key: cord-0875731-8zmummev
authors: Rice, Adrian; Verma, Mohit; Shin, Annie; Zakin, Lise; Sieling, Peter; Tanaka, Shiho; Balint, Joseph; Dinkins, Kyle; Adisetiyo, Helty; Morimoto, Brett; Higashide, Wendy; Anders Olson, C.; Mody, Shivani; Spilman, Patricia; Gabitzsch, Elizabeth; Safrit, Jeffrey T.; Rabizadeh, Shahrooz; Niazi, Kayvan; Soon-Shiong, Patrick
title: Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice
date: 2021-07-21
journal: Sci Rep
DOI: 10.1038/s41598-021-94364-5
sha: 529306888b0d117cc591cab69f2158f39c14e350
doc_id: 875731
cord_uid: 8zmummev

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity—including mucosal immunity—against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.

In response to the need for a COVID-19 vaccine that is safe, effective, and suitable for global distribution, we have developed the dual antigen hAd5 S-Fusion + N-ETSD vaccine including formulations for subcutaneous (SC), oral, and intranasal (IN) delivery. The vaccine comprises the SARS-CoV-2 spike (S) protein modified for enhanced cell surface expression (S-Fusion) to increase humoral responses and the nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to target N to the endosomal/lysosomal cellular compartment 1 to enhance MHC class I and II presentation.

The vaccine antigens are delivered using a human adenovirus serotype 5 (hAd5) vector with deletions in the E1, E2b, and E3 gene regions (hAd5 [E1-, E2b-, E3-]) 2 . Removal of E2b gene regions results in a reduction of late gene expression of viral protein such as the Ad5 viral fiber protein and allows for expression of inserted transgenes for extended periods of time even in the presence of pre-existing Ad5 immunity [3] [4] [5] [6] [7] . The platform therefore shows potential to be suitable for homologous prime-boost immunization and/or immunotherapy regimens [8] [9] [10] [11] [12] . Importantly, this next generation Ad vector has demonstrated safety in over 125 patients with solid tumors. In these Phase I/II studies in cancer patients, CD4 + and CD8 + antigen-specific T cells were successfully generated to multiple somatic antigens (CEA, brachyury, MUC1, PSA) even in the presence of pre-existing Ad immunity 8, 11, 12 .

SARS-CoV-2 is an enveloped positive sense, single-strand RNA β coronavirus primarily composed of four structural proteins-S, N, membrane (M), and envelope (E)-as well as the viral membrane and genomic RNA. The S glycoprotein [13] [14] [15] is displayed as a trimer on the viral surface, whereas N is located within the viral particle. Spike initiates infection by the SARS-CoV-2 virus by interaction of its receptor binding domain (RBD) with human host angiotensin-converting enzyme 2 (ACE2) expressed on the surface of cells in the respiratory system, including alveolar epithelial cells 16 , as well as cells in the digestive tract.

The majority of current SARS-CoV-2 vaccines under development deliver only the S antigen because antibodies raised against S RBD are expected to neutralize infection [17] [18] [19] . Reliance on S as the sole vaccine antigen www.nature.com/scientificreports/ is not without risk, however, particularly in the face of the rapidly dominating variants including the B.1.351 variant expressing E484K, K417N, and N501Y mutations 20 ; the B.1.1.7 variant (N501Y) 21, 22 ; and the Cal.20C L452R variant 23 all of which have altered RBD sequences that may not be as effectively recognized by antibodies generated in response to first-wave sequence S-based vaccines [24] [25] [26] .

To lessen the risk of single-antigen delivery and to broaden protective immune responses, we included the N protein in our hAd5 S-Fusion + N-ETSD vaccine. N is a highly conserved and antigenic SARS-CoV-2-associated protein that has been studied previously as an antigen in coronavirus vaccine design for SARS-CoV [27] [28] [29] [30] . N associates with viral RNA and has a role in viral RNA replication, virus particle assembly, and release 31, 32 . Studies have shown that nearly all patients infected with SARS-CoV-2 have antibody responses to N 33, 34 . Furthermore, another study reported that most, if not all, COVID-19 survivors tested were shown to have N-specific CD4 + T-cell responses 19 .

The ability of N to elicit vigorous T-cell responses highlights another advantage of the addition of N. A robust T-cell response to vaccination is at least as important as the production of antibodies 35 and should be a critical consideration for COVID-19 vaccine efficacy. First, humoral and T-cell responses are highly correlated, with titers of neutralizing antibodies being proportional to T-cell levels, suggesting the T-cell response is necessary for an effective humoral response 36 . It is well established that the activation of CD4 + T helper cells enhances B-cell production of antibodies. Second, virus-specific CD4 + and CD8 + T cells are widely detected in COVID-19 patients 37 , based on findings from patients recovered from the closely-related SARS-CoV, and there are reports that such T cells persist for at least 6-17 years, suggesting that T cells may be an important part of long-term immunity [38] [39] [40] . These T-cell responses were predominantly to N, as described in Le Bert et al., who found that in all 36 convalescent COVID-19 patients in their study, the presence of CD4 + and CD8 + T cells recognizing multiple regions of the N protein could be demonstrated 40 . They further examined blood from 23 individuals who had recovered from SARS-CoV and found that the memory T cells acquired 17 years ago also recognized multiple proteins of SARS-CoV-2. These findings emphasize the importance of designing a vaccine with the highly conserved N present in both SARS-CoV and SARS-CoV-2. Third, recovered patients exposed to SARS-CoV-2 have been found without seroconversion, but with evidence of T-cell responses 41 . T-cell based responses become even more critical given the finding in at least one study that neutralizing antibody titers decline in some COVID-19 patients after about 3 months 42 . The importance of both S and N was highlighted by Grifoni et al. 19 who identified both antigens as a priori potential epitopes that are predicted to induce both B and T cell responses to the SARS-CoV virus that is similar to SARS-CoV-2.

While we find the evidence that cell-mediated protection is a key element for vaccine efficacy, we note its role, and specifically the contribution of N-elicited T-cell responses, to protection against SARS-CoV-2 infection has not been established in animals models.

Additional considerations for vaccine design beyond the choice of antigens include the ability to generate mucosal immunity that provides the highest probability of preventing transmission. IN delivery offers the potential to confer mucosal immunity. SARS-CoV-2 is a mucosal virus 43, 44 that in most instances, initiates infection by entry to the nose and mouth. Similarly, it's most efficient route of transmission is by respiratory droplets that are then transmitted to other persons 45 . Thus a vaccine that also elicits protective mucosal responses mediated by IgA is more likely to reduce transmission as compared to systemic, IgG-only humoral and T-cell responses 46 .

It was our goal in the studies presented herein to confirm enhanced cell surface expression of S-Fusion as compared to S-wild type (S-WT) in in vitro studies, then compare humoral and T cell responses after vaccination with either hAd5 S-WT or S-Fusion + N-ETSD in in vivo studies in CD-1 mice. Here, after we established generation of anti-S antibodies and S-reactive T-cell responses were greater with hAd5 S-Fusion + N-ETSD than hAd5 S-WT, we then compared SC and IN prime delivery of the hAd5 S-Fusion + N-ETSD vaccine. In a third experiment, the two routes of delivery were combined in a single prime to ascertain if together optimal immune responses could be achieved that may not necessarily be dependent upon a boost.

In all three study paradigms-SC prime with SC boost study, SC versus IN prime with boost, and combined SC plus IN prime with or without boost-immunization of CD-1 mice with the hAd5 S Fusion + N-ETSD vaccine elicited Th1 biased humoral responses against S. Both CD4 + and CD8 + T-cell responses to SARS-CoV-2 S and N peptide pools were also seen, with cytokine production being greater overall in response to N peptides. Potent neutralization of SARS-CoV-2 by sera from vaccinated mice in all studies was confirmed by a surrogate neutralization assay 47 . While all dosing paradigms produced broad immune responses, perhaps the most significant and compelling finding was that a single prime administration by combined SC and IN dosing generated immune responses that were at least as great as dosing regimens that included a boost.

tionally-relevant S RBD as compared to S-WT. Before initiation of in vivo studies in mice, our goal of enhancing cell-surface display of S was confirmed by transduction (infection) of HEK-293T cells with hAd5 S-WT, S-WT + N-ETSD, S-Fusion alone, and S-Fusion + N-ETSD followed by flow cytometric analysis of anti-S RBD antibody binding. As shown in Fig. 1 , there was very little binding of anti-S RBD-specific antibodies to the surface of HEK 293T cells transduced with hAd5 S-WT (Fig. 1a) or hAd5 S-WT + N-ETSD (Fig. 1b) constructs; antibody binding to hAd5 S-Fusion was higher (Fig. 1c) . The highest cell-surface expression of RBD was detected after transduction with dual antigen hAd5 S-Fusion + N-ETSD (Fig. 1d) .

Similar results were seen for recombinant angiotensin converting-enzyme 2 (ACE2)-Fc binding to HEK 293T cells transfected with hAd5 S-WT, S-Fusion or S-Fusion + N-ETSD; with ACE2 showing higher binding to S-Fusion than S-WT and the dual antigen construct showing the highest binding ( Fig. 1e- www.nature.com/scientificreports/ These findings support our rationale for modification of S with the fusion sequence that was predicted to increase cell-surface display of physiologically-relevant S. Generation of IgG2a and 2b anti-S antibodies was significantly greater for mice that received the hAd5 S-Fusion + N-ETSD vaccine as compared to those that received hAd5 S-WT (Fig. 2b) . Sera from mice that received the hAd5 S-Fusion + N-ETSD vaccine showed higher inhibition of rACE2 binding to S RBD in the Genscript cPass surrogate assay 47 than sera from hAd5 S-WT inoculated mice (Fig. 2c) . Inhibition of binding in this assay suggests the presence of anti-S RBD neutralizing antibodies and inhibition of 30% or greater is correlated with neutralization of live SARS-CoV-2 virus.

Only mice receiving hAd5 S-Fusion + N-ETSD vaccination generated anti-N antibodies (Fig. 2d) , as expected. In hAd5 S-Fusion + N-ETSD mice, anti-N antibody levels were higher than anti-S antibody levels, given the dilution factor in the anti-S ELISA was 1:30 and was 1:90 for the anti-N ELISA.

All humoral responses showed a T helper cell 1 (Th1) bias (Fig. 2e) , based on determination of the IgG2a + IgG2b + IgG3 / IgG1 ratio (using ng equivalent values).

S-WT versus hAd5 S-Fusion + N-ETSD study using both SC prime and boost vaccination, Intracellular Cytokine Staining (ICS) revealed that CD4 + T cells were more responsive to the N peptide pool than either S peptide pools 1 or 2 ( Fig. 3a-c) . Responses to N peptides were only seen for hAd5 S-Fusion + N-ETSD vaccinated mice, as expected.

Conversely, the S1 peptide pool (containing S RBD) elicited higher interferon-γ (IFN-γ), IFN-γ/Tumor necrosis factor-α (TNF-α), and IFN-γ/TNF-α/interleukin-2 (IL-2) production in CD8 + T-lymphocytes than the N peptide pool (Fig. 3d-f ). All CD8 + T-cell responses were higher for hAd5 S-Fusion + N-ETSD mice as compared to the mice receiving hAd5 S-WT. www.nature.com/scientificreports/ In ELISpot analysis, IFN-γ secretion by T cells from hAd5 S-Fusion + N-ETSD mice in response to S peptide pool 1 and the N peptide pool was similar (Fig. 3g) . IFN-γ secretion by T cells from hAd5 S-WT mice was significantly lower. T-cell secretion of interleukin-4 (IL-4) was very low in response to all peptide pools for both vaccinated groups (Fig. 3h) , therefore T-cell responses, like humoral responses, showed a Th1 bias with the IFN-γ/IL-4 ratio being > 1 in all mice (Fig. 3i) . (Fig. 4b) . Sera were highly neutralizing as reflected by inhibition in the surrogate virus neutralization . T-cell responses to S are greater for hAd5 S-Fusion + N-ETSD as compared to hAd5 S-WT and CD4 + T cells were more responsive to N. Intracellular cytokine staining (ICS) of interferon-γ (IFN-γ), IFN-γ/tumor necrosis factor α (TNF-α), and IFN-γ/TNF-α/interleukin-2 (IL-2) in response to S peptide pools 1 and 2 as well as the N peptide pool is shown for (a-c) CD4 + and (d-f) CD8β + T lymphocytes. CD4 + T cells were more responsive to N peptides and CD8 + T cells to S peptide pool 1 that contains the S RBD as compared to S peptide pool 2 or the N pool. Media and SIV nef are negative controls. ELISpot detection of (g) IFN-γ secretion by T-lymphocytes also reveals responses to S peptide pool 1 and the N pool. (h) Interleukin-4 (IL-4) secretion was very low. SIV nef is a negative control and Con A a positive control. www.nature.com/scientificreports/ assay in all but 3 mice in the SC > SC group (Fig. 4c) . Anti-N IgG was also detected in sera from all vaccinated mice, with the levels being very similar for vaccinated groups (Fig. 4d) . www.nature.com/scientificreports/ Anti-S IgG was also detected in lung homogenate of all vaccinated mice and was higher overall for the IN > IN group (Fig. 4e) . Lung homogenate from all IN > IN group mice showed high inhibition in the surrogate neutralization assay, whereas homogenate from 3 mice in the SC > SC boost group did not surpass the 30% level of inhibition that is associated with viral neutralization (Fig. 4f) . In lung homogenate, anti-N IgG showed a trend to be higher in the SC > IN group (Fig. 4g) . Not unexpectedly, both anti-S and anti-N IgA levels in lung homogenate were highest in the IN > IN boost group (Fig. 4h) . Furthermore, anti-S and anti-N IgG subclass analysis in both sera and lung showed Th1 bias for all vaccinated groups (Fig. 4i) .

Both CD4 + and CD8 + T-cell responses were greater to N than S, and higher with SC delivery. ICS of IFN-γ (Fig. 5a,d) ; IFN-γ, TNF-α (Fig. 5b,e) , and IFN-γ, TNF-α, interleukin-2 (IL-2) (Fig. 5c,f) showed the highest mean values for the SC > SC boost and SC > IN vaccinated groups with responses to the N peptide pool trending higher for both CD4 + and CD8 + T cells. This was somewhat in contrast with the findings of the first SC > SC study, where CD8 + T-cell responses were greater to the S1 peptide pool (Fig. 3d-f) , however, variation is expected in outbred CD-1 mice and robust CD8 + responses to both S and N were detected in SC > SC mice from each study. While the differences were not statistically significant due to variation among individual mice, www.nature.com/scientificreports/ overall the IN > IN boost group had a reduced population of CD8 + cells capable of accumulating cytokines in response to S and N peptide stimulation (Fig. 5d-f) . ELISpot findings were similar, with higher responses seen for the SC > SC and SC > IN groups when compared to the IN > IN group (Fig. 5g) . The highest responses were found to be specific to the N peptide pool. Interleukin-4 (IL-4) secretion in ELISpot was very low for all groups (Fig. 5h) , therefore the IFN-γ/IL-4 ratios were above 1 for almost all vaccinated mice in response to both S and N peptide pools (Fig. 5i) , indicating Th1 bias.

The findings in this study suggest an important contribution of SC delivery to T cell responses. This study design is shown in Fig. 6a . There were 5 groups of CD-1 mice: untreated, an SC prime at 1 × 10 10 viral particles (VP) without boost (SC > no boost), a combined 9 × 10 9 VP SC plus 1 × 10 9 All mice were euthanized on Day 35 and tissue including blood for serum, spleens for T cells, and lung tissue collected for analyses. Note this euthanasia day is one week later than the two studies described above (Figs. 2a,  4a) , which was a change meant to better characterize humoral responses at a time point at which we expected cell-mediated responses to remain high based on our prior work with this vaccine platform. The combined SC + IN > no boost regimen was just as effective in eliciting neutralizing anti-S IgG and anti-N IgG antibody production in both sera (Fig. 6b-d) and lung ( Fig. 6e-g) as either the SC + IN > IN or SC + IN > SC regimens. SC > no boost gave significantly lower humoral responses ( Fig. 6b-g) . All humoral responses showed a Th1 bias (Fig. 6h) based on the ratio of IgG2a, IgG2b, and IgG3 to IgG1 antibody subclasses.

Similar to the findings in the first study, ICS shows the N peptide pool stimulated cytokine production by CD4 + T lymphocytes from all vaccinated mice (Fig. 7a-c) , but CD8 + T cells from vaccinated mice responded to S peptide pool 1 which contains the S RBD (Fig. 7d-f ). The differences between vaccinated groups were not significant due to variability among mice, with SC + IN > no boost vaccinated mice having T-cell responses that were similar to those seen with mice that did receive boosts.

In ELISpot, the highest IFN-γ secretion in response to peptide pools differed by both peptide pool and vaccination regimen. As compared to the negative control (SIV nef), T-cell IFN-γ secretion was significantly greater for the combined SC + IN > SC group in response to the S1 peptide pool; greater for the SC + IN > IN group to the S2 peptide pool; and greater for the SC + IN > no boost group to the N peptide pool (Fig. 7g) .

IL-4 secretion was very low (Fig. 7h) , therefore the IFN-γ/IL-4 ratio was above 1 for all vaccinated mice with only one exception, reflecting Th1 bias of T-cell responses (Fig. 7i ).

Our hAd5 S-Fusion + N-ETSD vaccine was designed to overcome the risks of an S-only vaccine and elicit both T-cell immunity and neutralizing antibodies, leveraging the vital role T cells play in generating long-lasting antibody responses and in directly killing infected cells. The CD4 + and CD8 + T cell responses induced by this vaccine are multifunctional, and induction of such multifunctional T cells by vaccines is correlated with better protection against infection 48 . We postulated that enhanced CD4 + T-cell responses and Th1 predominance resulting from expression of an S antigen optimized for surface display and an N antigen optimized for endosomal/lysosomal subcellular compartment localization 1 and thus MHC I and II presentation, led to increased dendritic cell presentation, cross-presentation, B cell activation, and ultimately high neutralization capability as determined in the surrogate assay.

It is well established that the contemporaneous MHC I and MHC II presentation of an antigen by the antigen presenting cell activates CD4 + and CD8 + T cells simultaneously and is optimal for the generation of memory B and T cells. A key finding of our construct is that N-ETSD elicits a CD4 + T-cell response, a necessity for induction of memory T cells and helper cells for B cell antibody production. Others have also reported on the importance of lysosomal localization for eliciting the strongest T-cell IFN-γ and CTL responses, compared to natural N 49, 50 .

The T-cell responses to the S and N antigens expressed by hAd5 S-Fusion + N-ETSD were polycytokine, including IFN-γ, TNF-α, and IL-2, consistent with successful antimicrobial immunity in bacterial and viral infections [51] [52] [53] [54] [55] . Post-vaccination polycytokine T-cell responses have been shown to correlate with vaccine efficacy, including those with a viral vector 48 . Of relevance here, the polycytokine T-cell responses to SARS-CoV-2 N protein are consistent with those observed in recovered COVID-19 patients 27 , supporting the rationale that the dual antigen hAd5 S-Fusion + N-ETSD vaccine-induced immune responses against both SARS-CoV-2 and N antigens may provide vaccinated subjects with greater protections against SARS-CoV-2, including variants.

In future studies utilizing animal models of SARS-CoV-2 challenge, we hope to elucidate the role of S and N individually and in combination, as well as the relative importance of humoral and cell-mediated immune responses, in providing protection against SARS-CoV-2 infection.

The key finding here that prime-only vaccination delivered by combination SC and IN dosing results in broad humoral and systemic T-cell responses, along with the potential for enhanced mucosal immunity, supports the clinical testing of the hAd5 S-Fusion + N-ETSD vaccine by these simultaneous routes of immunization. The www.nature.com/scientificreports/ vaccine has currently completed Phase 1 testing as an SC prime and SC boost. Oral boost formulations that have shown efficacy in the ability to elicit immune responses that conferred complete protection against high-titer SARS-CoV-2 challenge in our pre-clinical studies in non-human primates 56 will soon also be tested in the clinic. To our knowledge, our vaccine is currently the only one available in SC, oral 57 , and IN formulations that offer expanded possibilities for efficient, feasible delivery across the globe. (Fig. 8b) .

The powerful cytomegalovirus (CMV) promoter 62 drives expression in the hAd5 construct (Fig. 8c ). On the final day of each study, blood was collected via the submandibular vein from isofluraneanesthetized mice for isolation of sera using a microtainer tube and then mice were euthanized for collection of spleen and lungs.

Spleens were removed from each mouse and placed in 5 mL of sterile media (RPMI/HEPES/Pen/Strep/10% FBS). Splenocytes were isolated within 2 h of collection and used fresh or frozen for later analysis.

Lungs were removed from each mouse, dissected in half and then immediately snap frozen on dry ice. Lung homogenates were generated by thawing one frozen lung half and homogenizing in 150 μL sterile PBS using a Fisher Scientific pestle drill. Homogenates were centrifuged at 13,000 rpm for 3 min and supernatants were utilized in ELISA and GenScript cPass surrogate neutralization assays. www.nature.com/scientificreports/ Intracellular cytokine stimulation (ICS). ICS assays were performed using 10 6 live splenocytes per well in 96-well U-bottom plates. Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of pools of overlapping peptide for S or N antigens at 2 μg/mL/peptide for 6 h at 37 °C in 5% CO 2 , with protein transport inhibitor, GolgiStop (BD) added two hours after initiation of incubation. The S peptide pool (JPT: Cat #PM-WCPV-S-1) is a total of 315 spike peptides split into two pools comprised of 158 and 157 peptides each. The N peptide pool (JPT; Cat # PM-WCPV-NCAP-1) was also used to stimulate cells. A SIV-Nef peptide pool (BEI Resources) was used as an off-target negative control. Stimulated splenocytes were then stained for a fixable cell viability stain followed by the lymphocyte surface markers CD8β and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-γ, TNF-α and IL-2 (in studies 2 and 3). Fluorescent-conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.

ELISpot assay. ELISpot assays were used to detect cytokines secreted by splenocytes from inoculated mice.

Fresh splenocytes were used on the same day, as were cryopreserved splenocytes containing lymphocytes. The cells (2-4 × 10 5 cells per well of a 96-well plate) were added to the ELISpot plate containing an immobilized primary antibody to either IFN-γ or IL-4 (BD), and were exposed to various stimuli (e.g. control peptides, target peptide pools/proteins) comprising 2 μg/mL peptide pools or 10 μg/mL protein for 36-40 h. After aspiration and washing to remove cells and media, extracellular cytokine was detected by a secondary antibody to cytokine conjugated to biotin (BD , or antimouse IgA HRP conjugated antibody (Sigma; Cat # A4789) was added to wells. For positive controls, a 100 μL of a 1/5000 dilution of rabbit anti-N IgG Ab or 100 μL of a 1/25 dilution of mouse anti-S serum (from mice immunized with purified S antigen in adjuvant) were added to appropriate wells. After incubation at room temperature for 1 h, the wells were washed with PBS-T and incubated with 200 μL o-phenylenediamine-dihydrochloride (OPD substrate (Thermo Scientific Cat # A34006) until appropriate color development. The color reaction was stopped with addition of 50 μL 10% phosphoric acid solution (Fisher Cat # A260-500) in water and the absorbance at 490 nm was determined using a microplate reader (SoftMax Pro, Molecular Devices).

curve of IgG was generated and absorbance values were converted into mass equivalents for both anti-S and anti-N antibodies. Using these values, we were able to calculate that hAd5 S-Fusion + N-ETSD vaccination generated a geometric mean value for S-and N-specific IgG per milliliter of serum. These values were also used to quantify the Th1/Th2 bias for the humoral responses by dividing the sum total of Th1 biased antigen-specific IgG subclasses (IgG2a, IgG2b and IgG3) with the total Th2 skewed IgG3, for each mouse. For mice that lack anti-S and/ or anti-N specific IgG responses, Th1/Th2 ratio was not calculated. Conversely, some responses, particularly for anti-N responses in IgG2a and IgG2b (both Th1 biased subclasses), were above the limit of quantification with OD values higher than those observed in the standard curve. These data points were reduced to values within the standard curve, and thus will reflect a lower Th1/Th2 bias than would otherwise be reported.

GenScript cPass neutralizing antibody detection. The GenScript cPass assay (https:// www. gensc ript.

com/ cpass-sars-cov-2-neutr aliza tion-antib ody-detec tion-Kit. html) for detection of neutralizing antibodies was used according to the manufacturer's instructions 47 . The kit detects circulating (sera) neutralizing antibodies against SARS-CoV-2 that block the interaction between the S RBD with the ACE2 cell surface receptor. It is suitable for all antibody subclasses and appropriate for use with in animal models without modification. www.nature.com/scientificreports/ Statistical analyses and graph generation. All statistical analyses were performed and graphs used in figures were generated using GraphPad Prism software. Statistical tests for each graph are described in the figure legends. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Immune responses to T-cell epitopes of SARS CoV-N protein are enhanced by N immunization with a chimera of lysosome-associated membrane protein

HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8(+) T cells producing both interferongamma and tumor necrosis factor-alpha

HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells

Shaping successful and unsuccessful CD8 T cell responses following infection

T cell responses to viral infections-Opportunities for peptide vaccination

T-cell quality in memory and protection: Implications for vaccine design

Complete protection of nasal and lung airways against SARS-CoV-2 challenge by antibody plus Th1 dominant N-and S-specific T-cell responses to subcutaneous prime and thermally-stable oral boost bivalent hAd5 Vaccination in an NHP study

Use of adenovirus as a model system to illustrate a simple method using standard equipment and inexpensive excipients to remove live virus dependence on the cold-chain

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: A doseescalation, open-label, non-randomised, first-in-human trial

Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: Implications for gene therapy

Structural genomics of SARS-CoV-2 indicates evolutionary conserved functional regions of viral proteins

Promoter strength in adenovirus transducing vectors: down-regulation of the adenovirus E1A promoter in 293 cells facilitates vector construction

We thank Phil Yang of ImmunityBio, Inc. for his coordination of project updates for this study. All methods used for in vivo studies in mice are in accordance with ARRIVE guidelines.

A.R. and M.V. designed and, with S.M., J.B., and K.D.; performed in vivo studies and co-analyzed data. A.R., M.V., and J.B. co-wrote the manuscript; A.S., assisted by B.M., generated the vaccine construct, protein purification, and flow cytometry studies; S.T. performed protein purification. L.Z. performed cell culture; WH and CAO generated additional constructs used for comparison; P.S. contributed to study design; H.A. performed assays for the in vivo studies; P.S. co-analyzed data, generated graphs and figures and co-wrote the manuscript; E.G. contributed to the platform (hAd5) and vaccine design; J.T.S. contributed to study design and immunological analyses; S.R. contributed to vaccine and study design; K.N. co-designed the vaccine and reviewed data; P.S.-S. originated the project, contributed to vaccine and study design and reviewed all data. All authors reviewed the manuscript.

All authors are affiliated with ImmunityBio, Inc. and have designed, tested, or supported studies of the vaccine described, a potential commercial product.

Correspondence and requests for materials should be addressed to P.S.-S.