Intradermal administration of DNA vaccine targeting Omicron SARS-CoV-2 via pyro-drive jet injector provides the prolonged neutralizing antibody production via germinal center reaction

Emerging SARS-CoV-2 Omicron variants are highly contagious with enhanced immune escape mechanisms against the initially approved COVID-19 vaccines. Therefore, we require stable alternative-platform vaccines that confer protection against newer variants of SARS-CoV-2. We designed an Omicron B.1.1.529 specific DNA vaccine using our DNA vaccine platform and evaluated the humoral and cellular immune responses. SD rats intradermally administered with Omicron-specific DNA vaccine via pyro-drive jet injector (PJI) thrice at 2-week intervals elicited high antibody titers against the Omicron subvariants as well as the ancestral strain. Indeed, the Omicron B.1.1.529-specific antibody titer and neutralizing antibody were higher than that of other strains. Longitudinal monitoring indicated that anti-spike (ancestral and Omicron) antibody titers decreased toward 30 weeks after the first vaccination dose. However, neutralization activity remained unaltered. Germinal center formation was histologically detected in lymph nodes in rats immunized with Omicron DNA vaccine. Ancestral spike-specific immune cell response was slightly weaker than Omicron spike-specific response in splenocytes with Omicron-adapted DNA vaccine, evaluated by ELISpot assay. Collectively, our findings suggest that Omicron targeting DNA vaccines via PJI can elicit robust durable antibody production mediated by germinal center reaction against this new variant as well as partially against the spike protein of other SARS-CoV-2 variants.


Intradermal administration of DNA vaccine targeting Omicron SARS-CoV-2 via pyro-drive jet injector provides the prolonged neutralizing antibody production via germinal center reaction
Hiroki Hayashi 1* , Jiao Sun 1 , Yuka Yanagida 1 , Takako Otera 1,8 , Jiayu A. Tai 2 , Tomoyuki Nishikawa 2 , Kunihiko Yamashita 2,9 , Naoki Sakaguchi 9 , Shota Yoshida 1,3 , Satoshi Baba 1,3 , Chin Yang Chang 4 , Munehisa Shimamura 4,5 , Sachiko Okamoto 10 , Yasunori Amaishi 10 , Hideto Chono 10 , Junichi Mineno 10 , Hiromi Rakugi 3 , Ryuichi Morishita 6 & Hironori Nakagami 1,7 Emerging SARS-CoV-2 Omicron variants are highly contagious with enhanced immune escape mechanisms against the initially approved COVID-19 vaccines.Therefore, we require stable alternative-platform vaccines that confer protection against newer variants of SARS-CoV-2.We designed an Omicron B.1.1.529specific DNA vaccine using our DNA vaccine platform and evaluated the humoral and cellular immune responses.SD rats intradermally administered with Omicronspecific DNA vaccine via pyro-drive jet injector (PJI) thrice at 2-week intervals elicited high antibody titers against the Omicron subvariants as well as the ancestral strain.Indeed, the Omicron B.1.1.529specificantibody titer and neutralizing antibody were higher than that of other strains.Longitudinal monitoring indicated that anti-spike (ancestral and Omicron) antibody titers decreased toward 30 weeks after the first vaccination dose.However, neutralization activity remained unaltered.Germinal center formation was histologically detected in lymph nodes in rats immunized with Omicron DNA vaccine.Ancestral spike-specific immune cell response was slightly weaker than Omicron spike-specific response in splenocytes with Omicron-adapted DNA vaccine, evaluated by ELISpot assay.Collectively, our findings suggest that Omicron targeting DNA vaccines via PJI can elicit robust durable antibody production mediated by germinal center reaction against this new variant as well as partially against the spike protein of other SARS-CoV-2 variants.
The development of effective vaccines has been the critical agenda in combating SARS-CoV-2 since the onset of the global COVID-19 (Coronavirus Disease 2019) pandemic 1 .Several companies have produced SARS-CoV-2 vaccines based on new technologies, such as mRNA-based and adenovirus-based vaccines, with a high preventive effect [2][3][4] .However, the continued evolution of SARS-CoV-2 has generated new variants, resulting in reduced efficiency of the originally approved vaccines or monoclonal antibodies [5][6][7] .However, since November 2021, the Omicron DNA vaccine induces humoral immune responses.Next, we evaluated whether the Omicron DNA vaccine induced antibody production in vivo.We intradermally administered the Omicron DNA vaccine into SD rats using a pyro-drive jet injector (PJI) 21 thrice every other week (Fig. 2a) and analyzed antibody titers for ancestral and omicron variants, including BA.1, BA.2, and BA.5.Six weeks after the first vaccination, the antibody titer was increased for all variants; indeed, the antibody titer for BA.1 was the highest, compared with that of the other strains (Fig. 2b and Supplementary Fig. 2).Neutralization activity against BA.1 was also higher than the other variants, analyzed by pseudo-typed virus assay (Fig. 2c).Antibody levels were observed during follow-ups with a high antibody titer being detected even 30 weeks after the first dose of vaccination (Fig. 2d).However, antibody titers for BA.1 and BA.2 were significantly decreased (Fig. 2e).The DNA vaccineinduced IgG subclass were IgG2b dominant, or Th1-based immune response (Supplementary Fig. 3).We also analyzed neutralization activity at 30 weeks using a pseudo-typed virus.Notably, the neutralization activities of all variants remained similar without any decrease (Fig. 2f and Supplementary Fig. 4).These results suggest that the DNA vaccine could induce long-term protection of neutralizing antibody.

Germinal center formation was detected by PJI-mediated Omicron DNA vaccine.
To produce long-lived antibody from B cells or memory B cells, germinal center formation is known to be critical in peripheral lymphoid organ in infection or vaccination [22][23][24] .We analyzed the germinal center formation in lymph node by histological analysis.Lymph nodes was collected and stained with hematoxylin and eosin (HE) at 7 weeks after 1st dose of DNA vaccine (at 3 weeks after 3rd dose).In control group, there are only primary follicles.By contrast, many secondary (activated) follicles with germinal center formation in Omicron DNA vaccine group (Fig. 3a-d and Supplementary Fig. 5a-d).By immunohistochemical analysis, cells in germinal centers in DNA vaccine group were stained with BCL6 (follicular B and T cells), CD20 (B cells), and CD3 (T cells) (Fig. 3e-i).These suggested that PJI-injected Omicron DNA vaccine induced germinal center responses, leading to longlasting antibody production.
Omicron DNA vaccine elicits cellular immune responses.SARS-CoV-2 spike-specific cellular immune responses evoked by the DNA vaccine were monitored via PJI-mediated intradermal administration 20,21 .We evaluated the omicron variant-specific cellular immune responses using the ELISpot assay.The splenocytes responded to S1 and S2 peptide pools from ancestral and Omicron BA.1 to produce IFNγ (Fig. 4a, b).However, the total number of spots (S1 + S2) belonging to ancestral spike protein was lower than those generated by Omicron BA.1 (Fig. 4c).Collectively, this data suggests that the Omicron DNA vaccine could elicit a cellular immune response in all the tested strains; however, it was relatively weaker in case of the ancestral spike peptide pool.

Discussion
Omicron B.1.1.529-adaptedDNA vaccine elicited a humoral immune response, with the BA.1-specific antibody titer being highest at 6 weeks after the first vaccination dose; the response eventually tapered toward 30 weeks.Antibody titers for BA.1 and BA.2 were significantly decreased between 6 and 30 weeks.However, neutralization activity did not decrease even at 30 weeks.In lymph nodes, the affinity of some antibodies against antigens is enhanced by affinity maturation and somatic mutation in the germinal center [25][26][27][28] .The generation of memory B cells and long-lived plasma cells for prolonged neutralizing antibody production from antigen-specific germinal center reactions is required for a vaccine to exert its effectivity 29 .Recent studies indicate that approved SARS-CoV-2 mRNA vaccines induce a germinal center reaction in humans and mice [30][31][32][33] .In this study, Omicron DNA vaccine via PJI induced secondary (activated) follicles with germinal center formation, which are mainly containing germinal center B cells, stained with BCL6, CD20 (Fig. 3).By contrast, less number of primary follicles, which are mostly composed on naïve B cells, were detected (Fig. 3 and Supplementary Fig. 5) 34 .Taken together, we speculate that Omicron-adapted DNA vaccine developed based on our DNA vaccine platform may induce the prolonged antibody production, and affinity maturation.However, further investigation is needed to evaluate follicular helper T cells, supporting germinal center reaction 35 , and germinal center B cells 24 induced by DNA vaccine.
Cross-reactive cellular immune response was also activated by the Omicron-adapted DNA vaccine, similar to the original DNA vaccine.However, the Omicron-variant DNA vaccine elicited a relatively weaker activation against the ancestral stain (Fig. 4).Omicron spike-specific T-cell response was significantly decreased in peripheral blood mononuclear cells from individuals who had received ancestral stain-targeted vaccines [36][37][38] .In the spike protein, 88.4% of T cell epitopes are conserved between the ancestral and Omicron (B.1.1.529)variants 36 , which affects spike-specific T cell responses.In our study, rat splenocytes vaccinated with the Omicron-adapted DNA vaccine exhibited reduced activation against ancestral spike stimulation, especially for S2 peptides pool (Fig. 4).The further analysis of T cell epitope will be necessary to explain these results.However, ancestral spike-specific T cell activation was maintained, suggesting that the Omicron-targeted DNA vaccine might also induce protective T cell immunity against infection by potential re-emerging previous VOC-related variants with highly conserved T cell epitopes 39 .
It is known that intradermal delivery of vaccine is effective than others, due to enriched population of immune cells such as antigen presenting cells (APCs) 40,41 .The advantage of PJI, needle-less injector, using pyro-technology is well-controlled injection by two types adjustable explosives 42 .It was reported that PJI precisely and effectively delivered plasmid DNA into intradermal region, leading to enhanced antigen expression and humoral immune response, compared with a conventional intradermal injection using needle-syringe delivery 20,43 .Our results in this study shows that PJI-mediated DNA vaccine robustly induced germinal center responses (Fig. 3), where activated B cells undergo clonal evolution by somatic mutations, affinity maturations, with the help of follicular helper T cells and follicular dendritic cells to produce more durable and broadly reactive antibodies against SARS-CoV-2 variants 24,44 .Although further investigations are needed to elucidate the effect of PJI on innate immune responses, the efficient intradermal antigen expression induced by PJI confers strong humoral immune responses including germinal center reactions for broadly protection.
The result of a phase I study to evaluate the safety and immunogenicity of intradermal DNA vaccine targeting ancestral strain via pyro-drive jet injector in healthy adults has been reported with unfavorable consequence 45 .In this clinical study, pVAX1-based DNA vaccine targeting ancestral strain was intradermally administrated via PJI into healthy individuals at 2 doses, low-dose (0.2 mg) or high-dose (0.4 mg) twice at 2-weeks interval.While, Their DNA vaccine received emergency use authorization in India 46 .In their clinical trial, DNA vaccine was administrated at low-dose (1 mg), or high-dose (2 mg) three times (total amount of DNA vaccine: 6 mg (2 mg × 3 times), which lead to humoral and cellular immune responses 47 .Those suggested that the weak humoral response may be because of insufficient antigen expression 45 .The enhancement of antigen expression might be one of ways to induce better humoral response.We have developed DNA vaccine by modified spike (amino acid substitutions at K986/V987P, 19 amino acid deletion at C-terminal), resulting in strengthened antigen expression on cell surface level, which leads to better humoral responses compared with original DNA vaccine that was used in clinical trial 21 .Although the difference in immune system between experimental animals and human needs to be considered, introduction of modification in DNA vaccine to enhance antigen expression might be efficient approach for future DNA vaccine development.
As a limitation of this study, we did not performed live SARS-CoV-2 challenge test to evaluate how maintained neutralization activity of antibodies impact on the pathogenicity of live SARS-CoV-2 including circulating or next variants.
In summary, we designed the Omicron B.1.1.529specific DNA vaccine based on our improved SARS-CoV-2 spike DNA vaccine platform 21 and evaluated vaccine-induced humoral and cellular immune responses against ancestral and Omicron sub lineages.Our findings confirmed that the Omicron B.1.1.529strain-adapted DNA vaccine successfully induced germinal center reaction, leading to high antibody titers, which were cross-reactive and durable for 30 weeks, and elicited pseudo virus neutralization activity.
Western blotting.After transfection with plasmid DNA, the cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Roche).Cell lysates were sonicated and centrifuged at 20,000×g for 5 min at 4 °C.The supernatant was collected and stored at − 80 °C until further use.The cell lysate was quantified, and the sample was mixed with Laemmli sample buffer (Bio-Rad) along with β-mercaptoethanol.The boiled samples were separated on a 4-20% gradient gel (Daiichi Gel).The separated samples were then transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane.The PVDF membranes were blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBS-T) for 1 h at RT.The membrane was then incubated with primary antibodies (SARS-CoV-2 spike #42172, Cell Signaling Technology, GAPDH MAB374, Millipore) overnight at 4 °C.After the membrane was washed with PBS-T, it was incubated with a secondary antibody tagged with horseradish peroxidase (HRP, GE Healthcare) for 1 h at RT.The membrane was washed and developed using a HRP substrate (Chemi-Lumi One L, Nacalai Tesque).The signals were detected using a ChemiDoc™ Touch imaging system (Bio-Rad).
Immunocytochemistry. Cells were seeded on glass-bottomed plates (Matsunami Glass).After transfection with the plasmid DNA, the cells were washed with PBS and fixed with 4% paraformaldehyde.Fixed cells were blocked with PBS containing 5% skim milk (Wako) for 1 h at RT.The cells were incubated with primary antibodies for the SARS-CoV-2 Spike (BLSN-005P, Beta Lifescience) or control IgG (Thermo Fisher) at 4 °C overnight.The following day, cells were washed and incubated with a secondary antibody labeled with Alexa Fluor 488 (Molecular Probes) for 1 h at RT. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Roche).The cells were observed under a confocal microscope (FV10i; Olympus).
Animals and immunization protocol.Female Crl: Caesarean Derived (CD) Sprague Dawley (SD) rats (7 weeks old; Charles River) were housed with free access to food and water in a temperature-and light-cyclecontrolled facility.DNA vaccines were intradermally administered using a pyro-drive jet injector (PJI, Actranza Lab, Type: Rat, provided by Daicel.Co.).For the vaccination, 240 μg of DNA vaccine was administered into flank regions of rats by PJI thrice at 2-week intervals (2 mg/mL of DNA vaccine, 30 μL/injection × 4 sites/animal).Control group: N = 3.DNA vaccine group: N = 6.All animal experiments were performed under isoflurane-, or three types of mixed anesthetic agents (medetomidine, midazolam, and butorphanol)-mediated anesthesia.All studies involving animals are reported in accordance with ARRIVE guidelines.

Determination of antibody titers using enzyme-linked immunosorbent assay (ELISA).
Antispike antibody titers were measured using ELISA as previously described 19 .Briefly, 96-well plates were coated with 1 μg/ml recombinant spike protein (ancestral spike; SPN-C52H9, Omicron BA.1 spike; SPN-C52Hz, Omicron BA.2 spike; SPN-C5223, Omicron BA.5 spike; SPN-C522e, Acro Biosystems) and kept at 4 ℃ overnight.After blocking with 5% skim milk PBS for 2 h at RT, the diluted sera (ranging from 10-to 31,250-fold dilution with 5% skim milk PBS) were added to the wells and incubated at 4 °C overnight.The following day, the wells were washed with PBS-T and incubated with HRP-conjugated anti-IgG antibody (GE Healthcare) for 3 h at RT.The wells were developed using the peroxidase chromogenic substrate 3,3′-5,5′-tetramethyl benzidine (TMB, Sigma) for 30 min at RT.The reaction was quenched by adding sulfuric acid (0.5 N).The absorbance of the samples in the wells was measured immediately at 450 nm using a microplate reader (Bio-Rad).The half-maximum antibody titer of the sample was determined from the highest absorbance in the dilution range (GraphPad Prism 8 software).For IgG subclass determination, anti-IgG1 (Abcam), IgG2a (Abcam), IgG2b (Abcam), and IgG2c (Southern Biotech) antibodies were used as the secondary antibodies.
Enzyme-linked Immunospot (ELISpot) assay.SARS-CoV-2 spike-specific cellular immune responses were evaluated by ELISpot assay using an Immunospot® kit (Cellular Technology Limited).Briefly, PVDF membrane-bottomed 96-well plates were activated with methanol and then coated with an anti-rat interferon γ (IFNγ) capture antibody at 4 °C overnight.On the following day, the splenocytes, collected from vaccinated or control rats, were incubated with ancestral or Omicron spike peptide pools (Ancestral; PM-WCPV-S-SU1-1 and SU2-1, Omicron BA.1; PM-SARS2-SMUT08-1, JPT peptide technologies) for 24 h at 37 °C in a humidified CO 2 incubator.After washing, the anti-IFNγ-detecting antibody was added to the wells and incubated for 2 h at RT. IFNγ spots were developed using the Tertiary solution and Blue developer solution.Finally, the number of IFNγ-spots (IFNγ-releasing splenocytes) was counted using the Immunospot analyzer.The spot values were subtracted from background spots for analysis.
For the neutralization assay, serially diluted serum samples were incubated with the pseudo-typed virus and added to Vero cells.After 24 h at 37 °C, the cells were lysed and activated using the luciferase assay system (Promega).Luciferase activity was measured using Synergy LC (BioTek).The neutralization value was analyzed using GraphPad Prism 8 based on 50% inhibitory dilution (ID50).

Histology and immunohistochemistry.
For histological analysis, inguinal lymph nodes were collected at 7 weeks after the first vaccination and fixed in 4% paraformaldehyde.Fixed tissues were embedded in paraffin, cut and stained with hematoxylin and eosin (HE).
For immunohistochemical analysis of CD3 (T cells), CD20 (B cells), and Bcl6 (follicular B cells and T cells), the deparaffinized sections were heated for antigen retrieval.After blocking with Antibody Diluent with Background Reducing Components (Dako), the sections were incubated with primary antibodies (CD3: ab16669 abcam, CD20: ab64088 abcam, Bcl6: ab272859 abcam) for overnight at 4 °C.After washing, the sections were treated with HRP-labelled pokymer anti-rabbit (Dako Envision system, Dako) for 30 min at RT, and developed with the ImmPACT DAB system (Vector) for 5 min.The sections were counterstained with Mayer's hematoxylin.Stained tissue sections were observed under a BZ-X810 microscope (Keyence).

Statistical analysis.
All values are presented as mean ± SEM.To assess significant differences, Student's t-test, as well as one-and two-way ANOVA, were performed.These were followed by Tukey's or Bonferroni's multiple comparison test using GraphPad Prism 8 software.Finally, statistical significance was set at p < 0.05.

Statements.
All methods in this study were performed in accordance with the relevant guidelines and regulations.

Ethics approval. All experiments were approved by the Ethical Committee for Animal Experiments of
Osaka University Graduate School of Medicine.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Development of DNA vaccine targeting SARS-CoV-2 Omicron variant.(a) Schematic representation of plasmid construction for SARS-CoV-2 Omicron DNA vaccine.K986P/V987P mutation and deletion of 19 aa at the C-terminal were introduced to improve cell-surface antigen expression are highlighted in red.Optimized-Omicron spike sequences were inserted into the pVAX1 vector.(b) Expression of Omicron spike induced by Omicron DNA vaccine was detected in HEK293 cells.Upper and lower bands represent the full-length and S1 domain of the spike, respectively.GAPDH was used as a control.The images were cropped from the data shown in Supplementary Fig. 1.(c) Cell surface localization of spike protein was evaluated by immunocytochemical analysis in HEK293 cells.pVAX1 control plasmid (upper panels) and Omicron DNA vaccine (lower panels) were transfected into HEK293 cells.Scale bar = 10 μm.See also Supplementary Fig. 1.

Figure 4 .
Figure 4. Cellular immune responses induced by SARS-CoV-2 Omicron DNA vaccine.(a) Representative image of ELISpot assay.Spike-specific T-cell responses were analyzed by ELISpot assay.At 7 weeks after the first dose of DNA vaccine, the vaccinated or control splenocytes were isolated and stimulated with spike peptide pool from ancestral as well as Omicron BA.1.(b) Quantitative analysis of IFNγ-releasing cells from ELISpot assay.The number of spots were analyzed after subtraction from background spots (no peptide stimulation) of each group.*p < 0.05 versus control (c) Quantitative data of the total number of spots (S1 + S2) of IFNγ-releasing cells from each strain (S1 and S2 peptide pool).*p < 0.05 versus ancestral spike.N = 3.The experiments were repeated twice.Data are presented as mean ± SEM.