Induction of Th1/Th2-balanced protection against SARS-CoV-2 through mucosal delivery of an adenovirus vaccine expressing an engineered spike protein Different influence the efficacy of the against

We developed a series of recombinant human type 5 adenoviruses that express the full-length or membrane-truncated spike protein (S) of SARS-CoV-2 (AdCoV2-S or AdCoV2-SdTM, respectively). We tested the immunoprotective efficacy against SARS-CoV-2 via intranasal (i.n.) or subcutaneous (s.c.) immunization in a rodent model following two-dose immunizations. Mucosal delivery of adenovirus (Ad) vaccines could induce anti-SARS-CoV-2 IgG and IgA in the serum and in the mucosal, respectively as indicated by vaginal wash (vw). Serum anti-SARS-CoV-2 IgG but not IgA was induced in the vw by s.c. injection of AdCoV2-S. Intranasal administration of AdCoV2-S was able to induce higher anti-SARS-CoV-2 antibody levels than s.c. injection. Immunization with AdCoV2-SdTM induced a lower antibody response than AdCoV2-S. In addition, the degree of neutralization of clinically isolated SARS-CoV-2 in the serum correlated with the above anti-SARS-CoV-2 responses; the most potent neutralizing activity was observed in the AdCoV2-S i.n. group, and less viral neutralizing activity was observed in response to AdCoV2-S s.c. and AdCoV2dTM i.n. Novelty, S-specific IgG1 which represented Th2-mediated humoral response was dominantly induced in Ad i.n.-immunized serum in contrast to more IgG2a which represented Th1-mediated cellular response found in Ad s.c.-immunized serum. The activation of S-specific IFN-ɣ and IL-4 in Th1 and Th2 cells, respectively, was observed in the AdCoV2s i.n. and s.c. groups, indicating the Th1/Th2-balenced immunity was activated. During the protection study, two doses of i.n. AdCoV2-S or i.n. The reaction was halted with 100 µl of 2N H 2 SO 4 . The optical density (O.D.) measurements were determined at 450 nm using a microplate reader. The endpoint serum dilution was calculated using the O.D. values and the cutoff value of serially diluted sera was set to twofold the background signal. IL-2 IL-4, using the and Results are presented as the of the


Introduction
COVID-19 is an emerging respiratory infectious disease that caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is efficiently transmitted from person to person and has thus been able to spread rapidly across all continents globally. To date, no effective medications are used clinically; therefore, the development of a safe and effective treatment for COVID-19 is urgently needed.
There are three coronaviruses that cause deadly pneumonia in humans: severe acute respiratory syndrome coronavirus (SARS-CoV) 1 , Middle-East respiratory syndrome coronavirus (MERS-CoV) 2 , and SARS-CoV-2 3 . SARS-CoV, MERS-CoV, and SARS-CoV-2 all belong to the β-coronavirus genus. The genome of SARS-CoV-2 is 96.2% identical to that of bat CoV RaTG13, whereas it is 79.5% identical to that of SARS-CoV 4 . The coronavirus is an enveloped virus containing a positive single-stranded RNA associated with a lipid membrane derived from the host cell.
The coronavirus has the largest RNA genome among all the known RNA viruses 4 .
Coronavirus encodes the spike (S) protein, which forms homotrimers that protrude from the surface of viral particles and is used for entry into host cells 5 . During viral replication in the infected cell, translated premature S protein is cleaved at the boundary between the S1 and S2 subunits, which remain noncovalently bound in the prefusion conformation 6 . S1 is responsible for binding to the host cell receptor, and S2 is responsible for the fusion of the viral and cellular membranes after S1-receptor interactions occur 6,7 . Recent studies have indicated that SARS-CoV and several SARS-related coronaviruses, including SARS-CoV-2, interact directly with angiotensin-converting enzyme 2 (ACE2) via S1 to enter target cells 8 .
A replication-incompetent adenoviral vector (Ad) with a recombinant E1-deficient Ad carrying a transgene has been shown to be a potential vaccine vector in multiple successful preclinical and clinical studies 9-11 . Ad is a strong DC activator 5 that can coordinate and stimulate distinct subsets of T helper (Th) cells, such as Th1 cells, to activate B cells for antibody secretion or Th2 cells to trigger cellular immunity-mediated cytotoxic T-cell responses 11,12 . The Ad vector can be delivered by different routes such as systemic or mucosal site administration, which makes vaccines convenient for immunization against respiratory pathogens that preferentially initiate infection at a mucosal site or in the respiratory tract 9, 11,13 .
Here, we characterize the immunogenicity of recombinant Ad expressing S and S-engineered proteins of SARS-CoV-2 in animals. The immune responses induced by Ad vaccine mucosal immunization indicated significant Th1/Th2-biased immunity and a reduction in pulmonary viral loads and the associated inflammation induced by SARS-CoV-2 infection. The increased efficacy of the Ad vaccine against SARS-CoV-2 when administered intranasally rather than systemically was investigated. These results support the future development of mucosal Ad vaccines for clinical use to control SARS-CoV-2.

Ethics statement
The study was carried out in compliance with the ARRIVE guidelines. All animal experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of the National Health Research Institutes (LAC-NHRI) in Taiwan. The animal use protocols were reviewed and approved by the NHRI Institutional Animal Care and Use Committee (Approval Protocol No. NHRI-IACUC-109073-M1-A-S01). To perform immunization or a viral challenge, the animals were placed in an anesthetic inhalator inhalation chamber containing isoflurane (initial phase: 5%; maintenance phase: 3%-4%) for 1 min before vaccine administration or SARS-CoV-2 challenge. 6 After the investigation, the animals were euthanized by 100% CO2 inhalation for 5 min followed by cervical dislocation to minimize suffering.

Cell lines and viruses
Human embryonic kidney cells (293A) were purchased from Invitrogen (Cat. R70507), and green monkey kidney cells (Vero) were purchased from the American Type Culture Collection (ATCC No. CCL-81). The 293 cells were cultured, grown and maintained in DMEM (HyClone, Cat. SH300) supplemented with 10% FBS in an incubator at 37°C with 5% CO2. Vero cells were cultured, grown and maintained in M199 medium (GIBCO-BRL) supplemented with 5% FBS in an incubator at 37°C with 5% CO2. The SARS-CoV-2 strain (hCoV-19/Taiwan/4/2020) was isolated from the Taiwan Centers for Disease Control and propagated in Vero cells. The propagation of SARS-CoV-2 was performed in the P3-grade laboratory, which was maintained in accordance with the regulations and approved by the Taiwan CDC inspection service.
The viral stocks were stored at -80°C. Viral stock titers were tested by cytopathic effect (CPE), and the TCID50 values were calculated by Reed-Muench method.

Animals, immunization, and live SARS-CoV2 challenge
BALB/c mice and Syrian hamsters (purchased from the National Laboratory Animal Center, Taiwan) aged six to ten weeks were maintained in pathogen-free cages at the LAC-NHRI throughout the animal study. The BALB/c mice were primed with 1×10 7 pfu/50 µL of Ad intranasally (i.n.) or subcutaneously (s.c.). After 14 days, the mice were boosted with the same dose of the respective immunogens administered via the same route. The mice were bled at 14 days and 1, 2, and 3 months after the booster immunization. The serum was analyzed by ELISA for the binding activity to recombinant S expressed in Sf9 insect cells (GenScript, Cat. 7 Z0.481-100) and for neutralizing activity against SARS-CoV-2 by the neutralization-TCID50 assay. For the challenge studies performed in the P3 laboratory, 1×10 5 TCID50/hamster of live SARS-CoV-2 was administered i.n. 1 month after the second immunization. The hamsters were monitored for 6 days to record their body weights or sacrificed on days 3 and 6 after viral challenge, and the lung tissues were isolated for plaque assays and histochemistry.
Whole lungs were excised from the hamsters and homogenized, clarified, and titrated by the TCID50 on Vero cells in 96-well plates. The cells were inoculated with serially diluted lung homogenate in M199 with 5% FBS and incubated for 5 days. The cells were visualized when 50% were lysed by microscopy and used as sets of diluted lung homogenates according to the calculation of the virus titer in the sample.
For histochemistry, the lung tissues were placed directly into 10% formalin

Production of recombinant Ads
AdCoV2 which encodes the condon-optimized SARS-CoV-2's S (AdCoV2-S) gene and transmembrane-deleted S (AdCoV2-SdTM) gene, the mutant S (AdCoV2-S(GA)) gene that has 2 amino acids mutated from LY to GA at 611 and 612 residues of S, and the condon-optimized SARS-CoV's S (AdSARS-S) gene, were cloned and generated 8 with the pAd⁄CMV⁄V5-DEST™ Gateway® Vector Kit (purchased from Cat. V49320, ThermoFisher Scientific) 15,16 . AdCoV2-S(GA) and AdSARS-S that are not related to this study are shown in the supplementary Figure 1. Ad-LacZ, which encodes the reporter gene bacterial β-galactosidase (LacZ), was also constructed as a control vector. The propagation of AdCoV2-S, AdCoV2-SdTM, and Ad-LacZ was performed in adherent 293A cells in the presence of 10% FBS 9 . Three days after Ad infection, cell pellets were harvested for freezing and thawing twice at -80°C for 30 min and 37°C for 1 min. The lysates were centrifuged at 3500 rpm for 15 min at room temperature (RT), and the supernatants that contained Ad were collected. Recombinant Ad was purified and concentrated using Vivapure adenoPACK 100RT (Satorius Stedin Biotech). The purified viral titer was determined using a modified standard plaque assay 9 . Cat. After 1 h of incubation, the membrane was washed three times with PBS-T, the Millipore ECL substrate (Millipore, Cat. WBKLS0500) was layered onto the membrane, and then the signal was detected using an Amersham Imager 600.

Neutralizing assay
One hundred microliters of serially diluted serum (reconstituted with preimmunized normal mouse serum to equal amounts of serum) was mixed with 200 TCID50/100 µl of SARS-CoV-2 and incubated for 2 h at 37°C. The mixture was then added to 2.4×10 4 Vero cells in a 96-well plate, and each dilution was repeated 11 four times. Vero cell cultures that had been treated with the same dose of virus without sera were used as a positive control. Five days later, the CPE of the cells was visualized by microscopy at the set point of the diluted-fold sera according to the calculation of the titer of neutralizing antibody possessed by the sera.
3-amine-9-ethylcarbazole (AEC; Sigma-Aldrich, Cat. AEC101-KT) substrate was added to react for cytokine-specific immunospot generation. The generated spots were scored using an immunospot counting reader (Immunospot, Cellular Technology Ltd.). The obtained number of spots was the substrate to the number of spots gained from the respective well without restimulation (medium only). The results are expressed as the number of cytokine-secreting cells per 5 × 10 5 splenocytes seeded in the initial well, and the assay limit is higher than 1 cytokine-secreting cell per 5 × 10 5 splenocytes.

Statistical analysis.
One-way ANOVA test was used to analyze the results from Fig. 2, 3B, 4, 5, 6B, 7, and 8. Two-way ANOVA test was used to analyze the results from Fig. 3A and 6A. The results were considered statistically significant when P<0.05. The symbols *, **, and ***are used to indicate P <0.05, P < 0.01, and P <0.001, respectively.

Expression of S and SdTM of SARS-CoV-2 in Ad-infected cells.
We had constructed an Ad5 genome that carried the full-length and transmembrane-deleted S genes of the SARS-CoV-2, AdCoV2-S and AdCoV2-SdTM (Fig. 1A). To detect the expression of spike protein in Ad-infected cells, the immunoblotting of the culture medium and lysates with anti-S antibody was performed. The bands located at ~185 kDa and ~182 kDa corresponded to S-and SdTM-expressed proteins, respectively, and some degraded S proteins were detected in the cytosolic fraction of the lysates (Fig. 1B and supplementary Fig. 1A).
S and SdTM were also detected in the membrane fraction of the lysate (  Fig. 1A, 1B, and 1C, respectively) were detected independently as sample controls. These results indicated that either intact S or SdTM could be expressed in the respective Ad vector-infected cells. In addition, the membrane-deleted S that formed as a secretory protein was expected.

Induction of SARS-CoV-2-specific antibody responses by AdCoV2 vaccines
To investigate the immunogenicity of AdCoV2-S and AdCoV2-SdTM via systemic or mucosal administration, we immunized i.n. or s.c. BALB/c mice with 1x10 7 pfu of AdCoV2s on days 1 and 14. The sera were serially collected at 2 weeks and 1, 2, and 3 months after the second immunization. The anti-SARS-CoV-2 IgG response and the binding titer in the sera were analyzed. The specific IgG antibody was significantly detected as early as 2 weeks (titer = approximately 12800~25600; Fig. 2A) and serum. The antibody titers were approximately 3200, 25600, 102400, and 25600 at 2 weeks and 1, 2, and 3 months, respectively, in the AdCoV2-SdTM-immunized i.n.
serum. Antibody titers of approximately 6400, 25600, 51200, and 25600 were detected in the AdCoV2-S-immunized s.c. serum at 2 weeks and 1, 2, and 3 months, respectively. No anti-SARS-CoV-2 IgG was detected at any of the time points in the Ad-LacZ-immunized serum (Fig. 2). In parallel, SARS-CoV-2-specific IgA was observed in the mucosal site of the vaginal wash (vw), which was collected from the mice immunized i.n. with AdCoV2-S at 2 weeks and 1 month after the second immunization, compared to the lower level of SARS-CoV-2-specific IgA found in the vw obtained from the AdCoV2-SdTM-immunized i.n. mice. The vw from AdCoV2-S s.c.
A higher or lower antigen-specific IgG1/IgG2a ratio in immunized sera that represented an immune response biased to Th2-mediated humoral or Th1-mediated cellular immunity, respectively, was reported 18 . SARS-CoV-2-specific IgG1 and IgG2a from all the AdCoV-2 vaccine sera were detected, in which the high to low sequence of IgG1 and IgG2a levels were AdCoV-2-S i.n. > AdCoV2-S s.c. > AdCoV2-SdTM-S i.n. (Fig. 3B). Interestingly, a higher IgG1 titer than that of IgG2a was observed in the i.n. groups administered AdCoV2-S and AdCoV2-SdTM, in contrast to the s.c. of AdCoV2-S, in which the IgG2a titer was higher than the titer of IgG1 (Fig. 3B).
Therefore, the IgG1/IgG2a ratio from individual samples was calculated, and we found that AdCoV2-S i.n. induced a higher ratio score than AdCoV2-S s.c., but AdCoV2-S i.n. was similar to AdCoV2-SdTM i.n. (Fig. 3C). This finding reveals that different immunization routes might affect the immune reaction, whereas i.n.
immunization favors the Th2 response, in contrast to Th1 responses, which are favored by s.c. immunization.

Induction of Th1/Th2-mediated cellular immunity by the AdCoV2 vaccine
Cellular immunity is important for providing protective efficacy during the development of SARS-CoV-2 vaccines 19 . To examine which type of cellular immunity was induced by AdCoV2 vaccines, splenocytes were isolated from the Ad-immunized spleen at 2 months after the boost, followed by in vitro restimulation with medium only or recombinant S from SARS-CoV-2, and then assayed for cytokines by ELISPOT. Ad-LacZ-immunized mice was observed (Fig. 4B). The IFN-γ/IL-4 ratio was also calculated, and the ratio obtained from the AdCoV2-S i.n. group was equal to that of the AdCoV2-S s.c. group (Fig. 4C), thereby indicating that balanced Th1/Th2 responses were activated and that there was no difference in the activity needed to induce S-specific cellular immunity in response to AdCoV2-S i.n. and AdCoV2-S s.c.
These results indicated that neutralizing antibodies against SARS-CoV-2 could be effectively induced and reached a peak at 3 months post vaccination with AdCoVs.

The increase in protective antibodies elicits a long-term pattern in
AdCoV2-immunized subjects. This finding is consistent with the SARS-CoV-2 binding activity results showing that AdCoV2-S possesses stronger immunogenicity than AdCoV-2-SdTM in inducing the anti-SARS-CoV-2 antibody response. In addition, the i.n. route is better than the s.c. route for AdCoV2 immunization.

Potency of AdCoV2 vaccine in protection against SARS-CoV-2 infection
A previous study showed that hamsters were an animal model for SARS-CoV and SARS-CoV-2 20 . Therefore, we assessed the protective effect of AdCoV2 vaccines 16 against SARS-CoV-2 in this animal model. Administration of AdCoV2-S or AdCoV2-SdTM via the i.n. route in hamsters following a challenge with live SARS-CoV-2 was performed. The animals were then sacrificed, and the pulmonary viral loads and lung tissue sections and histochemical staining were examined at days 3 and 6 post-viral challenge. On day 3 post-challenge, there was significant inhibition (10000 ~1000-fold reduction) of SARS-CoV-2 in the lungs of hamsters that were immunized with AdCoV2-S or AdCoV2-SdTM in comparison to Ad-LacZ-vaccinated tissues that contained high amounts of SARS-CoV-2 (Fig. 6A). Similarly, very little or no SARS-CoV-2 was detected in AdCoV2-S-and AdCoV2-SdTM-immunized lungs compared to some virus that was detected in Ad-LacZ-immunized lungs on day 6 post-challenge (Fig. 6B). Additionally, Ad-LacZ was not able to prevent SARS-CoV-2-induced weight loss, while AdCoV2-S and AdCoV2-SdTM could prevent severe weight loss in mice (Fig. 6C).
To evaluate the inhibition of SARS-CoV-2-induced lung inflammation and the safety of AdCoV2 vaccine administration in animals upon viral infection, we examined the lung pathogenesis of hamsters immunized with AdCoV2-S or AdCoV2-SdTM followed by SARS-CoV-2 infection. H/E-stained lung tissues from Ad-LacZ-immunized hamsters showed severe inflammation associated with lymphocyte infiltration among the alveoli, as expected. No or very mild inflammation was found in AdCoV-S-immunized tissues and mild to moderate inflammation was observed in AdCoV-SdTM-immunized tissues (Fig. 7A). We scored the degree of inflammation in the lungs of each visualized section. The sections from Ad-LacZ-immunized mice exhibited the highest score (average = 3.75) for inflammation in the lung sections. By contrast, the lowest inflammation score (average = 0.5) was observed in AdCoV-S-immunized tissues. A medium inflammation score (average = 3) was found in AdCoV-SdTM-immunized tissues (Fig. 7A). These pathologic results correlated with 17 the antibody response showing that AdCoV2-S i.n. was better than AdCoV2-SdTM i.n. at inducing and neutralizing antibodies. Additionally, AdCoV2-S was better than

AdCoV2-SdTM at inhibiting SARS-CoV-2-induced lung inflammation in hamsters.
To further examine the induction of lung inflammation by AdCoV2 vaccine immunization, DMEM medium (Mock) or AdCoV2-immunized mice were sacrificed, and individual BALFs were prepared on 1 week after the two-dose immunization for detecting the secretion of proinflammatory cytokines. IFN-γ was significantly induced in the BALF from AdCoV2-S in. and Ad-CoV2-SdTM i.n.-immunized groups but was not induced in the BALF from AdCoV2-S s.c. or Ad-LacZ i.n.. It indicates that i.n. delivery of AdCoV2 vaccine can induce Th1-biased responses in the lungs (Fig. 8A). IL-1, IL-2, and IL-4 ( Fig. 8B) were measured and all of them were not induced after Ad vaccines immunization. The amounts of these cytokines were detected as background level as Mock group, suggesting that s.c. and i.n. of AdCoV2 vaccine did not induce the proinflammatory cytokines and was safe during the immunization in animals. studies also showed that AdCoV2s elicit strong antibody response (eg., AdCoV2-S; Fig.   2) and active the Th1 and Th2-comparable cellular immunity (eg., IFN-γ and Il-4 release respectively, shown in Fig. 4, and IgG2a and IgG1 induction, shown in Fig. 4) in response to AdCoV2-S and AdCoV2-SdTM. Indeed, AdCoV2-SdTM induced a weaker neutralizing antibody response than AdCoV2-S (Fig. 5). The intact S protein expressed by AdCoV2-S-infected cells might highly mimic the prefusion form of S protein on the viral particle that could activate an adequate host antibody to recognize the natural virus. In other words, the structure could be changed after the deletion of the membrane domain from the S protein, which might induce inadequate antibodies and subsequently affect binding to the virus. Second, the virus-binding titer and neutralizing antibody titer in the serum after i.n. AdCoV2-S administration was higher than that the serum after s.c. AdCoV2-S administration ( Fig. 2 and Fig. 5, respectively). These findings indicated that mucosal administration intranasal vaccine of Ad-PR.8.HA was more potent than when administered epicutaneously 29 . However, the third event in this study reveals that AdCoV2-S administration via the i.n. or s.c. route, or AdCoV2-SdTM via the i.n. route, all 19 showed sufficient efficacy in preventing SARS-CoV-2 infection, as evidenced by the effect on hamsters that were immunized following challenge with SARS-CoV-2 (Fig.   6). This result demonstrated that not only neutralizing antibodies but also Th1/Th2-mediated cellular immunity contribute to inhibiting SARS-CoV-2 in the host.

Antibody-dependent enhancement (ADE) has been observed in coronaviruses
Several studies have shown that mucosal but not systemic immunization with an Ad vaccine induced long-term immunity against pathogens, such as the Ad vector-expressing glycoprotein B of herpes simplex virus type 2 (HSV-2) against HSV-2 30 . Long-lasting anti-respiratory syncytia virus (RSV) immunity was induced by a nasal Ad vector expressing the fusion protein Ad-RSV-F 31 . Our results also confirmed that the neutralizing antibody against SARS-CoV-2 induced by i.n. delivery of AdCoV2-S was even continuingly increased up to highest titer after three months of the second booster (Fig. 5).
Pre-existing anti-Ad host immunity is always an issue to track when assessing the efficacy of Ad-based vaccines. A high titer of anti-influenza HI antibody was induced in subjects who had a high pre-existing antibody titer to Ad by Ad-PR.8 HA vaccine immunization. This finding indicated that the Ad-PR.8. HA vaccine showed no correlation with pre-existing neutralizing antibodies to Ad and the potency of Ad vaccines in a human trial 29 . Nasal injection of an Ad5-expressing Ebola Zaire glycoprotein (Ad5-ZGP), which bypassed the influence of the pre-existing anti-Ad antibody, fully protected mice from a lethal challenge with Ebola 32 . The anti-Ad antibody did not influence either anti-RSV antibody induction by Ad-RSV-F via i.n.
immunization or the induction of protection against RSV challenge in mice 9 . Thus, intranasal delivery of the AdCoV2 vaccine has the advantage of expressing full protection against SARS-CoV-2.
There were clinical observations of COVID-19 patients who had severe inflammatory responses in the lungs corresponding to viral burdens in the pulmonary 20 epithelial cells and resulting in respiratory failure 33 . This finding also correlated with our result that pulmonary inflammation was recapitulated by Ad-LacZ immunization following a challenge with SARS-CoV-2 in animals. No or very mild inflammation in the lungs of mice that received AdCoV2-S was observed (Fig. 7). This consequence is corresponding to the Th1 cytokine and no proinflammatroy cytokine secretions in the lungs of mice which received AdCoV2-S i.n. (Fig. 8).
Collectively, this evidence indicates that the protective immunogenicity of   using IgG anti-recombinant intact S protein-immobilized ELISA. Anti-mouse IgG conjugated with HRP was used as the detection antibody. The titer of specific antibody in the serum was calculated as a fold dilution of the tested sera, whose detected value at OD450 nm of absorbance > 2 times the value at OD450 nm of absorbance obtained from the medium alone. A one-way ANOVA test was used.   Sera collected from AdCoV vaccine-immunized BALB/c mice at 14 days (A), 1 month (B), and 3 months (C) post-immunization as described in the legend of Fig. 2 were applied to the neutralizing assay, which is described in the Materials and Methods.
The experimental protocol was followed for the regulation of the RG-3 level during laboratory operation. One-way ANOVA test was used. post-infection, the hamsters were sacrificed and the lung tissues were homogenized for the TCID50 assay. One and two-way ANOVA test were used for statistical analysis.
(C) The body weight of AdCoV2-immunized hamsters followed by SARS-CoV-2 challenge was recorded. When Ad-LacZ i.n. compares with AdCoV2-S i.n., the symbols * are used to indicate P <0.05. When Ad-LacZ i.n. compares with AdCoV2-SdTM i.n., the symbols # are used to indicate P <0.05. Two-way ANOVA test were used for statistical analysis.