key: cord-0814313-pl294poq
authors: Cao, Huibi; Mai, Juntao; Zhou, Zhichang; Li, Zhijie; Duan, Rongqi; Watt, Jacqueline; Chen, Ziyan; Bandara, Ranmal Avinash; Li, Ming; Ahn, Sang Kyun; Boon, Betty; Christie, Natasha; Gray-Owen, Scott; Kozak, Rob; Mubareka, Samira; Rini, James M.; Hu, Jim; Liu, Jun
title: Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2
date: 2021-04-08
journal: bioRxiv
DOI: 10.1101/2021.04.08.439006
sha: 2f1be9ee13b7fd0bd9316089cfb8095ab8076c94
doc_id: 814313
cord_uid: pl294poq

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) 1 , is ongoing and has resulted in more than 120 million confirmed cases and at least 2.8 million deaths worldwide. The development of safe and effective vaccines against SARS-CoV-2 is a global health priority and a number of vaccine platforms have already been tested 2 . These include inactivated virus 3, 4 , naked DNA delivered by electroporation 5 , mRNA delivered by lipid nanoparticles [6] [7] [8] [9] , viral vectors such as nonreplicating adenovirus 10-17 , replication-competent vesicular stomatitis virus (VSV) 18 With the roll out of approved vaccines in many countries, several limitations of the firstgeneration of COVID-19 vaccines became increasingly apparent. The requirement of ultra-cold temperature for transportation and storage of the mRNA vaccines is a major limitation especially for global distribution in developing countries. In addition, all the approved vaccines are delivered by intramuscular injections, which may not be able to prevent the infection at the upper respiratory tract and stop viral shredding and transmission 26 .

Here, we investigate the performance of a COVID-19 vaccine developed in a novel platform, the helper-dependent adenoviral (HD-Ad) vector. HD-Ad, also known as gutless adenovirus, is the latest (third) generation of adenoviral vectors and has primarily been used for in vivo gene delivery in preclinical tests for the treatment of inherited genetic diseases 27, 28 .

HD-Ad was constructed with all the adenoviral coding sequences deleted and this eliminates the expression of unwanted adenoviral proteins 27, 29 . This characteristic minimizes the host immune response to the vector and allows for long-term expression of the transgene in host tissues or organs [30] [31] [32] [33] [34] . In addition, HD-Ad does not integrate into the host genome, thereby eliminating the risk of introducing chromosomal mutations. Besides its excellent safety profile, HD-Ad has a high cloning capacity (up to 36 kb) for transgenes, which makes it possible to deliver large genes or multiple genes. All these features make HD-Ad an attractive platform for vaccine construction and delivery. In this study, we delivered the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) to mice intranasally using an HD-Ad vector and examined its immunogenicity and protective efficacy.

The RBD sequence of SARS-CoV-2 was codon optimized (Table S1 ) and expressed from the chicken beta-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer, to increase the transcription, and the first intron of the human UbC gene to increase mRNA stability (Fig. 1A) . A DNA sequence encoding the 20-amino acid signal peptide of the human cystatin S protein was included upstream of the coding sequence of the RBD, which allows for RBD secretion. The BGH poly A tail was used to terminate the transcription.

To examine the expression and secretion of the RBD, epithelial cells (A549 and IB3) were transfected with HD-Ad_RBD at different dosages and cell lysates and culture supernatants were prepared and subjected to Western blot analysis. The result showed that the RBD was expressed at high levels in both cell lines and in a dose-dependent manner (Fig. 1B) .

Importantly, we estimated that about 90% of the RBD was detected in the culture supernatant, indicating that the majority of the RBD was secreted into the culture media. The secreted transgene product delivered by HD-Ad vectors is known to reach both airway fluid and the blood circulation system 28 , thus the secreted RBD is expected to reach antigen presenting cells, locally and systemically, to induce antigen-specific immune responses.

To examine the immune responses induced by HD-Ad_RBD, we intranasally immunized BALB/c mice (n=5) with three different doses of HD-Ad_RBD; three weeks later these mice were sacrificed and sera collected ( Fig. 2A) . ELISA analysis of the sera showed high levels of RBD-specific IgG in all three groups of mice immunized with HD-Ad_RBD, whereas low, if any, levels of IgG were detected in mice immunized with the HD-Ad control, which does not carry a transgene for protein expression (Fig. 2B) . Animals vaccinated with 10 8 , 5´10 9 and 10 10 HD-Ad_RBD viral particles had reciprocal geometric mean titers (GMT) of 30,314, 459,479, and 378,929, respectively. This result indicates a dose-dependent response and that 5´10 9 of HD-AD_RBD is the optimal dosage for vaccination.

We next tested the prime-boost intranasal vaccination regimen. Based on the results of the single dose vaccinations, we chose 10 8 and 5´10 9 HD-Ad_RBD viral particles for the prime immunizations, followed 3 weeks later by boost vaccinations at the same dosage ( Fig. 2A) . A lower boost dosage (10 8 viral particles) was also tested in the 5´10 9 -primed group. Three weeks after the second vaccination, the animals were sacrificed and sera and bronchoalveolar lavages (BALs) were collected.

Boosting with 10 8 HD-Ad_RBD viral particles increased the IgG titer ~1.5-fold relative to the single 10 8 vaccination, and the IgG reciprocal GMTs for the 10 8 prime-boost group, and the 5´10 9 -prime/10 8 -boost group were 50,476 and 688,862, respectively (Fig. 2C) . Remarkably, the IgG reciprocal GMT in the 5´10 9 prime-boost group increased 4-fold compared to that of the single vaccination, reaching 1,837,920 (Fig. 2C) .

High levels of RBD specific IgA were also detected in the sea of the boosted animals and the highest level was detected in the 5´10 9 prime-boost group, reaching a reciprocal GMT of 18,379 (Fig. 2D) .

We also detected high levels the RBD-specific IgG and IgA in BLAs. For the 5´10 9 primeboost group, the IgG and IgA reciprocal GMTs were 18,379 and 8,000, respectively ( Fig. 2E & F) .

Similarly, there was a dose-dependent increase in the neutralizing activity of the sera against the SARS-CoV-2 virus (Fig. 2G) . The reciprocal 50% inhibition dilution (ID50) GMTs of the neutralizing antibody in the 5´10 9 -prime/10 8 -boost group and the 5´10 9 prime-boost group were 378 and 948, respectively.

Finally, we also detected an increase of IFN-g producing CD4 + T cells in the lungs of animals vaccinated with 5´10 9 HD-Ad_RBD viral particles (prime and boost) compared to the control groups, indicating that the Th1 response was activated (Fig 2H) .

To examine the protective efficacy of HD-Ad_RBD, we intranasally immunized K18-hACE2 transgenic mice with HD-Ad_RBD (n=17) or the HD-Ad vector alone (sham control, n=18).

SARS-CoV-2 does not infect regular mice due to the lack of the right receptor. The K18-hACE2 transgenic mouse model was generated by McCray et al. 35 by expressing the human ACE2, the receptor for SARS-CoV-2, using the K18 gene expression cassette developed by one of our laboatories 36 . The hACE2 mice received a prime immunization of 5´10 9 viral particles of HD-Ad_RBD or HD-Ad at day 1. At day 21, the mice received a boost immunization of the same dose of the vaccine or the control. At day 21 after the second immunization, hACE2 mice were intranasally challenged with 10 5 50% tissue culture infectious dosage (TCID50) of SARS-CoV-2. At day 1, 3 and 5 post-infection, mice were euthanized and lungs, spleen and heart were harvested for viral burden and cytokine analysis (Fig. 3A) .

Notably, there were no detectable infectious virus in the lungs of 16 mice immunized with HD-Ad_RBD as determined by the TCID50 assay, whereas high levels of the infectious virus were detected in all mice (n=18) vaccinated with the vector control (Fig. 3B) . Only one mouse immunized with HD-Ad_RBD showed a detectable but low level of infectious virus, which may be an outlier due to improper vaccination. We noticed that mice sometimes sneezed out the vaccine solution during intranasal inoculation. Using the primers that are specific to the sequence of the SARS-CoV-2 E gene, we detected very high levels of viral RNA (10 8 to 10 9 copies/mg) in the lungs of mice vaccinated with the HD-Ad vector control. In contrast, the viral RNA levels were reduced by >4 log10 in 16 out of 17 mice vaccinated with HD-Ad_RBD (Fig. 3C) .

The very low RNA levels in the lungs of mice vaccinated with HD-AD_RBD may reflect the input and nonreplicating virus. This is consistent with two observations. First, comparing mice of the control group at day 1 and 3 post-infection, there was a significant increase (>1 log10) in both the infectious viral titer and the viral RNA copy number in the lungs at day 3 compared to day 1, indicating substantial viral replication in these mice ( Fig. 3B & C) . However, this trend was not observed in the mice vaccinated with HD-Ad_RBD as no infectious virus was detected in these mice and the viral RNA level remained constant at day 1 and 3 post-infection ( Fig. 3B & C) .

Second, similar low SARS-CoV-2 RNA levels (~10 4 copies/mg) were observed at these time points in BALB/c and C57BL/6 mice lacking hACE2 receptor expression, which represent the input, nonreplicating virus 37 .

Remarkably, intranasal delivery of HD-Ad_RBD provided effective protection of the upper respiratory tract as judged by the absence of measurable viral RNA in the oropharyngeal swabs (Fig. 3D) . Only one mouse vaccinated with HD-Ad_RBD showed a detectable level of viral RNA, which is the same abovementioned mouse that showed infection in the lungs. All mice in the control group exhibited high levels of viral RNA (10 6 copies/swab). We also detected no measurable or very low levels of viral RNA in the heart and spleen of mice vaccinated with HD-Ad_RBD ( Fig. 3E & F) .

The protection of HD-Ad_RBD vaccinated animals against SARS-CoV-2 is likely due to high neutralizing antibody levels. To test this, we collected the sera of these viral challenged animals and measured the neutralizing antibody levels. As expected, since the sera were collected shortly after the challenge by SARS-CoV-2 (day 1 to 5 post-infection), the control group mice did not have enough time to develop detectable levels of neutralizing antibody (Fig.   3G ). In animals vaccinated with HD-Ad_RBD, high levels of neutralizing antibody against SARS-CoV-2 were detected, with the reciprocal ID50 GMTs ranging from 328-640 (Fig. 3G) , which is in the same range of neutralizing antibody detected in the vaccinated but not challenged animals ( Fig. 2G) .

We next examined the effect of the vaccine on lung inflammation. The lung tissues at day 3 post-infection were chosen for this analysis and the mRNA levels of several proinflammatory cytokines and chemokines were measured and normalized against the same samples prepared from the naïve mice, which were mice that were vaccinated with the HD-Ad vector control but not challenged by SARS-CoV-2.

In the mice that were vaccinated with the HD-Ad vector control and challenged by SARS-CoV-2, there was a dramatic increase on the mRNA levels of IL-6, CXCL10, and CXCL11 (Fig. 4) .

Remarkably, in mice that were vaccinated with the HD-Ad_RBD and challenged by SARS-CoV-2, these proinflammatory cytokines and chemokines were reduced to the levels of naïve mice.

The mRNA levels of CXCL1, IFN-g, IL-1b and IL-11 were also significantly lower in the lung tissues of animals immunized with HD-Ad_RBD compared to the HD-AD control (Fig. 4) .

Taken together, our results demonstrate that immunization with HD-Ad_RBD decreases both viral infection and consequent inflammation in the lungs of animals infected with SARS-CoV-2.

Although several COVID-19 vaccines have been approved for emergency use in humans, Key to controlling COVID-19 will be the development of vaccines that provide long-term protection, a property requiring long-term T cell responses 44 . It is known that adenovirus-based vaccines can elicit strong T cell memory 45 and since our HD-Ad vaccine shares the same capsid proteins as regular adenovirus-based vaccines, we expect long-term T cell memory from our HD-Ad_RBD vaccine as well. Indeed, spleen cell samples from mice 6.5 months after a singledose (5´10 9 viral particles) vaccination with HD-Ad_RBD show high levels of antigen-specific IFN-g (~14,000 pg/ml), an indication that our vaccine elicits a strong and long-term T cell response.

In additional to its superior safety profile, HD-Ad also has a large cloning capacity (up to 36 kb) for transgenes and this makes it possible to deliver large genes or multiple genes. These features, together with the remarkable success of the HD-Ad_RBD vaccine demonstrated in this

The SARS-CoV-2 virus was isolated from local patients at Toronto in March 2020. All work with infectious SARS-CoV-2 was performed in the Containment Level 3 (CL-3) facilities at University of Toronto using appropriate protective equipment and procedures approved by the Institutional Biosafety Committee.

BALB/c and K18-hACE2 C57BL/6 mice were purchased from The Jackson Laboratory.

K18-hACE2 mice were bred in house and each mouse was genotyped before use.

All of the animal procedures were approved by the University of Toronto Animal Care 

A viral vector genome expressing a secreted form of the RBD of SARS-CoV-2 spike protein was constructed by multiple steps. We started with a plasmid, which is based on pBluscript containing the chicken beta actin gene (CBA) promoter and the ploy A signal from bovine growth gene (BGHpA), by inserting UbC gene intron 1 into the plasmid between CBA promoter and BGHpA by In-fusion cloning. To build a plasmid expressing RBD from the CBA promoter, we inserted the RBD sequence with the signal peptide of the human cystatin S gene at its 5' end after the UbC intron between the restriction sites, EcoRV and ApaI. Finally, we inserted the RBD-expression cassette as AscI fragment from the RBD expressing plasmid into viral vector, pC4HSU-NarD at AscI site, resulting in the new plasmid, pC4HSU-NarD-RBD for the vaccine production.

The vaccine production was carried out using 116 cells 46 . A helper-virus, NG163, was used to provide vector DNA replication and the production of viral capsid proteins. The packaging signal sequence in the helper-virus was flanked by two loxP sites. During vector production, the host cells expressed the Cre recombinase which cleaved off the packaging signal of the helper virus.

Thus, only HD-Ad vector particles were assembled. The large-scale production of HD-Ad vectors was carried out in suspension cells using 3L Bioreactors. The vector particles were harvested from the cell lysate and purified through two rounds of CsCl gradient centrifugation. 

Heat-inactivated serum was serial diluted (2-fold) in DMEM and incubated with 200 TCID50 of SARS-CoV-2 for 2 h at 37°C. For each dilution, there were six technical replicates. After the 2h neutralizing, the serum-virus mixture was then incubated with 20,000 Vero-E6 cells supplied with 2% FBS at 37°C. CPE of each well was examined at day 5. The highest dilution of serum that can protect 50% of cells from SARS-CoV-2 infection is considered as the neutralizing antibody titer, as described in 4 .

Six-to eight-week-old K18-hACE2 C57BL/6 mice (female and male at equal ratio) were intranasally immunized with 5 ´ 

The infectious virus number was determined by a cytopathogenic efficiency (CPE) assay. Vero-E6 cells (30, 000) were seeded into a 96-well plate one day before the inoculation. Collected tissues were weighed and homogenized with stainless steel beads (Qiagen, #69989) in 1 ml of DMEM with 2% FBS. Lung homogenates were centrifugated at 3,000 g for 5 min and the supernatants were collected. Serial 10-fold dilutions of the lung homogenates were then added into the monolayer Vero-E6 cells. For each dilution, there were six technical replicates. After 5 days of culture, the CPE of each well was examined, and the virus titer (TCID50) was calculated according to the Karber method 47 and normalized by the organ weight.

The viral RNA copy number was measured by one-step real-time quantitative PCR (qRT-PCR) as described in 48 

RNA of the lung homogenates was extracted and qRT-PCR were performed as mentioned above. Primers used in this experiment were listed in Table S2 . The mRNA level of cytokines and chemokines were normalized to GAPDH. Fold change was calculated using the 2 -ΔΔCq method by comparing SARS-CoV-2 infected mice to uninfected mice. 

HD-Ad an ideal platform for the construction of multivalent vaccines targeting SARS-CoV-2 and its emerging variants, work which is now underway in our laboratories

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and VS-1-17553138 (to JL, JH and JR).