T-cell responses are insufficient to generate protective immunity to SARS-CoV-2

T-cell responses are insufficient to generate protective immunity to SARS-CoV-2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article T-cell responses are insufficient to generate protective immunity to SARS-CoV-2 Soung-Chul Cha, Szymon J Szymura, Aaron Anderson, Zhenyuan Dong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8158863/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Neutralizing antibodies are established correlates of protection against SARS-CoV-2, yet T-cell responses are also thought to contribute substantially to limiting disease severity and enhancing durability of protection. To examine whether cellular immunity alone can confer protection, we engineered DNA vaccines encoding modified Spike proteins, including C-terminal truncation (SARS-CoV-2-ΔC, ΔC) and cleavage-site–deleted, linker-inserted (SARS-CoV-2-Linker-ΔT, Linker-ΔT) variants, with or without genetic fusion to MIP3α, which has been shown to enhance targeting of antigen-presenting cells (APC) and preferentially induce T-cell responses. In BALB/c mice, ΔC constructs induced non-neutralizing Spike- and RBD-binding antibodies across variants, as well as robust CD4 + and CD8 + T cell responses, whereas Linker-ΔT elicited strong Th1-skewed cellular immunity in the absence of humoral responses. In K18-hACE2 mice antibody neutralizing activity was not detected by any of the vaccines, and none conferred protection following lethal virus challenge, despite robust specific T-cell cytokine responses. These results support vaccine designs incorporating chemokine fusion to enhance T-cell priming, but cellular responses alone are insufficient for SARS-CoV-2 protection. Integrating such APC-targeting strategies with structural modifications that preserve pre-fusion neutralizing epitopes may be worthwhile. Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The development of safe and effective vaccines against SARS-CoV-2 has relied largely on immunogens capable of eliciting high titers of neutralizing antibodies, as exemplified by mRNA and adenoviral platforms. Neutralizing antibody levels strongly correlate with protection in both non-human primates and humans, serving as the principal mechanistic correlate of immunity 1 , 2 . However, accumulating evidence suggests that T-cell responses also play an important role, particularly in limiting disease severity and sustaining long-term protection as antibody titers wane 3 – 5 . The extent to which T-cell immunity can compensate for deficient neutralizing antibody responses remains a central unresolved question. This issue has particular clinical relevance for patients with B-cell malignancies or those receiving B-cell–depleting therapies, in whom antibody formation after SARS-CoV-2 vaccination is frequently impaired while T-cell responses are often preserved. Understanding the balance between these immune mechanisms is therefore critical for guiding vaccine design and optimizing protective strategies in immunocompromised populations. DNA vaccines provide a flexible platform for rapid antigen engineering and have shown the capacity to induce both humoral and cellular responses in preclinical and clinical settings 3 , 6 . Nevertheless, the immunogenicity of DNA vaccines is often lower than that of mRNA vaccines, and strategies to improve antigen delivery and immune activation are needed. One such approach involves chemokine fusion, which targets antigens to antigen-presenting cells (APCs), thereby enhancing priming of adaptive immunity 7 . Our group has previously demonstrated that chemokine–antigen fusion DNA vaccines can elicit potent immune responses against infectious pathogens and tumors in preclinical models 7 – 10 , and clinical safety has been established in a first-in-human trial of a lymphoma DNA vaccine 11 . Consistent with prior work in infectious disease and cancer models, MIP3α fusion DNA vaccination drives a Th1-skewed immune profile, with dominant IFNγ and TNFα secretion and minimal induction of Th2-associated cytokines 12 . Therefore, we selected MIP3α as a driver for immature dendritic cells to enhance cellular immunity. We hypothesized that MIP3α fusion to the Spike glycoprotein enhances APC targeting and T-cell priming, supporting cellular immunity as the primary protective mechanism against SARS-CoV-2. Here, we applied this APC-targeted DNA vaccine strategy to SARS-CoV-2. We engineered multiple DNA vaccine constructs encoding the spike glycoprotein (including variants with C-terminal truncation (ΔC), protease cleavage site deletion and linker insertion (Linker-ΔT), with and without fusion to the chemokine MIP3α. By evaluating humoral and cellular immunity in BALB/c and K18-hACE2 mouse models, followed by viral challenge in the latter, we sought to dissect the relative contributions of binding antibodies, neutralizing antibodies, and T cells in vaccine-mediated protection against SARS-CoV-2. Methods Vaccine constructs DNA vaccine plasmids were generated using a preclinical-grade pVax-based backbone. The SARS-CoV-2 Spike (isolate Wuhan-Hu-1; GenBank accession MN908947.3) sequence was codon-optimized for mammalian expression. Three Spike variants were engineered: (i) ΔC, with a 19–amino acid cytoplasmic tail deletion; (ii) Linker-ΔT, with deletion of the S1/S2 and S2′ protease cleavage sites and insertion of a (G4S)₂ linker with truncation of the transmembrane and cytoplasmic domains. For APC targeting, murine MIP3α was fused to the N-terminus of each construct via a flexible linker. An HA-tag was included at the C-terminus for detection. An irrelevant control construct encoding MIP3α fused to A20-scFv was also generated. Plasmids were purified under endotoxin-free conditions (Qiagen EndoFree kit) and validated by sequencing and restriction digest. Animals and immunization The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute at City of Hope (COH) approved protocol 20032, which is assigned to this study. All study procedures were conducted in strict compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Mice were maintained on a 12-hour light/12-hour dark cycle at 22–24°C and 30–70% humidity, with ad libitum access to food and water. Six-week-old BALB/c (BALB/cJ, 000651) or B6.Cg-Tg (K18-ACE2)2Prlmn/J (K18-hACE2, 034860) transgenic mice were obtained from The Jackson Laboratory. Vaccinations were administered via intramuscular injection into the quadriceps using the Tropis injector (PharmaJet), delivering 50 µg or 100 µg of DNA in 50 µL of sterile PBS per dose. A three-dose schedule was implemented on days 0, 14, and 28. Blood samples for humoral immune analysis were collected prior to each immunization, and the mice were euthanized for blood and spleen collection two weeks following the final vaccination. Splenocytes for cellular immune analysis were collected two weeks after booster immunization and isolated using standard procedures following humane euthanasia of the animals. Serological assays Sera were collected retro-orbitally prior to each immunization and two weeks post-final immunization. Antibody binding to recombinant SARS-CoV-2 Spike and variant RBD proteins (WT, Alpha, Beta, Gamma, Delta; BEI Resources) was measured by ELISA. Plates were coated with antigen (2 µg/mL), blocked with BSA, and incubated with serially diluted sera. Bound IgG was detected with HRP-conjugated anti-mouse IgG and developed with TMB substrate. Endpoint titers and area under the curve (AUC) were calculated. SARS-CoV-2 pseudovirus production and neutralization assay COVID 19 Spike Coronavirus Pseudovirus (MyBioSource, Cat:MBS434275) input was standardized by 100X signal above cell-only background. Heat-inactivated mouse sera were pooled, serially diluted (1:20–1:640), and incubated 1 hour at 37°C with SARS-CoV-2 spike pseudoviruses. HEK293T-ACE2 cells (2 × 10^4/well) were infected in the presence of 5 µg/mL polybrene, and luciferase activity was quantified 48 h later using Promega reagents. We plotted the data using luminescence vs. virus dilution. Neutralization was calculated as NT = [1 − (RLU immune sera / RLU control)] × 100, with NT90 values interpolated from dilution curves. The method was adapted from Crawford, H.D. et al 13 and Chiuppesi F et al 14 . T-cell analysis Splenocytes were collected two weeks after the final immunization and prepared by mechanical dissociation followed by red blood cell lysis. Cells were rested overnight in complete RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 2 mM L-glutamine, and 50 µM 2-mercaptoethanol. For functional assays, 1–2 × 10⁶ splenocytes per well were stimulated with overlapping peptide pools covering the SARS-CoV-2 Spike protein (Miltenyi Biotec; S or S1 peptide pools; 1–2 µg/mL per peptide) or with an irrelevant CMV peptide pool as a negative control. Stimulations were performed in the presence of brefeldin A and monensin for 5–6 h at 37°C with 5% CO₂. Following stimulation, cells were stained with fixable viability dye and antibodies against CD3, CD4, and CD8. After fixation and permeabilization (BD Cytofix/Cytoperm), intracellular cytokine staining was performed with antibodies specific for IFN-γ, TNF-α, IL-2, IL-4, and IL-5. Data were acquired on a BD LSRFortessa flow cytometer, and ≥ 100,000 lymphocyte events were collected per sample. FlowJo (Tree Star) was used for analysis. Gating was applied sequentially on singlets, live CD3⁺ T cells, and CD4⁺ or CD8⁺ subsets, with cytokine-positive populations quantified. Boolean gating was used to define polyfunctional subsets expressing one or more cytokines simultaneously. Concurrently, T cell responses were evaluated using cytokine ELISA, with splenocytes from immunized mice plated at a density of 5 x 10 6 cells per well in 24-well plates. The cells were stimulated with overlapping peptide pools spanning the SARS-CoV-2 Spike protein (Miltenyi Biotec; S or S1 peptide pools; 1–2 µg/mL per peptide) or with an irrelevant CMV peptide pool as a negative control, and incubated at 37°C for 3 days. The supernatant was collected and analyzed to assess cytokine production. Mouse IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5 were quantified by ELISA using the corresponding antibody sets (Invitrogen) in accordance with the manufacturer’s instructions. SARS-CoV-2 wildtype virus neutralization assay and challenge in K18-hACE2 mice All procedures were carried out in strict compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at Bioqual, Inc. Bioqual acquired 64 K18-hACE2 mice (32 males and 32 females) from Jackson Laboratory, which were subsequently randomly assigned to four experimental groups (n = 16 per group). The animals were immunized on Study Days (SD) 0, 14, and 28, in accordance with the prescribed vaccination schedule. On SD41, six mice per group (three males and three females) were humanely euthanized for terminal blood collection and splenectomy, while the remaining animals underwent interim blood sampling. Serum samples were subsequently analyzed by Bioqual through a plaque reduction neutralization test (PRNT). A subset of these samples was forwarded to City of Hope for confirmatory testing of Spike protein recognition. Surviving mice in SD48 were intranasally inoculated with 2.8 × 10³ PFU of live SARS-CoV-2 WAS-Calu-3 (lot #12152020-1235). During the study period, mice were monitored for weight loss and clinical manifestations of disease. On SD64, all remaining animals were humanely euthanized, and their spleens were collected for subsequent flow cytometry and ELISpot analyses. Statistical analysis Data were analyzed with GraphPad Prism v9. ELISA titers, cytokine levels, and flow cytometry data were compared by two-tailed unpaired Student’s t-test or one-way ANOVA with Tukey’s post hoc correction, as appropriate. Survival curves were compared using the log-rank (Mantel–Cox) test. P < 0.05 was considered statistically significant. All data are presented as mean ± SEM. Results SARS-CoV-2 DNA Vaccine design The Spike glycoprotein (S-protein) of the SARS-CoV-2 (isolate Wuhan-Hu-1; MN_908, 947.3; 15 ) virion was codon optimized and inserted into a pVax-based DNA vaccine vector (Fig. 1A). A leader sequence derived from the interferon-induced protein 10 (IP-10) was engineered and inserted at the N-terminus of the spike glycoprotein to facilitate its secretion through the rough endoplasmic reticulum membrane during protein synthesis 7 . Additionally, a hemagglutinin tag (HA-tag) was incorporated into each design to serve as a universal epitope tag. We designed DNA vaccines to encode four codon optimized SARS-CoV-2 DNA spike proteins, with and without murine MIP3α fusion. First, we constructed a full-length spike protein construct with 19 amino acids (KFDEDDSEPVLKGVKLHYT) removed from the CT (pSARS-CoV-2-ΔC) and a fusion of (pMIP3α-SARS-CoV-2-ΔC). We also deleted the TM and CT, further hypothesized that deleting the protease sites and inserting (G 4 S)₂ linkers would disrupt epitope folding and thereby abrogate antibody induction, with MIP3α ( pMIP3α-SARS-CoV-2-Linker-ΔT ) or without MIP3α ( pSARS-CoV-2-Linker-ΔT ). A DNA vaccine encoding A20 lymphoma scFv with irrelevant specificity (pMIP3α-A20-scFv) was used as a negative control 10 . pSARS-CoV-2-ΔC DNA vaccine immunogenicity in BALB/c mice We first characterized immunogenicity of the various DNA vaccines in BALB/c mice. Doses of either 50 µg or 100 µg of pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC vaccines were administered by Tropis injector to deliver uniform vaccine administration. Mice were serially immunized three times, with a 2-week interval between each vaccination. Blood samples were collected before each immunization, and the mice were euthanized for blood and spleen collection 2 weeks after the final vaccination (Fig. 2A). Endpoint humoral responses were measured by ELISA and demonstrated antibody binding to the SARS-CoV-2 Spike protein by all mice in both groups (Fig. 2B). Area under the curve calculations revealed no significant differences between pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC vaccines at either dose level (Supplementary Fig. 1A). Because SARS-CoV-2 recognizes host cell receptors through its receptor-binding domain (RBD), immune sera were also analyzed for their ability to recognize various RBD variants (Supplementary Fig. 1B). The results demonstrated that serum antibodies derived from pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC effectively distinguish between variant RBDs without any substantial differences. While antibodies generated by both vaccines exhibited reactivity against Spike proteins from multiple variants (Fig S1 A), they failed to induce neutralizing antibody responses against SARS-CoV-2 pseudovirus (Supplementary Fig. 1C). T cell responses to the SARS-CoV-2 were analyzed using splenocytes collected from vaccinated mice stimulated with either the Miltenyi Biotec S peptide pool, S1 peptide pool, or a CMV peptide pool as a negative control. Splenic T cells demonstrated a robust specific cytokine responses, including production of IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5 in culture supernatants by ELISA (Fig. 2C). Furthermore, analysis by intracellular staining revealed statistically significant expression of IFNγ and TNFα, but not IL-2, by both CD4 and CD8 T cells stimulated with the SARS-CoV-2 spike peptide pool across both constructs and doses without any obvious differences, compared with control stimulation (CD4 T cells: Fig. 2D; CD8 T cells: Fig. 2E). pSARS-CoV-2-Linker-ΔT DNA vaccine immunogenicity in BALB/c mice Our central hypothesis was that cellular immunity, rather than humoral immunity, constitutes the dominant protective mechanism against SARS-CoV-2. Linker-ΔT constructs were designed to disrupt optimal prefusion spike conformation, thereby potentially diminishing antibody titers while preserving, or even enhancing, T cell–mediated immunity. Humoral and cellular immunity of pSARS-CoV-2-Linker-ΔT vaccines, with and without MIP3 fusion, were also evaluated at 50 µg and 100 µg doses. As expected, antibody binding to the SARS-CoV-2 spike protein was not detected in the serum samples collected from mice vaccinated with either construct or at either dose (Fig. 3B). In contrast, Linker-ΔT vaccines induced specific T-cell responses. Splenic T cells from mice vaccinated with either construct produced significant levels of IFNγ, IL-6, IL-2, and TNFα in culture supernatants by ELISA, with no substantial differences between the two constructs of doses (Fig. 3C). Intracellular cytokine analysis of peptide-restimulated splenocytes revealed a predominantly Th1-skewed response, especially in mice vaccinated with the MIP3 fusion (Fig. 3DE). For example, pMIP3α-SARS-CoV-2-Linker-ΔT vaccine induced significant TNFα and IFNγ production in CD4⁺ and CD8⁺ T cells at either dose, while less IL-2 production was observed. Moreover, in CD4⁺ T cells, significant IFNγ production was observed exclusively in the pMIP3α groups at both doses (Fig. 3D). . Vaccine immunogenicity in K18-hACE2 mice With the eventual goal of testing for virus protection, we extended immunogenicity experiments to K18-hACE2 transgenic mice that express human angiotensin-converting enzyme 2 (ACE2), the receptor exploited by SARS-CoV-2 for cellular entry. Female k18-hACE2 mice (N = 5) were first immunized with 100 µg of the pMIP3α-SARS-CoV-2-ΔC or pSARS-CoV-2-ΔC DNA vaccines diluted in 50µL of sterile phosphate buffered saline (PBS) on days 0, 14, and 28, as before (Fig. 4A). Compared with pSARS-CoV-2-ΔC, mice vaccinated with pMIP3α-SARS-CoV-2-ΔC exhibited a trend towards higher serum antibody responses against the S protein (Fig. 4B). Moreover, serum antibody responses against wild-type RBD, Alpha(UK), Beta(SRA), Delta(Indian), and Gamma(Brazil) RBD variants were more robust in the pMIP3α-SARS-CoV-2-ΔC group (Supplementary Fig. 2A). However, neutralizing antibodies were not consistently detected in mice vaccinated with either pMIP3α-SARS-CoV-2-ΔC or pSARS-CoV-2-ΔC (Fig. 4C). Robust specific splenic T cell responses were detected in both groups, as analyzed by cytokine production by ELISA. Specifically, splenocytes from mice vaccinated with either pSARS-CoV-2-ΔC or pMIP3α-SARS-CoV-2-ΔC and stimulated with the SARS-CoV-2 Spike peptide pool produced statistically significant levels of IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5, compared with those stimulated with negative controls (CMV peptide pools; Fig. 4D). Specific IFNγ production by stimulated splenocytes from both pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC groups was also detected by intracellular staining, particularly from CD8 + cells (Fig. 4E). T-cell immunity without neutralizing antibodies was insufficient for protection against SARS-CoV-2 We designed a second experiment to investigate both immunogenicity and virus protection in K18-hACE2 mice, including both vaccines above and adding the pMIP3α-SARS-CoV-2-Linker-ΔT vaccine (Fig. 5A). Sixteen mice per group were vaccinated with 100 micrograms pSARS-CoV-2-ΔC, pMIP3α-SARS-CoV-2-ΔC, pMIP3α-SARS-CoV-2-Linker-ΔT, or pMIP3α-A20-scFv (negative control) in three doses administered as above. Two weeks after the final vaccination, six mice from each experimental group were euthanized for terminal bleeding and splenocyte isolation. The remaining ten mice were retro-orbitally bled, subsequently challenged with 2.8 × 10³ PFU of WAS-Calu-3 (LOT #12152020-1235) ten days later, and then monitored for variations in body weight and survival. As anticipated, mice vaccinated with both SARS-CoV-2-ΔC vaccines, but especially those vaccinated with the MIP3 fusion, demonstrated significant increases in serum antibody titers against the S protein (Fig. 5B, S3 Fig). However, corresponding neutralizing antibody activity against live SARS-CoV-2 virus was not observed in any of the groups (Fig. 5C). T-cell responses analyzed by intracellular cytokine staining for TNFα, IL-2, and IFNγ showed significant responses after stimulation with SARS-CoV-2 Spike protein peptide pools (S), compared with negative control stimulation (CMV) by both CD4 + and CD8 + T cells (Fig. 5D). Specific IFNγ production was also detected by all three vaccines by ELISPOT (Fig. 5E). Compared with negative controls, none of the experimental vaccines protected against weight loss or death (Fig. 5F and G). Our results indicate that T cell immunity was not sufficient to provide SARS-CoV-2 viral protection in K18-hACE2 mice. Discussion Building upon our previous studies demonstrating that the genetic fusion of chemokines to tumor antigens enhances antigen cross-presentation and induces robust CD8⁺ and Th1-type T-cell immunity in cancer models, we have applied this principle to the design of a vaccine for infectious diseases by leveraging MIP3α-mediated targeting of immature dendritic cells to augment cellular immunity against SARS-CoV-2. To test the hypothesis that T-cell responses against SARS-CoV-2 are necessary and sufficient for protection against this virus, we investigated the immunogenicity of several different SARS-CoV-2 DNA vaccine prototypes, designed to primarily induce T-cell immunity. All vaccine candidates induced robust CD4+ and CD8+ T cell responses characterized by specific cytokine production. Moreover, Spike- and RBD-binding antibodies were consistently detected across constructs and recognized multiple viral variants; however, none of the vaccines elicited neutralizing antibodies or conferred protection against lethal viral challenge. These findings suggest that neutralizing antibodies may be required for virus protection . Our findings align with large-scale correlates of protection analyses, which demonstrate that neutralizing antibody titers serve as the most reliable quantitative predictors of vaccine efficacy against SARS-CoV-2 infection. For example, Khoury and colleagues demonstrated that neutralizing antibody titers exhibited a strong correlation with protection from symptomatic infection 16 . This finding suggests that T-cell immunity alone is insufficient to prevent viral entry. Our study's findings corroborate this conclusion, as robust T-cell cytokine responses were not protective in the absence of neutralizing antibodies. Our observations also corroborate findings in rhesus macaques, where passive transfer of IgG alone was sufficient to confer protection against challenge, while CD8+ T cells contributed only when neutralizing antibody titers were subprotective 17 . Nonetheless, the breadth and functionality of T-cell responses are likely to play crucial roles in mitigating disease severity following infection. Vaccine-induced T cells have been shown to maintain 70–80% of their epitope recognition across the Omicron variant and other variants, despite substantial reductions in neutralization capacity 18 thereby providing a critical immunological “backstop” against viral evolution. Clinical data further reinforce this paradigm: early and polyfunctional T-cell responses are correlated with milder clinical outcomes, and in B-cell–depleted patients, preserved T-cell responses following vaccination are associated with a reduced risk of severe disease 19 . The absence of neutralizing antibodies remains a critical vulnerability, addressed clinically through timed revaccination, monoclonal antibody prophylaxis, early antivirals, and occasionally convalescent plasma 20,21 . Furthermore, other DNA vaccine platforms have achieved protective efficacy in preclinical models when neutralizing antibodies were elicited. For example, Yu et al. showed that DNA vaccines encoding full-length spike induced both neutralizing antibodies and protection against SARS-CoV-2 in rhesus macaques 22 . Similarly, DNA vaccine delivered with electroporation, elicited both T-cell and neutralizing antibody responses in humans 23,24 . Interestingly, a recent study using an RBD–MIP3α construct demonstrated durable humoral responses lasting at least 12 months in mice, contrasting with the absent neutralization in our ΔC- and Linker-based constructs 25 These discrepancies likely arise from fundamental differences in antigen design, epitope presentation, and stabilizing mutations. Structure-guided stabilization of the full-length Spike ectodomain—exemplified by the S-2P and HexaPro variants—has been critical in locking the protein in its prefusion conformation, thereby exposing neutralization-sensitive epitopes and enhancing immunogenicity; these advancements have been fundamental to the success of mRNA-1273 and other commercial platforms, consistently eliciting high-titer neutralizing antibodies in both preclinical and clinical studies 26,27 . In contrast, our ΔC and Linker-ΔT constructs incorporated deletions and cleavage-site modifications without stabilizing mutations, alterations that may have compromised the conformational integrity of the RBD and, as a result, limited B cell recognition of neutralizing epitopes. Beyond stabilization, the multivalent display of antigens has emerged as a critical factor influencing vaccine efficacy. Self-assembling nanoparticles displaying multiple copies of the receptor-binding domain (RBD), such as the I53-50 two-component scaffolds, have been demonstrated to induce significantly higher neutralizing titers and broader recognition of variants compared to monomeric RBD or unstabilized Spike constructs 28,29 . Similarly, recombinant prefusion-stabilized Spike proteins, formulated with potent adjuvants such as the saponin-based Matrix-M employed in NVX-CoV2373, have exhibited robust neutralizing activity and demonstrated clinical efficacy 30 . Our DNA vaccine constructs lacked these design features—multimerization, prefusion stabilization, and optimized adjuvantation—and instead depended exclusively on plasmid-mediated expression of modified Spike antigens delivered via intramuscular injection. These discrepancies likely reflect differences in antigen design, epitope exposure, and stabilizing mutations. The protective efficacy of T cells in the absence of neutralization has been reported in other settings. For example, epitope-focused or ubiquitin-targeted DNA vaccines reduced morbidity and mortality in K18-hACE2 mice without detectable neutralizing antibodies 31 . Recent human studies have demonstrated that mRNA vaccination against SARS-CoV-2 induces a polyclonal, high-affinity T-cell response, a feature associated with long-lasting immunity. 32 In contrast, our MIP3α-targeted DNA vaccines elicited strong cytokine production but failed to protect, suggesting that the induced T cells lacked sufficient clonal diversity and receptor avidity. Future vaccine iterations should therefore aim to enhance T-cell quality through optimized antigen processing and presentation to achieve protective efficacy even in the absence of neutralizing antibodies. In addition to MIP3α, other chemokines have been investigated as antigen-delivery vehicles to APCs, with distinct immunological consequences. MCP-1 has been shown to stimulate IL-4 production, thereby driving Th2 polarization in a regulatory manner 33,34 . Similarly, the macrophage-derived chemokine CCL22 (MDC) selectively recruits Th2 cells to APCs 34 . MIP3α fusion preferentially induces Th1-driven cellular immunity, optimizing CD8⁺ effector T-cell responses and IFNγ output, whereas MCP-1 or MDC incorporation tends to bias immunity toward a Th2 phenotype with superior antibody production. Thus, rational chemokine selection provides a potential strategy to tailor immune polarization: Th1-inducing chemokines such as MIP3α may enhance antiviral T-cell responses, whereas Th2-inducing chemokines like MCP-1 or MDC may complement this by boosting antibody responses. In the context of SARS-CoV-2 vaccines, combining or sequentially deploying these chemokine-fusion strategies could theoretically broaden protective immunity by balancing cellular and humoral arms of the adaptive response. Our study demonstrates that while chemokine-targeted DNA vaccines elicit robust Th1-biased T-cell responses and broaden Spike-binding antibodies, neutralizing antibody induction remains indispensable for protection against SARS-CoV-2. These findings have direct implications for immunocompromised patients, such as those with B-cell depletion, in whom antibody formation is impaired but T-cell responses persist. Future vaccine strategies should combine Th1- and Th2-inducing chemokine fusions to optimize both neutralizing antibody quality and durable cellular immunity. Declarations Competing Interest The authors declare no competing interests. Funding: Not applicable. Contributions S.C. and L.W.K. designed the project, oversaw the experiments, and wrote the manuscript. S.C., S.J.S., and L.W.K. contributed to the design of the vaccine construct. S.J.S. and A.A. cloned the vaccine and conducted an analysis of the sequencing data. A.A., E.O., and S.C. optimized the mouse injection protocol and carried out the immunization of the mice. Z.D. developed and optimized assays for the production of SARS-CoV-2 pseudovirus and its neutralization. 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(A) Sera collected two weeks following the final immunization were assayed by ELISA against recombinant SARS-CoV-2 Spike protein, and antibody binding was quantified as the area under the curve (AUC) across serial dilutions. Left: comparison of pMIP3α-SARS-CoV-2-ΔC at 100 μg versus 50 μg; middle: comparison of pSARS-CoV-2-ΔC at 100 μg versus 50 μg; right: comparison between 100 μg doses of pMIP3α-SARS-CoV-2-ΔC and pSARS-CoV-2-ΔC. Data are presented as mean ± standard deviation, with individual animals represented by separate data points. Statistical analyses were conducted using unpaired two-tailed t-tests, and p-values are reported accordingly. No significant differences in AUC values were observed across different dose levels or between the constructs. (B) Induced Humoral Immunity was measured using binding antibody ELISA to the Spike protein, RBD W; Wild-type (Wuhan strain), RBD-RSA; Beta variant (South Africa), RBD-Brazil; Gamma variant (Brazil), RBD-India B.1; Delta variant (India, B1.167) , RBD-India B.1; Delta variant (India, B1.167.2) , and RBD-UK; Alpha variant (UK). (C) SARS-COV-2 pseudo-virus neutralization assays (using serum isolated from blood collected at experimental endpoint) SupplementaryFigure2.jpg Supplementary Figure 2. Antibody Binding to SARS-CoV-2 RBD Mutational Variants from Vaccinated K18-hACE2 mice. (A) K18-hACE2 mice were intramuscularly immunized every two weeks with three 100 µg doses of either pSARS-CoV-2-ΔC or pMIP3α-SARS-CoV-2- ΔC DNA Vaccine constructs using the Pharmajet Tropis Needleless Injector. Serum isolated from blood collected at experimental endpoint was used to assess ELISA antibody binding to wild type SARS-CoV-2 Spike Protein, RBD WT; Wild-type (Wuhan strain), RBD-RSA; Beta variant (South Africa), RBD-Brazil; Gamma variant (Brazil), RBD-India B.1; Delta variant (India, B1.167) , RBD-India B.1; Delta variant (India, B1.167.2) , and RBD-UK; Alpha variant (UK). (B) Quantitative analysis by area under the curve (AUC) confirmed antigen-specific binding in both vaccine groups. Data representative of two independent experiments. Values are presented as mean ± standard deviation. P values were determined using the Mann-Whitney test. SupplementaryFigure3.jpg Supplementary Figure 3. Comparative evaluation of antibody binding activity in K18-hACE2 mice, expressed as area under the curve (AUC) from serum ELISA. Serum binding antibodies were quantified by ELISA using serial dilutions against recombinant SARS-CoV-2 Spike protein. Quantitative analysis by area under the curve (AUC) confirmed antigen-specific binding in three vaccine groups compared to negative control group MIP3a-dC; pMIP3α-SARS-CoV-2- ΔC, MIP3a-linker; pMIP3α-SARS-CoV-2-linker-ΔT, ΔC: pSARS-CoV-2-ΔC, and MIP3a-A20; pMIP3α-A20-scFv DNA Vaccines. P values were determined using the Mann-Whitney test. Cite Share Download PDF Status: Published Journal Publication published 20 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Nov, 2025 Editor assigned by journal 20 Nov, 2025 Submission checks completed at journal 20 Nov, 2025 First submitted to journal 19 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":229075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of DNA Vaccines. (A) \u003c/strong\u003eSchematic diagram of the full-length Spike glycoprotein of the SARS-CoV-2 virion. The spike glycoprotein, composed of the S1 and S2 subunits, comprises the N-terminal domain (NTD), the receptor-binding domain (RBD), subdomain 1(SD1), subdomain (SD2), heptad repeat 1(HR1), central helix (CD), heptad repeat 2 (HR2), transmembrane domain (TM), and cytoplasmic tail (CT). \u0026nbsp;\u0026nbsp;\u003cstrong\u003e(B) \u003c/strong\u003eFour different DNA vaccines encoding variations of the SARS-CoV-2 Spike (S) protein were constructed: (i) full–length Spike (\u003cem\u003epSARS-CoV-2-ΔC;\u003c/em\u003e \u003cem\u003eΔC\u003c/em\u003e: KFDEDDSEPVLKGVKLHYT removed from the CT\u003cem\u003e \u003c/em\u003e), (ii) addition of the chemoattractant MIP3α to the \u003cem\u003eΔC\u003c/em\u003e Spike (\u003cem\u003epMIP3α-SARS-CoV-2-ΔC\u003c/em\u003e), (iii) the deletion of the TM and CT from Spike with the addition of two disulfide linkers (G4S)₂ \u0026nbsp;at the ΔS1/S2 and ΔS2’ cleavage sites of the Spike with MIP3α (\u003cem\u003epMIP3α-SARS-CoV-2-Linker-ΔT\u003c/em\u003e) and without MIP3α (\u003cem\u003ep SARS-CoV-2-Linker-ΔT\u003c/em\u003e). The pMIP3α-A20-scFv vaccine encodes the A20 mouse tumor idiotype as a negative control with irrelevant specificity. All DNA vaccine inserts were cloned into the pUCMVC3 DNA Vaccine backbone which included a CMV promoter.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/05b5953b6bb47a3a8ed98b2d.jpg"},{"id":96781272,"identity":"52d42d67-0f47-444a-b9a8-e4fe92e4f6dc","added_by":"auto","created_at":"2025-11-26 04:26:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":349086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 DNA vaccines drive cellular immunity and binding antibodies but no detectable neutralization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eBALBC/J wild type mice (N = 45) mice were intramuscularly immunized with 3 doses of either 50 or 100 µg at 2-week intervals using a mouse modified Pharmajet Tropis Needle free Injector with either the pMIP3α-SARS-CoV-2-ΔC or pSARS-CoV-2-ΔC. Mice were immunized with PBS as a negative control. The timeline shows the bleeding and immunization regiment. Humoral immune responses were assessed using binding antibody ELISA to the Spike protein on serum isolated from blood collected at experimental endpoint\u003cstrong\u003e. (B). \u0026nbsp;\u003c/strong\u003eHumoral responses in each group injected with different vaccine or dose measured by ELISA. Mice showing elicited Spike binding were color coded and matched to their corresponding number in the experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eCellular Immune response was measured using cytokine staining assays for splenocytes collected at experimental endpoint in response to pooled S peptides for IFNγ, IL-6, IL-2, TNFα, IL-4, IL-5m IL-10, and IL-13. Phorbol 12-myristate 13-acetate (PMA) was utilized as a positive control, Non-Treated (NT) as a negative control, and CMV as an irrelevant control. \u0026nbsp;As cytokine levels in each experimental group increased similarly to those in the PBS PMA control (gray color), only the PBS data are presented in the graph for the other groups, with the PMA data omitted.\u003cstrong\u003e (D, E) \u003c/strong\u003eTNFα, IL-2, and\u003cstrong\u003e \u003c/strong\u003eIFNγ Intracellular staining for CD4+ and CD8+ T cells collected at experimental endpoint for assessing cellular immune responses induced by the DNA vaccines. Data representative of two independent experiments. Values are presented as mean ± standard deviation. \u003cem\u003eP\u003c/em\u003evalues were determined using the Mann-Whitney test. * 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, *** 0.001 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. **** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/193cb6278db6fc575efdfed8.jpg"},{"id":96781276,"identity":"e2c0b382-536a-4d76-a59f-885589f374fa","added_by":"auto","created_at":"2025-11-26 04:26:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":316690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 DNA Linker-ΔT Vaccine induces T cell Responses but not Humoral Responses to SARS-CoV-2 S Protein. (A)\u003c/strong\u003e BALBC/J wild type mice (N = 50) mice were intramuscularly immunized with 3 doses of either 50 or 100 µg of either the pMIP3α-SARS-CoV-2-Linker-ΔT or the pSARS-CoV-2-Linker- ΔT DNA Vaccine at 2-week intervals using a mouse modified Pharmajet Tropis Needle free Injector. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe Linker-ΔT construct encodes for a disulfide linker to be inserted at the ΔS1/S2 and ΔS2’ cleavage sites of the spike protein. Mice were immunized with Phosphate buffered saline as a negative control. The timeline shows the bleeding and immunization regiment. Humoral immune responses were assessed using binding antibody ELISA to the Spike protein using serum isolated from blood collected at experimental endpoint\u003cstrong\u003e. (B) \u003c/strong\u003eEndpoint humoral antibody binding titers in response to the SARS-CoV-2 spike was not detected in the serum by ELISA\u003cstrong\u003e. (C) \u003c/strong\u003eCellular Immune response was measured using cytokine staining assays for splenocytes collected at experimental endpoint in response to pooled S peptides for IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5. Phorbol 12-myristate 13-acetate (PMA) was utilized as a positive control, Non-Treated (NT) as a negative control, and CMV as an irrelevant control. As cytokine levels in each experimental group increased similarly to those in the PBS PMA control (gray color), only the PBS data are presented in the graph for the other groups, with the PMA data omitted. \u003cstrong\u003eD) \u003c/strong\u003eTNFα, IL-2, and\u003cstrong\u003e \u003c/strong\u003eIFNγ intracellular staining for CD4+ and CD8+ T cells collected at experimental endpoint also assessed cellular immune responses induced by the DNA vaccine constructs. Data representative of two independent experiments. Values are presented as mean ± standard deviation. \u003cem\u003eP\u003c/em\u003e values were determined using the Mann-Whitney test. * 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, *** 0.001 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/1aa3fd3f6addc923a83608d7.jpg"},{"id":96781286,"identity":"c97f7568-1854-44b2-9fae-4d8320a2d7f8","added_by":"auto","created_at":"2025-11-26 04:26:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":308783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMIP3α-fusion DNA vaccines induced T-cell and antibody responses without neutralization antibody in K18-hACE2 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eK18-hACE2 transgenic mice (N=15) were intramuscularly immunized with 3 doses of 100 µg at 2-week intervals using a mouse modified Pharmajet Tropis Needle free Injector with either the pMIP3α-SARS-CoV-2-ΔC, pSARS-CoV-2-ΔC, or pMIP3α-A20-scFv DNA Vaccines. Mice were immunized with PBS as a negative control. One day prior to each immunization, mice were retro-orbitally (RO) bled. The timeline shows the bleeding and immunization regimen. Humoral immune responses were assessed using binding antibody ELISA to the Spike protein using serum isolated from blood collected at experimental endpoint.\u003cstrong\u003e (B)\u003c/strong\u003e Endpoint humoral responses measured by ELISA in each group. Mice that elicited highest Spike binding or neutralizing antibodies were color coded and matched to their corresponding number in the experimental group.\u003cstrong\u003e (C) \u003c/strong\u003eand SARS-COV-2 pseudo-virus neutralization assays. \u003cstrong\u003e(D) \u003c/strong\u003eCellular immune response was measured using cytokine staining assays for splenocytes collected at experimental endpoint in response to pooled S peptides for IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5. Phorbol 12-myristate 13-acetate (PMA) was utilized as a positive control, Non Treated (NT) as a negative control, and CMV as an irrelevant control. As cytokine levels in each experimental group increased similarly to those in the PBS PMA control (gray color), only the PBS data are presented in the graph for the other groups, with the PMA data omitted.\u003cstrong\u003e (E) \u003c/strong\u003eIFNγ intracellular staining for CD4+ and CD8+ T cells collected at experimental endpoint assessingcellular immune responses induced by the DNA vaccines. Data representative of two independent experiments. Values are presented as mean ± standard deviation. \u003cem\u003eP\u003c/em\u003e values were determined using the Mann-Whitney test. * 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, *** 0.001 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. **** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/6d66fbac10047a83bb3e2f1a.jpg"},{"id":96781282,"identity":"3e05f6cf-081a-49e3-b17a-c58719435f71","added_by":"auto","created_at":"2025-11-26 04:26:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":394505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT Cell Immunity Alone Does Not Induce Protection against Live Virus in K18-hACE2 Mice (A) \u003c/strong\u003eK18-hACE2 (N=64) mice were intramuscularly immunized with 3 doses of 100 µg at 2-week intervals using a mouse modified Pharmajet Tropis Needle free Injector with either the pMIP3α-SARS-CoV-2-ΔC, pSARS-CoV-2-ΔC, pMIP3α-SARS-CoV-2-linker-ΔT, or pMIP3α-A20-scFv DNA vaccines. On Day 44 of the experiment, two weeks after the third and final vaccination N=24 mice were terminally bled, and their spleen was collected for analysis. The remaining N=40 mice were then challenged with 2.8 e3 PFU of the WAS-Calu-3 SARS-CoV-2 live virus, and their survival was recorded over the course of 10 days. Humoral immune responses for N = 64 mice were assessed using binding antibody ELISA to the Spike protein \u003cstrong\u003e(B) \u003c/strong\u003eand SARS-CoV-2 live virus (WAS-Calu-3 Strain) neutralization assays \u003cstrong\u003e(C) \u003c/strong\u003eusing serum isolated from blood collected at experimental endpoint.\u003cstrong\u003e \u003c/strong\u003eThe SARS-CoV-2 neutralization assay included a virus only negative control, Rabbit Spike neutralizing antibody positive control, and a no virus control. \u003cstrong\u003e(D) \u003c/strong\u003eCellular immune response to the vaccine was assessed via Intracellular staining cytokine analysis for N = 24 mouse CD4+ and CD8+ T cells collected at Day 44 in response to pooled S peptides for IFNγ, TNFα, and IL-2. \u003cstrong\u003e(E) \u003c/strong\u003eIFNγ ELISPOT assay also measured the cellular immune response induced by the vaccine in response to stimulation from CMV peptide pool irrelevant control, S protein peptide pools, or Concanavalin A (ConA) a positive control. Survival data was graphed over the course of 10 days post challenge in % Body Weight Loss \u003cstrong\u003e(F) \u003c/strong\u003eand % Survival \u003cstrong\u003e(G) \u003c/strong\u003eafter viral challenge with 2.8 x 10\u003csup\u003e3\u003c/sup\u003e PFU dose of WAS-Calu-3 strain of the live SARS-CoV-2 virus. Data representative of two independent experiments. Values are presented as mean ± standard deviation. \u003cem\u003eP\u003c/em\u003e values were determined using the Mann-Whitney test. * 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** 0.01 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, *** 0.001 \u0026lt; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/1ea1138e4e15b9d759439166.jpg"},{"id":105223356,"identity":"98761c36-3c46-46d8-9d57-d4fd08a395a2","added_by":"auto","created_at":"2026-03-23 16:04:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2585070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/dead0390-e529-4467-8c88-207a3749da09.pdf"},{"id":96781300,"identity":"ab473a77-eb49-4cf3-a878-47593bbe7c24","added_by":"auto","created_at":"2025-11-26 04:26:17","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":326736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Antibody Binding to SARS-CoV-2 spike protein and RBD Mutational Variants from Vaccinated BALBc/J mice. (A) \u003c/strong\u003eSera collected two weeks following the final immunization were assayed by ELISA against recombinant SARS-CoV-2 Spike protein, and antibody binding was quantified as the area under the curve (AUC) across serial dilutions. Left: comparison of pMIP3α-SARS-CoV-2-ΔC at 100 μg versus 50 μg; middle: comparison of pSARS-CoV-2-ΔC at 100 μg versus 50 μg; right: comparison between 100 μg doses of pMIP3α-SARS-CoV-2-ΔC and pSARS-CoV-2-ΔC. Data are presented as mean ± standard deviation, with individual animals represented by separate data points. Statistical analyses were conducted using unpaired two-tailed t-tests, and p-values are reported accordingly. No significant differences in AUC values were observed across different dose levels or between the constructs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eInduced Humoral Immunity was measured using binding antibody ELISA to the Spike protein, RBD W; Wild-type (Wuhan strain), RBD-RSA; Beta variant (South Africa), RBD-Brazil; Gamma variant (Brazil), RBD-India B.1; Delta variant (India, B1.167) , RBD-India B.1; Delta variant (India, B1.167.2) , and RBD-UK; Alpha variant (UK). \u003cstrong\u003e(C) \u003c/strong\u003eSARS-COV-2 pseudo-virus neutralization assays (using serum isolated from blood collected at experimental endpoint)\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/655587857dcab5d2d6b648dd.jpg"},{"id":96916047,"identity":"5034bfe5-0820-4e1c-8756-955e191e7c4c","added_by":"auto","created_at":"2025-11-27 14:07:54","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":125404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Antibody Binding to SARS-CoV-2 RBD Mutational Variants from Vaccinated K18-hACE2 mice. (A) K18-hACE2 \u003c/strong\u003emice were intramuscularly immunized every two weeks with three 100 µg doses of either pSARS-CoV-2-ΔC or pMIP3α-SARS-CoV-2- ΔC DNA Vaccine constructs using the Pharmajet Tropis Needleless Injector. Serum isolated from blood collected at experimental endpoint was used to assess ELISA antibody binding to wild type SARS-CoV-2 Spike Protein,\u003c/p\u003e\n\u003cp\u003eRBD WT; Wild-type (Wuhan strain), RBD-RSA; Beta variant (South Africa), RBD-Brazil; Gamma variant (Brazil), RBD-India B.1; Delta variant (India, B1.167) , RBD-India B.1; Delta variant (India, B1.167.2) , and RBD-UK; Alpha variant (UK). \u003cstrong\u003e(B) \u003c/strong\u003eQuantitative analysis by area under the curve (AUC) confirmed antigen-specific binding in both vaccine groups. Data representative of two independent experiments. Values are presented as mean ± standard deviation. \u003cem\u003eP\u003c/em\u003e values were determined using the Mann-Whitney test.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/dc0867ac776a7174bd36dc7a.jpg"},{"id":96781287,"identity":"06db1a8a-26e4-4bc4-b694-825737881959","added_by":"auto","created_at":"2025-11-26 04:26:03","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":269504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Comparative evaluation of antibody binding activity in K18-hACE2 mice, expressed as area under the curve (AUC) from serum ELISA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum binding antibodies were quantified by ELISA using serial dilutions against recombinant SARS-CoV-2 Spike protein. Quantitative analysis by area under the curve (AUC) confirmed antigen-specific binding in three vaccine groups compared to negative control group MIP3a-dC; pMIP3α-SARS-CoV-2- ΔC, MIP3a-linker; pMIP3α-SARS-CoV-2-linker-ΔT, ΔC: pSARS-CoV-2-ΔC, and MIP3a-A20; pMIP3α-A20-scFv DNA Vaccines. \u003cem\u003eP\u003c/em\u003e values were determined using the Mann-Whitney test.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8158863/v1/b14df9c52e1d6da1f7809ed2.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"T-cell responses are insufficient to generate protective immunity to SARS-CoV-2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe development of safe and effective vaccines against SARS-CoV-2 has relied largely on immunogens capable of eliciting high titers of neutralizing antibodies, as exemplified by mRNA and adenoviral platforms. Neutralizing antibody levels strongly correlate with protection in both non-human primates and humans, serving as the principal mechanistic correlate of immunity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, accumulating evidence suggests that T-cell responses also play an important role, particularly in limiting disease severity and sustaining long-term protection as antibody titers wane\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The extent to which T-cell immunity can compensate for deficient neutralizing antibody responses remains a central unresolved question. This issue has particular clinical relevance for patients with B-cell malignancies or those receiving B-cell\u0026ndash;depleting therapies, in whom antibody formation after SARS-CoV-2 vaccination is frequently impaired while T-cell responses are often preserved. Understanding the balance between these immune mechanisms is therefore critical for guiding vaccine design and optimizing protective strategies in immunocompromised populations.\u003c/p\u003e\u003cp\u003eDNA vaccines provide a flexible platform for rapid antigen engineering and have shown the capacity to induce both humoral and cellular responses in preclinical and clinical settings\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the immunogenicity of DNA vaccines is often lower than that of mRNA vaccines, and strategies to improve antigen delivery and immune activation are needed. One such approach involves chemokine fusion, which targets antigens to antigen-presenting cells (APCs), thereby enhancing priming of adaptive immunity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Our group has previously demonstrated that chemokine\u0026ndash;antigen fusion DNA vaccines can elicit potent immune responses against infectious pathogens and tumors in preclinical models \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and clinical safety has been established in a first-in-human trial of a lymphoma DNA vaccine \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consistent with prior work in infectious disease and cancer models, MIP3α fusion DNA vaccination drives a Th1-skewed immune profile, with dominant IFNγ and TNFα secretion and minimal induction of Th2-associated cytokines\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Therefore, we selected MIP3α as a driver for immature dendritic cells to enhance cellular immunity. We hypothesized that MIP3α fusion to the Spike glycoprotein enhances APC targeting and T-cell priming, supporting cellular immunity as the primary protective mechanism against SARS-CoV-2. Here, we applied this APC-targeted DNA vaccine strategy to SARS-CoV-2. We engineered multiple DNA vaccine constructs encoding the spike glycoprotein (including variants with C-terminal truncation (ΔC), protease cleavage site deletion and linker insertion (Linker-ΔT), with and without fusion to the chemokine MIP3α. By evaluating humoral and cellular immunity in BALB/c and K18-hACE2 mouse models, followed by viral challenge in the latter, we sought to dissect the relative contributions of binding antibodies, neutralizing antibodies, and T cells in vaccine-mediated protection against SARS-CoV-2.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eVaccine constructs\u003c/h2\u003e\u003cp\u003eDNA vaccine plasmids were generated using a preclinical-grade pVax-based backbone. The SARS-CoV-2 Spike (isolate Wuhan-Hu-1; GenBank accession MN908947.3) sequence was codon-optimized for mammalian expression. Three Spike variants were engineered: (i) ΔC, with a 19\u0026ndash;amino acid cytoplasmic tail deletion; (ii) Linker-ΔT, with deletion of the S1/S2 and S2\u0026prime; protease cleavage sites and insertion of a (G4S)₂ linker with truncation of the transmembrane and cytoplasmic domains. For APC targeting, murine MIP3α was fused to the N-terminus of each construct via a flexible linker. An HA-tag was included at the C-terminus for detection. An irrelevant control construct encoding MIP3α fused to A20-scFv was also generated. Plasmids were purified under endotoxin-free conditions (Qiagen EndoFree kit) and validated by sequencing and restriction digest.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals and immunization\u003c/h3\u003e\n\u003cp\u003e The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute at City of Hope (COH) approved protocol 20032, which is assigned to this study. All study procedures were conducted in strict compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Mice were maintained on a 12-hour light/12-hour dark cycle at 22\u0026ndash;24\u0026deg;C and 30\u0026ndash;70% humidity, with ad libitum access to food and water. Six-week-old BALB/c (BALB/cJ, 000651) or B6.Cg-Tg (K18-ACE2)2Prlmn/J (K18-hACE2, 034860) transgenic mice were obtained from The Jackson Laboratory. Vaccinations were administered via intramuscular injection into the quadriceps using the Tropis injector (PharmaJet), delivering 50 \u0026micro;g or 100 \u0026micro;g of DNA in 50 \u0026micro;L of sterile PBS per dose. A three-dose schedule was implemented on days 0, 14, and 28. Blood samples for humoral immune analysis were collected prior to each immunization, and the mice were euthanized for blood and spleen collection two weeks following the final vaccination. Splenocytes for cellular immune analysis were collected two weeks after booster immunization and isolated using standard procedures following humane euthanasia of the animals.\u003c/p\u003e\n\u003ch3\u003eSerological assays\u003c/h3\u003e\n\u003cp\u003eSera were collected retro-orbitally prior to each immunization and two weeks post-final immunization. Antibody binding to recombinant SARS-CoV-2 Spike and variant RBD proteins (WT, Alpha, Beta, Gamma, Delta; BEI Resources) was measured by ELISA. Plates were coated with antigen (2 \u0026micro;g/mL), blocked with BSA, and incubated with serially diluted sera. Bound IgG was detected with HRP-conjugated anti-mouse IgG and developed with TMB substrate. Endpoint titers and area under the curve (AUC) were calculated.\u003c/p\u003e\n\u003ch3\u003eSARS-CoV-2 pseudovirus production and neutralization assay\u003c/h3\u003e\n\u003cp\u003eCOVID 19 Spike Coronavirus Pseudovirus (MyBioSource, Cat:MBS434275) input was standardized by 100X signal above cell-only background. Heat-inactivated mouse sera were pooled, serially diluted (1:20\u0026ndash;1:640), and incubated 1 hour at 37\u0026deg;C with SARS-CoV-2 spike pseudoviruses. HEK293T-ACE2 cells (2 \u0026times; 10^4/well) were infected in the presence of 5 \u0026micro;g/mL polybrene, and luciferase activity was quantified 48 h later using Promega reagents. We plotted the data using luminescence vs. virus dilution. Neutralization was calculated as NT = [1 \u0026minus; (RLU immune sera / RLU control)] \u0026times; 100, with NT90 values interpolated from dilution curves. The method was adapted from Crawford, H.D. et al\u003csup\u003e13\u003c/sup\u003e and Chiuppesi F et al \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eT-cell analysis\u003c/h3\u003e\n\u003cp\u003eSplenocytes were collected two weeks after the final immunization and prepared by mechanical dissociation followed by red blood cell lysis. Cells were rested overnight in complete RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin\u0026ndash;streptomycin, 2 mM L-glutamine, and 50 \u0026micro;M 2-mercaptoethanol. For functional assays, 1\u0026ndash;2 \u0026times; 10⁶ splenocytes per well were stimulated with overlapping peptide pools covering the SARS-CoV-2 Spike protein (Miltenyi Biotec; S or S1 peptide pools; 1\u0026ndash;2 \u0026micro;g/mL per peptide) or with an irrelevant CMV peptide pool as a negative control. Stimulations were performed in the presence of brefeldin A and monensin for 5\u0026ndash;6 h at 37\u0026deg;C with 5% CO₂. Following stimulation, cells were stained with fixable viability dye and antibodies against CD3, CD4, and CD8. After fixation and permeabilization (BD Cytofix/Cytoperm), intracellular cytokine staining was performed with antibodies specific for IFN-γ, TNF-α, IL-2, IL-4, and IL-5. Data were acquired on a BD LSRFortessa flow cytometer, and \u0026ge;\u0026thinsp;100,000 lymphocyte events were collected per sample. FlowJo (Tree Star) was used for analysis. Gating was applied sequentially on singlets, live CD3⁺ T cells, and CD4⁺ or CD8⁺ subsets, with cytokine-positive populations quantified. Boolean gating was used to define polyfunctional subsets expressing one or more cytokines simultaneously.\u003c/p\u003e\u003cp\u003eConcurrently, T cell responses were evaluated using cytokine ELISA, with splenocytes from immunized mice plated at a density of 5 x 10\u003csup\u003e6\u003c/sup\u003e cells per well in 24-well plates. The cells were stimulated with overlapping peptide pools spanning the SARS-CoV-2 Spike protein (Miltenyi Biotec; S or S1 peptide pools; 1\u0026ndash;2 \u0026micro;g/mL per peptide) or with an irrelevant CMV peptide pool as a negative control, and incubated at 37\u0026deg;C for 3 days. The supernatant was collected and analyzed to assess cytokine production. Mouse IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5 were quantified by ELISA using the corresponding antibody sets (Invitrogen) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSARS-CoV-2 wildtype virus neutralization assay and challenge in K18-hACE2 mice\u003c/h2\u003e\u003cp\u003e All procedures were carried out in strict compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at Bioqual, Inc. Bioqual acquired 64 K18-hACE2 mice (32 males and 32 females) from Jackson Laboratory, which were subsequently randomly assigned to four experimental groups (n\u0026thinsp;=\u0026thinsp;16 per group). The animals were immunized on Study Days (SD) 0, 14, and 28, in accordance with the prescribed vaccination schedule. On SD41, six mice per group (three males and three females) were humanely euthanized for terminal blood collection and splenectomy, while the remaining animals underwent interim blood sampling. Serum samples were subsequently analyzed by Bioqual through a plaque reduction neutralization test (PRNT). A subset of these samples was forwarded to City of Hope for confirmatory testing of Spike protein recognition. Surviving mice in SD48 were intranasally inoculated with 2.8 \u0026times; 10\u0026sup3; PFU of live SARS-CoV-2 WAS-Calu-3 (lot #12152020-1235). During the study period, mice were monitored for weight loss and clinical manifestations of disease. On SD64, all remaining animals were humanely euthanized, and their spleens were collected for subsequent flow cytometry and ELISpot analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed with GraphPad Prism v9. ELISA titers, cytokine levels, and flow cytometry data were compared by two-tailed unpaired Student\u0026rsquo;s t-test or one-way ANOVA with Tukey\u0026rsquo;s post hoc correction, as appropriate. Survival curves were compared using the log-rank (Mantel\u0026ndash;Cox) test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSARS-CoV-2 DNA Vaccine design\u003c/h2\u003e\u003cp\u003eThe Spike glycoprotein (S-protein) of the SARS-CoV-2 (isolate Wuhan-Hu-1; MN_908, 947.3; \u003csup\u003e15\u003c/sup\u003e) virion was codon optimized and inserted into a pVax-based DNA vaccine vector (Fig.\u0026nbsp;1A). A leader sequence derived from the interferon-induced protein 10 (IP-10) was engineered and inserted at the N-terminus of the spike glycoprotein to facilitate its secretion through the rough endoplasmic reticulum membrane during protein synthesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Additionally, a hemagglutinin tag (HA-tag) was incorporated into each design to serve as a universal epitope tag.\u003c/p\u003e\u003cp\u003eWe designed DNA vaccines to encode four codon optimized SARS-CoV-2 DNA spike proteins, with and without murine MIP3α fusion. First, we constructed a full-length spike protein construct with 19 amino acids (KFDEDDSEPVLKGVKLHYT) removed from the CT (pSARS-CoV-2-ΔC) and a fusion of (pMIP3α-SARS-CoV-2-ΔC). We also deleted the TM and CT, further hypothesized that deleting the protease sites and inserting (G\u003csub\u003e4\u003c/sub\u003eS)₂ linkers would disrupt epitope folding and thereby abrogate antibody induction, with MIP3α (\u003cem\u003epMIP3α-SARS-CoV-2-Linker-ΔT\u003c/em\u003e) or without MIP3α (\u003cem\u003epSARS-CoV-2-Linker-ΔT\u003c/em\u003e). A DNA vaccine encoding A20 lymphoma scFv with irrelevant specificity (pMIP3α-A20-scFv) was used as a negative control\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003epSARS-CoV-2-ΔC DNA vaccine immunogenicity in BALB/c mice\u003c/h2\u003e\u003cp\u003eWe first characterized immunogenicity of the various DNA vaccines in BALB/c mice. Doses of either 50 \u0026micro;g or 100 \u0026micro;g of pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC vaccines were administered by Tropis injector to deliver uniform vaccine administration. Mice were serially immunized three times, with a 2-week interval between each vaccination. Blood samples were collected before each immunization, and the mice were euthanized for blood and spleen collection 2 weeks after the final vaccination (Fig.\u0026nbsp;2A).\u003c/p\u003e\u003cp\u003eEndpoint humoral responses were measured by ELISA and demonstrated antibody binding to the SARS-CoV-2 Spike protein by all mice in both groups (Fig.\u0026nbsp;2B). Area under the curve calculations revealed no significant differences between pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC vaccines at either dose level (Supplementary Fig.\u0026nbsp;1A). Because SARS-CoV-2 recognizes host cell receptors through its receptor-binding domain (RBD), immune sera were also analyzed for their ability to recognize various RBD variants (Supplementary Fig.\u0026nbsp;1B). The results demonstrated that serum antibodies derived from pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC effectively distinguish between variant RBDs without any substantial differences. While antibodies generated by both vaccines exhibited reactivity against Spike proteins from multiple variants (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), they failed to induce neutralizing antibody responses against SARS-CoV-2 pseudovirus (Supplementary Fig.\u0026nbsp;1C).\u003c/p\u003e\u003cp\u003eT cell responses to the SARS-CoV-2 were analyzed using splenocytes collected from vaccinated mice stimulated with either the Miltenyi Biotec S peptide pool, S1 peptide pool, or a CMV peptide pool as a negative control. Splenic T cells demonstrated a robust specific cytokine responses, including production of IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5 in culture supernatants by ELISA (Fig.\u0026nbsp;2C). Furthermore, analysis by intracellular staining revealed statistically significant expression of IFNγ and TNFα, but not IL-2, by both CD4 and CD8 T cells stimulated with the SARS-CoV-2 spike peptide pool across both constructs and doses without any obvious differences, compared with control stimulation (CD4 T cells: Fig.\u0026nbsp;2D; CD8 T cells: Fig.\u0026nbsp;2E).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003epSARS-CoV-2-Linker-ΔT DNA vaccine immunogenicity in BALB/c mice\u003c/h2\u003e\u003cp\u003eOur central hypothesis was that cellular immunity, rather than humoral immunity, constitutes the dominant protective mechanism against SARS-CoV-2. Linker-ΔT constructs were designed to disrupt optimal prefusion spike conformation, thereby potentially diminishing antibody titers while preserving, or even enhancing, T cell\u0026ndash;mediated immunity. Humoral and cellular immunity of pSARS-CoV-2-Linker-ΔT vaccines, with and without MIP3 fusion, were also evaluated at 50 \u0026micro;g and 100 \u0026micro;g doses. As expected, antibody binding to the SARS-CoV-2 spike protein was not detected in the serum samples collected from mice vaccinated with either construct or at either dose (Fig.\u0026nbsp;3B). In contrast, Linker-ΔT vaccines induced specific T-cell responses. Splenic T cells from mice vaccinated with either construct produced significant levels of IFNγ, IL-6, IL-2, and TNFα in culture supernatants by ELISA, with no substantial differences between the two constructs of doses (Fig.\u0026nbsp;3C). Intracellular cytokine analysis of peptide-restimulated splenocytes revealed a predominantly Th1-skewed response, especially in mice vaccinated with the MIP3 fusion (Fig.\u0026nbsp;3DE). For example, pMIP3α-SARS-CoV-2-Linker-ΔT vaccine induced significant TNFα and IFNγ production in CD4⁺ and CD8⁺ T cells at either dose, while less IL-2 production was observed. Moreover, in CD4⁺ T cells, significant IFNγ production was observed exclusively in the pMIP3α groups at both doses (Fig.\u0026nbsp;3D).\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eVaccine immunogenicity in K18-hACE2 mice\u003c/h2\u003e\u003cp\u003eWith the eventual goal of testing for virus protection, we extended immunogenicity experiments to K18-hACE2 transgenic mice that express human angiotensin-converting enzyme 2 (ACE2), the receptor exploited by SARS-CoV-2 for cellular entry. Female k18-hACE2 mice (N\u0026thinsp;=\u0026thinsp;5) were first immunized with 100 \u0026micro;g of the pMIP3α-SARS-CoV-2-ΔC or pSARS-CoV-2-ΔC DNA vaccines diluted in 50\u0026micro;L of sterile phosphate buffered saline (PBS) on days 0, 14, and 28, as before (Fig.\u0026nbsp;4A). Compared with pSARS-CoV-2-ΔC, mice vaccinated with pMIP3α-SARS-CoV-2-ΔC exhibited a trend towards higher serum antibody responses against the S protein (Fig.\u0026nbsp;4B). Moreover, serum antibody responses against wild-type RBD, Alpha(UK), Beta(SRA), Delta(Indian), and Gamma(Brazil) RBD variants were more robust in the pMIP3α-SARS-CoV-2-ΔC group (Supplementary Fig.\u0026nbsp;2A). However, neutralizing antibodies were not consistently detected in mice vaccinated with either pMIP3α-SARS-CoV-2-ΔC or pSARS-CoV-2-ΔC (Fig.\u0026nbsp;4C).\u003c/p\u003e\u003cp\u003eRobust specific splenic T cell responses were detected in both groups, as analyzed by cytokine production by ELISA. Specifically, splenocytes from mice vaccinated with either pSARS-CoV-2-ΔC or pMIP3α-SARS-CoV-2-ΔC and stimulated with the SARS-CoV-2 Spike peptide pool produced statistically significant levels of IFNγ, IL-6, IL-2, TNFα, IL-4, and IL-5, compared with those stimulated with negative controls (CMV peptide pools; Fig.\u0026nbsp;4D). Specific IFNγ production by stimulated splenocytes from both pSARS-CoV-2-ΔC and pMIP3α-SARS-CoV-2-ΔC groups was also detected by intracellular staining, particularly from CD8\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;4E).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eT-cell immunity without neutralizing antibodies was insufficient for protection against SARS-CoV-2\u003c/h2\u003e\u003cp\u003eWe designed a second experiment to investigate both immunogenicity and virus protection in K18-hACE2 mice, including both vaccines above and adding the pMIP3α-SARS-CoV-2-Linker-ΔT vaccine (Fig.\u0026nbsp;5A). Sixteen mice per group were vaccinated with 100 micrograms pSARS-CoV-2-ΔC, pMIP3α-SARS-CoV-2-ΔC, pMIP3α-SARS-CoV-2-Linker-ΔT, or pMIP3α-A20-scFv (negative control) in three doses administered as above. Two weeks after the final vaccination, six mice from each experimental group were euthanized for terminal bleeding and splenocyte isolation. The remaining ten mice were retro-orbitally bled, subsequently challenged with 2.8 \u0026times; 10\u0026sup3; PFU of WAS-Calu-3 (LOT #12152020-1235) ten days later, and then monitored for variations in body weight and survival. As anticipated, mice vaccinated with both SARS-CoV-2-ΔC vaccines, but especially those vaccinated with the MIP3 fusion, demonstrated significant increases in serum antibody titers against the S protein (Fig.\u0026nbsp;5B, S3 Fig). However, corresponding neutralizing antibody activity against live SARS-CoV-2 virus was not observed in any of the groups (Fig.\u0026nbsp;5C). T-cell responses analyzed by intracellular cytokine staining for TNFα, IL-2, and IFNγ showed significant responses after stimulation with SARS-CoV-2 Spike protein peptide pools (S), compared with negative control stimulation (CMV) by both CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;5D). Specific IFNγ production was also detected by all three vaccines by ELISPOT (Fig.\u0026nbsp;5E).\u003c/p\u003e\u003cp\u003eCompared with negative controls, none of the experimental vaccines protected against weight loss or death (Fig.\u0026nbsp;5F and G). Our results indicate that T cell immunity was not sufficient to provide SARS-CoV-2 viral protection in K18-hACE2 mice.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBuilding upon our previous studies demonstrating that the genetic fusion of chemokines to tumor antigens enhances antigen cross-presentation and induces robust CD8⁺\u0026nbsp;and Th1-type T-cell immunity in cancer models, we have applied this principle to the design of a vaccine for infectious diseases by leveraging MIP3α-mediated targeting of immature dendritic cells to augment cellular immunity against SARS-CoV-2.\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that T-cell responses against SARS-CoV-2 are necessary and sufficient for protection against this virus, we investigated the immunogenicity of several different SARS-CoV-2 DNA vaccine prototypes, designed to primarily induce T-cell immunity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll vaccine candidates induced robust CD4+ and CD8+ T cell responses characterized by specific cytokine production. Moreover, Spike- and RBD-binding antibodies were consistently detected across constructs and recognized multiple viral variants; however, none of the vaccines elicited neutralizing antibodies or conferred protection against lethal viral challenge. These findings suggest that neutralizing antibodies may be required for virus protection .\u003c/p\u003e\n\u003cp\u003eOur findings align with large-scale correlates of protection analyses, which demonstrate that neutralizing antibody titers serve as the most reliable quantitative predictors of vaccine efficacy against SARS-CoV-2 infection. For example, Khoury and colleagues demonstrated that neutralizing antibody titers exhibited a strong correlation with protection from symptomatic infection\u003csup\u003e16\u003c/sup\u003e. This finding suggests that T-cell immunity alone is insufficient to prevent viral entry. Our study's findings corroborate this conclusion, as robust T-cell cytokine responses were not protective in the absence of neutralizing antibodies. \u0026nbsp;Our observations also corroborate findings in rhesus macaques, where passive transfer of IgG alone was sufficient to confer protection against challenge, while CD8+ T cells contributed only when neutralizing antibody titers were subprotective\u003csup\u003e17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNonetheless, the breadth and functionality of T-cell responses are likely to play crucial roles in mitigating disease severity following infection. Vaccine-induced T cells have been shown to maintain 70–80% of their epitope recognition across the Omicron variant and other variants, despite substantial reductions in neutralization capacity \u003csup\u003e18\u003c/sup\u003e thereby providing a critical immunological “backstop” against viral evolution. Clinical data further reinforce this paradigm: early and polyfunctional T-cell responses are correlated with milder clinical outcomes, and in B-cell–depleted patients, preserved T-cell responses following vaccination are associated with a reduced risk of severe disease \u003csup\u003e19\u003c/sup\u003e.\u0026nbsp;The absence of neutralizing antibodies remains a critical vulnerability, addressed clinically through timed revaccination, monoclonal antibody prophylaxis, early antivirals, and occasionally convalescent plasma\u003csup\u003e20,21\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, other DNA vaccine platforms have achieved protective efficacy in preclinical models when neutralizing antibodies were elicited. For example, Yu et al. showed that DNA vaccines encoding full-length spike induced both neutralizing antibodies and protection against SARS-CoV-2 in rhesus macaques\u003csup\u003e22\u003c/sup\u003e. Similarly, DNA vaccine delivered with electroporation, elicited both T-cell and neutralizing antibody responses in humans\u003csup\u003e23,24\u003c/sup\u003e. Interestingly, a recent study using an RBD–MIP3α construct demonstrated durable humoral responses lasting at least 12 months in mice, contrasting with the absent neutralization in our ΔC- and Linker-based constructs\u003csup\u003e25\u003c/sup\u003e\u0026nbsp; These discrepancies likely arise from fundamental differences in antigen design, epitope presentation, and stabilizing mutations. Structure-guided stabilization of the full-length Spike ectodomain—exemplified by the S-2P and HexaPro variants—has been critical in locking the protein in its prefusion conformation, thereby exposing neutralization-sensitive epitopes and enhancing immunogenicity; these advancements have been fundamental to the success of mRNA-1273 and other commercial platforms, consistently eliciting high-titer neutralizing antibodies in both preclinical and clinical studies\u003csup\u003e26,27\u003c/sup\u003e. In contrast, our ΔC and Linker-ΔT constructs incorporated deletions and cleavage-site modifications without stabilizing mutations, alterations that may have compromised the conformational integrity of the RBD and, as a result, limited B cell recognition of neutralizing epitopes. Beyond stabilization, the multivalent display of antigens has emerged as a critical factor influencing vaccine efficacy. Self-assembling nanoparticles displaying multiple copies of the receptor-binding domain (RBD), such as the I53-50 two-component scaffolds, have been demonstrated to induce significantly higher neutralizing titers and broader recognition of variants compared to monomeric RBD or unstabilized Spike constructs\u003csup\u003e28,29\u003c/sup\u003e. Similarly, recombinant prefusion-stabilized Spike proteins, formulated with potent adjuvants such as the saponin-based Matrix-M employed in NVX-CoV2373, have exhibited robust neutralizing activity and demonstrated clinical efficacy\u003csup\u003e30\u003c/sup\u003e. Our DNA vaccine constructs lacked these design features—multimerization, prefusion stabilization, and optimized adjuvantation—and instead depended exclusively on plasmid-mediated expression of modified Spike antigens delivered via intramuscular injection.\u003c/p\u003e\n\u003cp\u003eThese discrepancies likely reflect differences in antigen design, epitope exposure, and stabilizing mutations. The protective efficacy of T cells in the absence of neutralization has been reported in other settings. For example, epitope-focused or ubiquitin-targeted DNA vaccines reduced morbidity and mortality in K18-hACE2 mice without detectable neutralizing antibodies\u003csup\u003e31\u003c/sup\u003e. Recent human studies have demonstrated that mRNA vaccination against SARS-CoV-2 induces a polyclonal, high-affinity T-cell response, a feature associated with long-lasting immunity.\u003csup\u003e32\u003c/sup\u003e In contrast, our MIP3α-targeted DNA vaccines elicited strong cytokine production but failed to protect, suggesting that the induced T cells lacked sufficient clonal diversity and receptor avidity. Future vaccine iterations should therefore aim to enhance T-cell quality through optimized antigen processing and presentation to achieve protective efficacy even in the absence of neutralizing antibodies.\u003c/p\u003e\n\u003cp\u003eIn addition to MIP3α, other chemokines have been investigated as antigen-delivery vehicles to APCs, with distinct immunological consequences. MCP-1 has been shown to stimulate IL-4 production, thereby driving Th2 polarization in a regulatory manner\u003csup\u003e33,34\u003c/sup\u003e. Similarly, the macrophage-derived chemokine CCL22 (MDC) selectively recruits Th2 cells to APCs \u003csup\u003e34\u003c/sup\u003e.\u0026nbsp; MIP3α fusion preferentially induces Th1-driven cellular immunity, optimizing CD8⁺\u0026nbsp;effector T-cell responses and IFNγ output, whereas MCP-1 or MDC incorporation tends to bias immunity toward a Th2 phenotype with superior antibody production. Thus, rational chemokine selection provides a potential strategy to tailor immune polarization: Th1-inducing chemokines such as MIP3α may enhance antiviral T-cell responses, whereas Th2-inducing chemokines like MCP-1 or MDC may complement this by boosting antibody responses. In the context of SARS-CoV-2 vaccines, combining or sequentially deploying these chemokine-fusion strategies could theoretically broaden protective immunity by balancing cellular and humoral arms of the adaptive response.\u003c/p\u003e\n\u003cp\u003eOur study demonstrates that while chemokine-targeted DNA vaccines elicit robust Th1-biased T-cell responses and broaden Spike-binding antibodies, neutralizing antibody induction remains indispensable for protection against SARS-CoV-2. These findings have direct implications for immunocompromised patients, such as those with B-cell depletion, in whom antibody formation is impaired but T-cell responses persist. Future vaccine strategies should combine Th1- and Th2-inducing chemokine fusions to optimize both neutralizing antibody quality and durable cellular immunity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.C. and L.W.K. designed the project, oversaw the experiments, and wrote the manuscript. S.C., S.J.S., and L.W.K. contributed to the design of the vaccine construct. S.J.S. and A.A. cloned the vaccine and conducted an analysis of the sequencing data. A.A., E.O., and S.C. optimized the mouse injection protocol and carried out the immunization of the mice. Z.D. developed and optimized assays for the production of SARS-CoV-2 pseudovirus and its neutralization. M.P. and K.A. prepared and organized all mouse samples and conducted the analysis of the antibody response to the S protein. S.S., Z.D., and A.A. performed FACS and subsequently analyzed the data. Z.D., J.B., S.J.S., and A.A. conducted the data analysis and contributed to the manuscript revision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKhoury, D. 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To examine whether cellular immunity alone can confer protection, we engineered DNA vaccines encoding modified Spike proteins, including C-terminal truncation (SARS-CoV-2-ΔC, ΔC) and cleavage-site\u0026ndash;deleted, linker-inserted (SARS-CoV-2-Linker-ΔT, Linker-ΔT) variants, with or without genetic fusion to MIP3α, which has been shown to enhance targeting of antigen-presenting cells (APC) and preferentially induce T-cell responses. In BALB/c mice, ΔC constructs induced non-neutralizing Spike- and RBD-binding antibodies across variants, as well as robust CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell responses, whereas Linker-ΔT elicited strong Th1-skewed cellular immunity in the absence of humoral responses. In K18-hACE2 mice antibody neutralizing activity was not detected by any of the vaccines, and none conferred protection following lethal virus challenge, despite robust specific T-cell cytokine responses. These results support vaccine designs incorporating chemokine fusion to enhance T-cell priming, but cellular responses alone are insufficient for SARS-CoV-2 protection. Integrating such APC-targeting strategies with structural modifications that preserve pre-fusion neutralizing epitopes may be worthwhile.\u003c/p\u003e","manuscriptTitle":"T-cell responses are insufficient to generate protective immunity to SARS-CoV-2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 04:25:58","doi":"10.21203/rs.3.rs-8158863/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-26T07:19:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-20T14:17:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T14:14:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-19T22:21:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8adf818b-d13d-4d6e-85bc-771d47b53b98","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58330731,"name":"Biological sciences/Immunology"},{"id":58330732,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-23T16:01:54+00:00","versionOfRecord":{"articleIdentity":"rs-8158863","link":"https://doi.org/10.1038/s41598-026-44391-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-20 15:58:29","publishedOnDateReadable":"March 20th, 2026"},"versionCreatedAt":"2025-11-26 04:25:58","video":"","vorDoi":"10.1038/s41598-026-44391-x","vorDoiUrl":"https://doi.org/10.1038/s41598-026-44391-x","workflowStages":[]},"version":"v1","identity":"rs-8158863","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8158863","identity":"rs-8158863","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}