TNF‐α production, consistent with a Th1‐associated cytokine profile. Notably, significant increases in IFN‐γ, IL‐2, and TNF‐α–secreting splenocytes were observed at day 30 post‐immunization, consistent with the well‐characterized Th1 bias of adenoviral vectors [24, 25]. Although the frequency of IFN‐γ–producing cells declined by day 90, sustained elevation of IFN‐γ cytokine levels in splenocyte culture supernatants indicated persistent systemic activation. While our results demonstrate significant cytokine production following splenocyte stimulation, we acknowledge that the current study characterizes cellular immunity primarily through bulk cytokine measurements and ELISpot assays. A more detailed phenotypic characterization is required to fully define the nature of this response [26]. Future studies should employ flow cytometry and intracellular cytokine staining (ICS) to specifically identify the contributions of CD4^+^ and CD8^+^ T‐cell subsets. Furthermore, investigating memory subsets, such as CD44^+^ CD62L^+^ central memory and CD44^+^ CD62L^−^ effector memory T cells, would be essential to determine the long‐term protective potential of the Ad5Spike vaccine candidate. The continued presence of IL‐2–secreting splenocytes suggests the maintenance of T cell proliferative potential, which is a component of long‐term immune persistence [27]. The transient nature of the TNF‐α response, peaking at day 30 and diminishing by day 90, is in line with the resolution of acute inflammatory responses, as previously observed in adenoviral vaccine studies [28]. Furthermore, our data are suggestive of a Th1‐dominant immune environment, which is generally considered favorable for controlling viral infections. Additional evaluation of Th2‐associated cytokines (such as IL‐4 and IL‐5) and IgG subclass profiling (e.g., the IgG2a/IgG1 ratio) would further strengthen and refine the characterization of Th1‐associated immune polarization.
In parallel, Ad5Spike vaccination stimulated significant and durable Spike‐specific humoral responses. By day 30, all vaccinated groups showed significantly elevated anti‐Spike IgG levels, which persisted through day 90 in a dose‐dependent manner, particularly in the 10^10^ and 10^1^ ^2^ vp groups. However, the present study focuses on functional antibody measurements, extending future analyses to include a more comprehensive characterization of the humoral immune architecture would further strengthen the understanding of this platform's long‐term memory potential. In particular, subsequent studies incorporating flow cytometric profiling of B‐cell subsets within secondary lymphoid organs—such as germinal center B cells (B220^+^ GL7^+^ Fas^+^), memory B cells, and antibody‐secreting plasma cells (CD138^+^)—will provide valuable insight into the durability and maturation of the vaccine‐induced response. Importantly, Ad5Spike elicited neutralizing antibodies (NAbs) capable of inhibiting SARS‐CoV‐2 pseudovirus infection. As this study utilized the ancestral Wuhan‐1 strain, the neutralization data provide a useful baseline for assessing vaccine‐induced antibody activity. Given the ongoing emergence of SARS‐CoV‐2 variants of concern (VOCs) with mutations in the RBD, future studies incorporating cross‐neutralization analyses will be valuable to further define the breadth and adaptability of the Ad5Spike‐induced antibody response. Nevertheless, the vp group showed significantly greater neutralizing activity at day 90 compared to both control and lower‐dose groups.
Despite these outcomes, several limitations should be acknowledged. First, the study was conducted exclusively in female BALB/c mice to maintain cohort consistency for this initial pilot investigation. While this approach is acceptable for an early‐stage preclinical assessment, immune responses can differ based on sex and genetic background; therefore, the findings may not fully capture the spectrum of vaccine‐induced responses across diverse populations. Future studies incorporating both sexes and expanded cohort sizes will be required to evaluate sex‐dependent differences in humoral and cellular immunity and to enhance the robustness and generalizability of the findings. Crucially, the evaluation of immune responses at day 30 and day 90 demonstrates the persistence of immune markers over a medium‐term period, supporting sustained immune activation. Incorporating memory recall experiments, such as antigen re‐challenge, in future studies would further clarify the capacity for rapid mobilization of functionally reactive memory populations. Another primary limitation is the absence of in vivo protection data using a SARS‐CoV‐2 challenge model. While pseudovirus neutralization assays provide a useful surrogate at the BSL‐2 level, they do not account for the complex interplay of the immune system during an active infection [29]. A critical next step will involve immunizing susceptible models, such as ACE2‐transgenic mice, followed by a SARS‐CoV‐2 challenge to assess protection against clinical disease, body‐weight changes, and lung viral load. Furthermore, while this study demonstrates systemic immune responses following intraperitoneal immunization, these findings provide a foundation for extending future investigations toward mucosal immunity. Given that SARS‐CoV‐2 is a respiratory pathogen, future studies incorporating intranasal administration in addition to systemic delivery will be valuable to determine whether Ad5Spike can elicit secretory IgA (sIgA) and tissue‐resident T cells capable of neutralizing the virus at the primary site of infection. Additionally, for clinical translation, the potential impact of pre‐existing anti‐Ad5 immunity in human populations must be considered [30]. Strategies such as using alternate serotypes or heterologous prime‐boost regimens should be explored in future optimization efforts [31].
In summary, our study demonstrates that Ad5Spike, a replication‐defective adenoviral vector expressing the full‐length SARS‐CoV‐2 Spike protein, induces measurable cellular immune responses associated with Th1‐related cytokine production, along with significant Spike‐specific IgG and neutralizing antibody responses in BALB/c mice. These findings support the continued development of the Ad5 platform as a modular system for COVID‐19 vaccine research and lay the groundwork for future preclinical evaluations and clinical translation.
Conclusion
5
In summary, our study provides a focused, early‐stage preclinical assessment of an Ad5‐based vaccine candidate encoding the ancestral SARS‐CoV‐2 Spike protein. While our results demonstrate that the platform is capable of inducing measurable antibody and cytokine responses in a female BALB/c mouse model, we emphasize that these findings represent an initial proof‐of‐concept. Substantial additional research is required before the translational potential of Ad5Spike can be fully realized. This includes, but is not limited to, detailed phenotypic T and B cell profiling via flow cytometry, cross‐neutralization assays against emerging VOCs, and rigorous in vivo protection studies in susceptible models such as ACE2‐transgenic mice. Furthermore, evaluating alternative administration routes to enhance mucosal protection remains a priority for future developmental iterations. Until such data are available, these results serve as a foundational demonstration of the platform's immunogenicity and provide a modular starting point for the continued development of adenoviral vector‐based strategies against evolving infectious disease threats.
Author Contributions
F. E. wrote the main manuscript text, conducted experiments, prepared figures, and performed statistical analysis. F. E. and S. S. conceived and designed the study. S. S. supervised the study. F. E., F. U., and S. S. interpreted the data. All authors reviewed the manuscript.
Funding
This research was funded by the KOSGEB R&D, Innovation and Support Program and additionally supported by the Akdeniz University Scientific Research Projects Coordination Unit under project number TDK‐2022‐6067.
Conflicts of Interest
The authors declare no conflicts of interest.
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