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GM SF Zip 001
To protect against SARS-CoV-2 infection, it is important to elicit neutralizing antibodies targeting the S1 RBD, S1 N-terminal domain, or the S2 region. These antibodies must block the binding of the RBD to the ACE2 receptor and prevent S2-mediated membrane fusion or entry into the host cell, thereby inhibiting viral infection [4,5]. Previous studies of vaccines for MERS-CoV (Middle East Respiratory Syndrome Coronavirus) [6] and SARS-CoV [7] showed that both humoral and cellular (cytotoxic) immune responses are important to inducing a protective immune response. To achieve these outcomes, one possible option is deoxyribonucleic acid (DNA) vaccination.
Among the various vaccination approaches against infectious diseases, such as human immunodeficiency virus (HIV), DNA vaccines have several advantages: they are easily produced, they provide opportunities for molecular engineering, they lack anti-vector immunity, and they have the potential to promote both cellular and humoral immune responses [8,9,10]. However, despite their high immunogenicity in murine models, DNA vaccines have shown poor efficiency in large animal models and in humans [11]. Interestingly, a study showed that a DNA vaccine encoding the S protein of SARS-CoV induced T cell and neutralizing antibody responses, which protected mice against an intranasal SARS-CoV challenge [12]. New strategies for the improvement of DNA vaccines include: optimization of transcriptional control elements; use of adjuvants such as toll-like receptor (TLR) ligands [13,14], cytokine-expressing plasmids [15,16,17], or apoptosis-based adjuvants [18]; and appropriate delivery systems, such as local electroporation (EP) [19,20,21].
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated significant adjuvant effect when included in DNA vaccines for many infectious diseases [22,23,24,25,26,27,28,29]. For example, the co-administration of plasmid-encoded GM-CSF with an HIV-1 DNA vaccine improved both the magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T cells which simultaneously produce interferon-γ, tumor necrosis factor-α, and interleukin-2 [30]. In a murine model of the Ebola virus, the inclusion of interleukin-12 or GM-CSF improved cell-mediated immunity. However, the GM-CSF adjuvant plasmid did not improve neutralizing antibody titers in this model [26]. The combination of GM-CSF with self-amplifying mRNA constructs encoding the influenza virus nucleoprotein significantly improved the magnitude of antigen-specific CD8+ T cell responses and increased recruitment of antigen-presenting cells at the vaccination site [27]. These results imply a local release of GM-CSF. Indeed, the delivery system of GM-CSF seems to have an impact on the initiation of immune response. Reali et al. [31] observed an increase in the immune response in mice following the local administration of GM-CSF. In contrast, systemic administration of GM-CSF results in immune suppression through the production of myeloid-derived suppressor cells (MDSCs) [32].
Vaccination Strategy. (A). Vaccination scheme. Three groups of 12 mice were immunized at Day 0 by an intradermal injection of DNA plasmid coding for SARS-CoV-2 spike protein. For two groups, the immunization was adjuvanted either with recombinant muGM-CSF or with muGM-CSF secreted by encapsulated cells. On the day of immunization, a submandibular blood puncture was performed for baseline serum isolation. On Days 10 and 28, 5 mice per group were sacrificed to assess T Cell and B Cell responses. At Day 28, the 2 remaining mice per group received a boost DNA spike plasmid injection and were kept until Day 56 for B Cell responses assessment. (B). Dermal SARS-CoV-2 Spike protein expression by western blot analysis. Skin punch biopsies were taken at the site of intradermal injection from all animals, digested, and analyzed by western blot for the presence of the SARS-CoV-2 spike protein. Three randomly selected mice per time points are represented. (C). muGM-CSF Adjuvant quantification by ELISA. The muGM-CSF secreted by encapsulated cells was quantified by ELISA before and after implantation in mice. **** p
To assess DNA electroporation efficiency, western blot analysis was performed on skin punch lysate for each animal included in the study. This analysis revealed the presence of SARS-CoV-2 spike protein in the skin of all animals at Days 10 and 56, and almost all of the animals at Day 28 (Figure 1B and Supplementary Figure S1 shows the uncropped western blot). Of note, we observed variations between samples due to the reliability of both localizing the injection site after several days and homogenizing the samples. Therefore, levels of SARS-CoV-2 spike protein could not be evaluated quantitatively.
We observed a significant increase in the percentage of TNF-α- and IFN-γ-secreting CD8+ T cells after spike peptide Pool 1 stimulation for the three vaccination strategies (DMSO without adjuvant: 0.018 0.007; Pool 1 without adjuvant: 0.078 0.014; DMSO with recombinant muGM-CSF: 0.037 0.010; Pool 1 with recombinant muGM-CSF: 0.115 0.027; DMSO with capsule muGM-CSF: 0.054 0.029; and Pool 1 with capsule muGM-CSF: 0.113 0.023). Since the background (DMSO condition) was slightly increased when muGM-CSF was added to the vaccine (recombinant muGM-CSF: 0.037 0.010 and capsule muGM-CSF: 0.054 0.029) compared with the non-adjuvanted group (without adjuvant: 0.018 0.007), no significant difference was observed between the three groups in response to spike peptide Pool 1 (Figure 2).
TNF-α and IFN-γ production by CD8+ T cells 10 days after vaccination. Mice were immunized with a single dose of spike DNA plasmid with or without GM-CSF (recombinant or secreted by encapsulated cells) as an adjuvant. T cells response was measured at Day 10 by intracellular cytokine staining (ICS) after stimulation of splenocytes with spike protein peptide pools or DMSO as a negative control. Data show the percentage of TNF-α or IFN-γ-secreting CD8+ T cells after stimulation. Data are represented as min-to-max boxes with individual values. ** p
Spike peptide Pool 2 induced significant cytokine secretion only when the DNA vaccine was adjuvanted with muGM-CSF. Indeed, in the group implanted with capsules, we observed a slight increase in the percentage of TNF-α-secreting CD8+ T cells after spike peptide Pool 2 stimulation (DMSO condition: 0.047 0.024 and Pool 2: 0.153 0.062). Moreover, adjuvanted immunization with recombinant muGM-CSF did not quantitatively amplify the cellular response compared with the non-adjuvanted group (Figure 2).
To further investigate whether the addition of muGM-CSF can modify the secretion profile of CD8+ T cells, the polyfunctionality of CD8+ T cells was analyzed. The results showed proportion of functional CD8+ T cells after stimulation (Figure 3). Moreover, we observed an increase in the polyfunctionality of the CD8+ T cells when they were isolated from animals immunized with an adjuvanted vaccine (Figure 3 and Table S2).
Polyfunctionality of Spike-specific CD8+ T cells at day 10 post-vaccination. (A). Histogram representing the percentage of functional CD8+ T cells with 1, 2, 3, 4 or 5 functions after pool 1 and pool 2 stimulation. (B). Pie charts representing the proportion of responding CD8+ T cells expressing different combinations of cytokines and degranulation markers after stimulation with spike peptide pool 1 or pool 2, PMA/ionomycin (positive control) or DMSO (negative control) stimulation. Histograms and Pie charts represents the mean of 5 animals per group.
Cytokines profile of spike-specific CD4+ T cells 10 and 28 days after vaccination. Mice were immunized with a single dose of spike DNA plasmid with or without GM-CSF (recombinant or secreted by encapsulated cells) as an adjuvant. T cells responses were measured at day 10 and 28 by intracellular cytokines staining (ICS) after stimulation of splenocyte with spike protein peptide pools or DMSO as a negative control. Data show the percentage of cytokines secreting CD4+ T cells after stimulation. Data show the percentage of responding CD4+ T cells after stimulation. Data are represented as min-to-max boxes with individual values. * p
Another interesting observation came from these data when comparing the two adjuvant methods. As previously described in the literature, we observed a deleterious effect of muGM-CSF delivered as recombinant protein at a high dose [32], in contrast with the immunostimulatory effect of the low, sustained, and local delivery by encapsulated cells [33,34]. Indeed, the fold-change of IgG, IgM, and IgA in the DNA spike + recombinant muGM-CSF group was lower than the group without adjuvant. The same pattern was observed for IgG alone.
Before addressing the effect of the adjuvant, we have to comment on the limited immunization capacity of the vaccine model. Indeed, i.d. immunization with electroporation of spike DNA plasmid in this murine model is suboptimal. Several aspects can explain the poor efficacy of this model. With very limited data available in the literature, the mouse is certainly not the most suitable animal model for SARS-CoV-2 immunization [37]. Firstly, SARS-CoV-2 does not use mouse ACE2 as its receptor [38] and wild-type mice are thought to be less susceptible to SARS-CoV-2 infection, although transgenic mice expressing human ACE2 have now been developed [39,40]. Secondly, the DNA plasmid in this study was administered by i.d. injection followed by electroporation. This method is not the most common delivery strategy as intramuscular injection is typically preferred, but was selected to allow proximity with the adjuvant source [12]. Finally, no optimization step for the plasmid sequence was performed and a plasmid coding for the full-length spike protein was used. 041b061a72