key: cord-1054997-ymuscsrx
authors: Hasanpourghadi, Mohadeseh; Novikov, Mikhail; Ambrose, Robert; Chekaoui, Arezki; Newman, Dakota; Ding, Jianyi; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Liu, Qin; Swagata, Kar; Ertl, Hildegund CJ
title: Prime-boost vaccinations with two serologically distinct chimpanzee adenovirus vectors expressing SARS-CoV-2 spike or nucleocapsid tested in a hamster COVID-19 model
date: 2022-03-18
journal: bioRxiv
DOI: 10.1101/2022.03.17.484786
sha: dc6373cf5fb80b9883f91e7579471a3491cca76a
doc_id: 1054997
cord_uid: ymuscsrx

Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing the spike (S) or nucleocapsid (N) proteins of an early SARS-CoV-2 isolate were tested individually or as a mixture in a hamster COVID-19 challenge model. The N protein, which was expressed as a fusion protein within herpes simplex virus glycoprotein D (gD) stimulated antibodies and CD8+ T cells. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gDN fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable that the S protein may potentiate and prolong protection achieved by the currently used genetic COVID-19 vaccines.

SARS-CoV-2 was first detected in humans towards the end of 2019. Since then, it has infected over 400 million humans and killed nearly 6 million. Although vaccines that are highly efficacious against disease caused by the initial isolates have been available for over a year, the virus is constantly evolving and one of the latest variants, called omicron, in part evades vaccine-induced virus neutralizing antibodies (VNAs) [1] .

Vaccines that are currently available are either genetic vaccines in form of mRNA vaccines [2, 3] , Ad vector vaccines [4] [5] [6] , or they are based on protein vaccines [7] or inactivated viral vaccines [8] .

Vaccines but for those using inactivated virus express only the SARS-CoV-2 S protein, which is the target antigen for VNAs [9] that were shown by passive transfer experiments to protect against an infection [10] . Two doses of mRNA vaccines were found to be initially over 90% effective in preventing symptomatic disease [2, 3] . The Ad vector-based Sputnik V vaccine given twice in a heterologous prime boost regimen is equally protective 6 while the single dose Ad vector vaccine from Johnson and Johnson (J&J) [4] and the AdC vector vaccine from AstraZeneca that uses the same vector for priming and boosting protect 63-67% of individuals against disease [5] , which is similar to the level of protection achieved with the protein vaccine 7 or the inactivated viral vaccines [8] . As has been studied most extensively with mRNA vaccines efficacy is not sustained necessitating booster immunizations after 6 months [11, 12] . Notwithstanding, even fully vaccinated individuals experience breakthrough infections [13, 14] especially with omicron [15] and although their symptoms are in general mild or nonexistent, they can spread the virus to others. SARS-CoV-2 will continue to evolve and infections of vaccinated or previously infected individuals will favor outgrowth of mutants that may escape vaccine-induced VNAs even further.

T cells induced by natural infections with common cold coronaviruses can cross-react with SARS-CoV-2 antigens [16] and they have been implicated to provide protection to so-called 'never COVID' individuals, who despite close contacts with SARS-CoV-2-infected individuals remain uninfected [17] . The N protein is one of the most abundantly expressed viral antigen in SARS-CoV-2 infected cells, which should make it an easy target for specific T cells. Antibodies to the N protein may also contribute to viral clearance through antibody dependent cellular cytotoxicity (ADCC) or complement-mediated lysis. N proteins potential role as a vaccine antigen to induce sustained and cross-reactive immunity to SARS-CoV-2 warrants further investigations. This was the goal of our study, which tested in a hamster challenge model, vaccines that not only express the S protein for induction of VNAs but also the more conserved N protein. We expressed each of the two SARS-CoV-2 proteins by two serologically distinct replication-defective AdC vectors of serotype SAdV-26, also called AdC6, and SAdV-24, also called AdC7 [18, 19] . Inserts were based on unmodified genes from an early viral isolate from Sweden, called SARS-CoV-2/human/SWE/01/2020 (AdC-SSWE, GenBank number: QIC53204), whose S and N protein sequences are identical to those of the original Wuhan isolate. The N protein was expressed as a fusion protein within herpes simplex virus gD, which through inhibition of the early B and T cell attenuator (BTLA) -herpes virus entry mediator (HVEM) checkpoint broadens SARS-CoV-2 N protein-specific CD8 + T cell responses [20, 21] and augments B cell responses [22] . Vaccines were given in a heterologous prime boost regimen either individually or as mixtures of the same vector backbones expressing the two different inserts. Our data show that vaccines that express the gD-N fusion protein induce N-protein-specific antibody and CD8 + T cell responses but provide robust protection against infection or disease. As expected, the S protein-expressing vaccines induce VNAs and reduce disease after SARS-CoV-2 challenge but fail to prevent infection. Best protection was achieved with the vaccine mixtures indicating that although N protein-specific immune mechanisms by themselves fail to lessen disease upon challenge with a high dose of SARS-CoV-2 they enhance protection mediated by S protein-immunity.

Sixteen female and sixteen male Syrian golden hamsters were enrolled and separated into 4 groups of 8 animals each (4 females, 4 males/group). The groups were primed by intramuscular (i.m.) injections as follows: group 1 was injected with 1 ´ 10 10 virus particle (vp) of AdC7-gDN, group 2 with 1 x10 10 vp of AdC7-S, group 3 with a mixture of 0.5 ´ 10 10 vp AdC7-gDN and 0.5 ´ 10 10 AdC6-S and group 4 with 1 ´ 10 10 vp of the control vector, called AdC7-HBV2, which expresses a sequence derived from hepatitis B virus. We only used half of the vaccine dose for each component of the mixture to keep the total amount of vector given to each animal constant, as in a clinical setting higher vaccine doses translate to increases in adverse events driven by innate responses to the Ad vector [23] . Animals were boosted 2 months after priming with AdC6 vectors expressing the same inserts and used at the same doses.

Hamsters were bled at baseline, 14 days after the prime and 14 days after the boost to determine SARS-CoV-2 S protein-specific antibody responses in groups 2-4. N-specific antibody and CD8 + T cell responses in groups 1, 3, and 4 were measured from blood 14 days after the boost ( Figure 1A ). Animals were challenged intranasally with SARS-CoV-2 4 weeks after the boost. After challenge animals were checked daily for clinical symptoms and weight loss. Oral swaps were collected on days 2, 4, 7, and 14 after viral challenge to determine levels of viral genomic and sub-genomic (sg)RNA. Four animals in each group were euthanized 4 days and the others 14 days after challenge. After euthanasia, lung viral titers were determined, and lung sections were screened for pathology.

Sera from hamsters of groups 2, 3, and 4 harvested at baseline and at 2 weeks after the prime or boost were tested by ELISA on plates coated with S1 and S2 proteins, which measures all S-binding antibodies. They were also tested in a neutralization assays using vesicular stomatitis virus (VSV) vectors pseudo-typed with SARS-CoV-2 S protein to detect VNAs, which can either be directed to the RBD sequence of S1 or the fusion peptide in S2. Sera harvested 2 weeks after the boost were in addition tested by an ELISA, which measures antibodies specific to the receptor binding domain (RBD) of S1 ( Figure 1A ). All pre-immunization samples and samples from the control groups harvested at either time point scored negative in either one of these assays. All but one of the sera from one hamster of groups 2 scored positive by the S1/S2-specific ELISA by 2 weeks after the prime. All hamsters seroconverted after the boost and overall responses showed significant increases ( Figure 1B ). More specifically geometric mean (GM) titers of group 2 females increased 1.6-fold, while those of males increased 6.8-fold; GM titers of group 3 females increased 1.5-fold while those of males increased 1.9-fold. There were no significant differences in responses of males compared to females although in group 2 males tended to have higher responses after the boost than females. All hamsters developed antibodies that neutralized the pseudotyped VSV-S virus with GM titers of 109 in group 2 and 167 in group 3 after the prime which changed to 166 and 365 after the boost, respectively. The superior booster response in group 3 was driven by female hamsters, which showed a 4-fold increase in VNA titers compared to the marginal 1.2fold increase in males. After the boost all animals scored positive in the RBD-specific ELISA ( Figure 1D) and there were no significant differences between males and females or groups 2 and 3.

VNAs were tested for reactivity against alpha, beta, delta, and omicron variants. Sera collected after the prime ( Figure 1E ) or after the boost ( Figure 1F ) showed significantly reduced titers comparing neutralization of VSV-S vectors expressing wild-type S protein to those with S proteins of variants.

Sera from hamsters of groups 1, 3, and 4 harvested after the boost were tested for antibodies to the N protein. All AdC-gDN vaccinated animals scored positive with no differences between groups 1 and 3 or males and females hamsters ( Figure 1G) .

Peripheral blood lymphocytes were tested two weeks after the boost for production of IFN-g by CD8 + T cells in response to three peptide pools representing the N protein sequence (Supplemental Table 1 ).

Most animals of groups 1 and 3 developed N protein-specific CD8 + T cell responses; 1 and 2 females of groups 1 and 3, respectively and 1 male of group 3 failed to respond ( Figure 1F ). Responses showed no preference to any of the 3 peptide pools representing different segments of the N protein.

Four weeks after the boost animals were challenged intranasally with SARS-CoV-2 ( Figure 2A ). Animals were scored twice daily for clinical symptoms. Most animals developed benign symptoms after challenge in form of mildly ruffled fur, hunched over posture and/or closed or squinted eyes. None of the vaccinated mice exceeded a COVID-19 disease score of 1 and only one female in the control group 4 was given a disease score of 2 on days 5-7 after challenge. One male animal in the control group died 7 days after challenge although it never exceeded a disease score of 1. His lung pathology suggested that his death was most likely caused by SARS-CoV-2.

Plotting days of clinical symptoms against days after challenge showed that most female animals developed symptoms by day 4 after challenge but for one group 3 animal, which became symptomatic on day 5. All males of group 4 and most of the males of the vaccine groups had symptoms by day 3 and by day 4 all male animals had visible signs of disease. Thereafter the disease course was markedly different for vaccinated female and male animals as has also been reported for humans [24] . In both gender groups unvaccinated hamsters exhibited symptoms till the day of their euthanasia. The same was seen for all males of the vaccine groups. With some fluctuations half of the females of group 1 and 2 remained symptomatic throughout the observation period while group 3 females became and remained symptom-free by days 5 or 6 ( Figure 2B ). Although this result is based on small numbers of animals is nevertheless suggests that vaccinated females recover more rapidly than vaccinated males upon breakthrough infections and that the combination vaccine compared to the individual vaccines accelerated recovery in females.

These data were mirrored by levels of weight loss. Control animals rapidly started losing weight after challenge ( Figure 2C ) with maximal weight loss around days 6-7 ( Figure 2D ). Group 1 animals which had only received AdC-gDN vectors lost weight with similar kinetics to the control group although their maximal weight loss by days 6 and 7 after infection tended to be lower. Hamsters of groups 2 and 3 lost only a minimal amount of weight early after challenge and started gaining weight by day 2 or 3 after challenge. On days 4 and 14 after challenge groups 2 and 3 showed significantly less weight loss compared to the control group 4; group 3 showed a significant difference to group 1 at both time points while group 2 only differed from group 1 by day 4 after infection. Females recovered their weight 1-2 days earlier than males (Suppl. Figure 1A) .

Viral titers were determined from oral swabs collected on days 2, 4 (8 animals for each group), 7 and 14 (4 animals for each group) ( Figure 3A ) and lung tissues collected from 4 animals of each group either on days 4 or 14 after challenge. Samples were screened for viral RNA and sgRNA, the latter has been suggested to determine titers of infectious virus more accurately [25] . Immunization with the vaccine mixture reduced oral viral RNA ( Figure 3A ) and sgRNA ( Figure 3B ) titers on day 2 after infection while immunization with the Ad-S vaccine only reduced sgRNA titers but not RNA titers on day 2. No significant differences were observed for the group 1 or the other time points. Vaccination with either the AdC-S or the AdC-S + AdC-gDN regimens reduced lung viral RNA titers ( Figure 3C ) tested on day 4 after challenge while AdC-S vaccination also reduced sgRNA titers at that time point ( Figure 3D ). By day 14 viral loads in lungs were markedly reduced in all groups and although group 3 continued to show lower RNA and sgRNA titers compared to the others this failed to reach significance.

Parts of the lungs were sectioned and stained after euthanasia for 4 animals of each group on day 4 and the remaining animals on day 14 after challenge and analyzed for pathological changes ( Figure 3E ).

Macroscopically lungs showed red or dark red patches, dark spotted, mottled/red lobes in groups 1, 2, and 4 but not 3 (Suppl. Figure 1B ). Tissue lesions were graded microscopically and lungs from most animals showed grade 1-3 histopathology. Especially on day 4 lungs showed perivascular edema, alveolar hemorrhage, bronchiolo-alveolar hyperplasia, mixed or mononuclear cell inflammation (bronchoalveolar, alveolar, and/or interstitial), mononuclear vascular/ perivascular inflammation, mesothelial hypertrophy, and/or pleural fibrosis ( Figure 3E ,F). In groups 2 and 3, lesions were milder and more multifocal than in groups 1 and 4 on day 4. The sum of the score for the individual symptoms showed on day 4 significant differences between groups 2 and 3 compared to the control animals of group 4. On day 14 lungs of the control animals continued to show marked pathology while group 3 animal lungs no longer showed any lesions; group 2 animals showed residual mild disease. Group 1 animals, which only recieved the AdC-gDN vaccine, also showed reduced pathology compared to group 4 animals ( Figure 3E ). Lung pathology showed gender specific differences ( Figure 3F ). By day 4 after infection the two group 3 females only showed grade 1 hyperplasia while the 2 males of this group had additional lesions. By day 14 both females of the control group showed a lessening of symptoms, which was not observed in the surviving male.

We analyzed the data for correlations by Spearman separately for hamsters that were tested for S protein specific antibody responses (groups 2 and 3) and/or N protein specific T and B cell responses (group 1, 3, and 4). T cell responses inversely correlated with oral RNA and sgRNA titers on day 2 (p = 0.037/0.02) while S protein specific antibody by the S1/S2 ELISA after the boost showed inverse correlations for oral viral RNA or sgRNA titers on day 4 (ELISA: p = 0.049/0.029). For other parameters such as relative weight after challenge, lung pathology or lung viral titers we analyzed animals that were euthanized on day 4 separately from those that were euthanized on day 14 again keeping the T and B cell groups separate. These analyses showed as expected direct correlations between lung viral loads and lung pathology. We did not observe significant correlations between T cell responses and any of the parameters of disease. In the early euthanasia group VNA titers after the boost inversely correlated with RNA and sgRNA lung viral titers on day 4 (RNA p = 0.01, sgRNA p = 0.006) while the late euthanasia group showed antibody responses by S1/S2 ELISA after the boost inversely correlated with oral viral RNA or sgRNA titers on day 4 (p = 0.01 for both). The most pronounced correlations were seen for antibody responses to the N proteins in groups 1, 3, and 4. N protein specific antibody responses showed for the analyses with all animals and the day 4 or 14 euthanasia group animals strong positive correlations with relative weight and for the former two analyses strong inverse correlations with RNA and sgRNA titers in saliva on day 4. The day 14 euthanasia animals also showed strong inverse correlations between N protein specific antibody titers and sgRNA and RNA titers in saliva on days 2 and 4, lung sgRNA titers and lung pathology ( Figure 4 ).

Our results show that vaccine induced S protein-specific immune responses reduce COVID-19 disease after SARS-CoV-2 infection and that addition of vaccines expressing the N protein has benefits. The S protein of SARS-CoV-2 has undergone extensive mutations, some of which increase viral transmission [26, 27] and affect neutralization by vaccine-induced antibodies [28] . Both the S and N protein have numerous MHC class I and II epitopes for recognition by CD8 + and CD4 + T cells (Suppl. Figs. 2-4) and such sequences may be more conserved than those of VNA binding epitopes. Using epitope prediction software (http://tools.iedb.org/main/tcell/), we analyzed HLA class I and II epitopes present within the SARS-CoV-2 S and N protein sequences and how these epitopes have been affected by mutations within variants. For HLA class I epitopes we used a cut-off score of 0.8 while for HLA class II epitopes a cut-off rank of ≤ 1 was used. The S protein is very rich in HLA class I epitopes and about 44% of the sequence can be recognized by CD8 + T cells from individuals with different HLAs while 24% of the sequence are covered by HLA class II epitopes. T cell epitopes are less abundant in the shorter N protein; 36% and 11% of the sequence scored as potential HLA class I or II epitopes respectively ( Figure   5A ). Of the mutations within the alpha or beta variants only a modest percentage affected HLA class I epitopes while within delta and omicron mutations were present in 38% or 48% of such epitopes. HLA class II epitopes within the S protein were less affected and showed mutations rates between 13-27% for the different variants. (Figure 5B ). Most of the epitope mutations within the variants resulted in a reduction or even loss of predicted binding to their corresponding restriction elements. Only one of the mutations within N present in the alpha variant affected an HLA class I epitope; this mutation resulted in increased HLA class I binding to the corresponding peptide ( Figure 5C ). HLA class II epitopes of N were not modified by any of the mutations ( Figure 5D ).

Several linear epitopes have been identified for SARS-CoV-2 S and N proteins [29] . Of the 4 within the S protein 3 are mutated in the variants; the 1 st epitope that stretches from amino acids 200-220 is mutated in all of the analyzed variants. A 2nd epitope from amino acids 544 to 562 is mutated in the alpha variant and the 3 rd epitope from amino acids 569 to 603 is mutated within omicron. The single linear epitope within the N protein is conserved between the variants.

In a remarkable achievement, SARS-CoV-2 vaccines were developed and approved for use in humans within less than a year after onset of the COVID-19 pandemic [2] [3] [4] [5] [6] [7] [8] . Nevertheless, even in countries with easy access to the vaccines the pandemic has been a roller coaster ride with cycles of reductions in cases followed by rapid increases due to the emergence and spread of more transmissible variants. This constant up and down in caseloads shows how fragile our control over SARS-CoV-2 remains. Available COVID-19 vaccines protect well against disease but largely fail to prevent infections, which allows for spread of the virus even in populations with high vaccine coverage [30] . In addition, protection induced by mRNA vaccines, one of the most widely distributed types of COVID-19 vaccines, wanes after a few months. Resistance to COVID-19 disease can be restored at least temporarily by a booster immunization but after a third dose of an RNA vaccine [31] , protection declines after four months [32] , and it is currently unknown if frequent boosts with such genetic vaccines within short intervals will continue to restore antibody titers. Long-lived plasma cells can maintain antibody responses often for years after vaccination or infection. Induction of long-lived plasma cells required T cell help within germinal centers and their maintenance requires continuous survival signals in specialized niches of the bone marrow or secondary lymphatic tissues [33] . Although COVID-19 mRNA vaccines were shown to result in germinal center formation [34, 35] , available evidence including the phenotypes of the vaccine-induced B cells [35] and the rapid declines in vaccine-induced antibody titers suggest that COVID-19 mRNA vaccines induce primarily short-rather than long-lived plasma cells [36] . This combined with the unceasing emergence of new variants that more and more escape vaccine-induced VNAs necessitates the development of second generation COVID-19 vaccines that induce sustained protection against a broad spectrum of current and future variants. SARS-CoV-2 has thus far mainly accumulated mutations within the S protein. As other viral proteins are less variable, we decided to explore vaccines that expressed the N protein in addition to the S protein vaccine. The N protein is a target for non-neutralizing antibodies that might play a role in combating a SARS-CoV-2 infection [37] . The N protein also induces CD8 + T cells, that by rapidly killing cells early after their infection, have also been implicated in contributing to protection against severe COVID-19 or to improve resistance in individuals with suboptimal VNA titers [38] [39] [40] .

For the hamster challenge study, we used two AdC vectors that belong to distinct serotypes to prevent that Ad capsid-specific antibody responses induced by the prime neutralized the Ad vector used for the boost. Humans only rarely have VNAs to AdC viruses and those who are seropositive have low titers [41] . VNAs to Ad viruses, directed mainly against the hypervariable loops of the hexon protein [42] , can prevent infection of cells with Ad vector vaccines and thereby expression of the transgene product. This in turn reduces the antigenic load and blunts immune responses to the vaccine antigen, which can be problematic for human serotype Ad vectors or for the repeated use of the same Ad vector [43, 44] . The AdC vectors for the S protein carry the full-length unmodified gene from an early SARS-CoV-2 isolate and as we showed earlier induce after a boost sustained antibody responses in mice [44] . These data were recapitulated in hamsters which developed binding as well as neutralizing antibodies after vaccination with the AdC-S vectors. A booster effect was seen for binding antibodies for groups 2 and 3 while VNA responses only increased in group 3 mice that received the AdC-S/AdC-gDN vector mixtures.

Whether this reflects increased T help due to responses to the N protein or a difference in vector dosing with group 2 hamsters receiving double the dose of the AdC-S vector is unknown. As expected, and reported by others, VNAs cross-reacted albeit with significantly reduced activity against viral variants of concern [45] [46] [47] .

The N protein is expressed by the AdC vectors as a fusion protein within HSV-gD, which as an inhibitor of the early BTLA-HVEM T cell checkpoint enhances B cell responses and broadens CD8 + T cell responses as we showed with several antigens including the SARS-CoV-2 N protein in mice [20, 21, 38] .

Expressing the N protein by an AdC vector within a heterologous viral protein that is expressed on the surface of an infected cells may promote B cell responses to the N protein's linear epitopes [29] but will likely distort the proteins' structure and destroy conformation-dependent epitopes. Nevertheless, we obtained a strong N protein specific antibody response after the boost in hamsters that received the AdC-gDN vaccines.

The N protein as expressed by the AdC vectors induces potent and broad CD8 + T cell responses in mice [38] . Responses are readily detectable but modest in hamsters, which may in part relate to the paucity of hamster-specific antibodies to T cell determinants and key cytokines, which only allowed us to test for circulating CD8 + lymphocytes producing IFN-g in response to the N peptide pools.

Hamsters were challenged after vaccination to assess if the vaccines protected against disease or infection. It is noteworthy that mirroring humans male hamsters were more susceptible to severe disease than female hamsters -one male hamster in the control group died and all male hamsters regardless of infected but they also transmitted the virus to unvaccinated hamsters that were placed into their cages as of day 4 after challenge (data not shown). Lung pathology mirrored lung sgRNA titers and again females had less lung disease compared to males and group 3 animals tended to recover faster than group 2 animals while group 1 animals showed no difference compared to control animals although addition of the AdC-gDN vaccine reduced disease and accelerated recovery in animals that were also vaccinated with the AdC-S vaccine. By itself the AdC-gDN vaccine only induced marginal protection, which contrasts data from another group; they reported T cell-mediated protection against COVID-19 disease and a reduction in viral loads upon intravenous immunization of hamsters with a human serotype 5 Ad vector expressing the N protein [48] . Differences in results may reflect the distinct immunization protocols or differences in the challenge.

In our study the contribution of the AdC-gDN vaccine to protection induced by the AdC-S vaccines was not solely mediated by T cells, which have the advantage that upon antigen-driven activation they persist for very long periods of time [49] , which would likely extend duration of protection well beyond the 4-6 months that current vaccines offer. Antibodies to the N protein strong positive correlations with relative weight of the animals and inverse correlations with disease parameters such as viral loads and lung pathology in the late euthanasia group indicating that the N protein-specific antibodies contribute to accelerated recovery from the infection.

Although available vaccines still avert serious COVID-19 disease and death, considering that we are entering the 3 rd year of the pandemic and the near certainty that additional variants with even more mutations that escape protection induced by current vaccines will evolve, new vaccines expressing additional SARS-CoV-2 antigens, such as the N protein to augment VNA mediated protection through additional immune mechanisms should be explored.

supplemented with 5% FBS and antibiotics. A549-hACE-TMPRSS2 (A549A/T) cells (InvivoGen, cat no. a549-hace2tpsa) were maintained in DMEM with 10%FBS, 300µg/ml of hygromycin, 0.5 µg/ml of puromycin and antibiotics. All cells were maintained at 37 o C in in a 5% CO2 incubator. Hamster challenge: Prior to challenge, hamsters were anesthetized with 80 mg/kg Ketamine and 5 mg/kg xylazine given i.m. The animals were challenged with 6 ´ 10 3 PFU (Vero76 cells) in 100 μL per animal (50 μl/nostril). Administration of virus was conducted as follows: Using a calibrated P200 pipettor, 50 μl of the viral inoculum was administered dropwise into each nostril. Anesthetized animals were held upright such that the nostrils of the hamsters were pointing towards the ceiling. The tip of the pipette was placed into the first nostril and virus inoculum was slowly injected into the nasal passage, and then removed. This was repeated for the second nostril. The animal's head was tilted back for about 20 seconds and then the animal was returned to its housing unit. ~20 min post challenged antisedan (0.04 ml, 1mg/kg) was given i.m. to all hamsters.

ELISA for anti-S1 and anti-S2 antibodies: Sera from individual hamsters were tested for S-specific antibodies by ELISA on plates coated overnight with 100 µl of a mixture of S1 and S2 (Native Antigen Company, Kidlington, UK) each diluted to 1 µg/ml in bicarbonate buffer. The next day plates were For the neutralization assay a dose of VSV-S vector that infected ~20-40 cells/well was selected.

A549A/T cells were plated into Terasaki plate wells as described above. The following day, sera were serially diluted in DMEM with 10% FBS. The VSV-S vector was diluted in medium containing 1 µg/ml of a mouse monoclonal antibody against VSV glycoprotein (sc-365019, Santa Crusz Biotechnology, Dallas, TX) and then incubated with the serum dilutions for 90 min at room temperature under gentle agitation starting with a serum dilution of 1 in 40. 5 µl aliquots of the mixtures were transferred onto Terasaki plate wells with A549A/T cells. Each serum dilution was tested in duplicates, an additional 4-6 wells were treated with VSV-S that had been incubated with medium rather than serum. Plates were incubated for 48 hours and then numbers of fluorescent cells were counted. Titers were set as the last serum dilution that reduced numbers of green-fluorescent cells by at least 50%.

ELISA for N protein-specific antibodies: Sera were tested for antibodies to the N protein by an ELISA on plates coated with 1 µg of a purified N protein (Abbexa, abx163973) using the same procedures as for antibodies to the S protein. A monoclonal antibody to N protein (Abbexa, MCA6373, lot# 156962) served as a positive control (data not shown). Genomic mRNA PCR Assay: The qRT-PCR assay utilizes primers and a probe specifically designed to bind to a conserved region of N gene of SARS-CoV-2. The signal is compared to a known standard curve and calculated to give copies per mL. For the qRT-PCR assay, viral RNA was first isolated from oral swab using the Qiagen MinElute virus spin kit (cat. no. 57704). For tissues it was extracted with RNA-STAT 60 (Tel-test"B")/ chloroform, precipitated and resuspended in RNAse-free water. To generate a control for the amplification reaction, RNA was isolated from the applicable SARS-CoV-2 stock using the same procedure. The amount of RNA was determined from an O.D. reading at 260, using the estimate that 1.0 OD at A260 equals 40 µg/mL of RNA. With the number of bases known and the average base of RNA weighing 340.5 g/mole, the number of copies was then calculated, and the control diluted accordingly. A final dilution of 10 8 copies per 3 µl was then divided into single use aliquots of 10 µl. For the master mix preparation, 2.5 mL of 2X buffer containing Taq-polymerase, obtained from the TaqMan RT-PCR kit (Bioline cat# BIO-78005), was added to a 15 mL tube. From the kit, 50 µl of the RT and 100 µl of RNAse inhibitor was added. The primer pair at 2 µM concentration was then added in a volume of 1.5 ml. Lastly, 0.5 ml of water and 350 µl of the probe at a concentration of 2 µM were added and the tube vortexed. For the reactions, 45 µl of the master mix and 5 µl of the sample RNA was added to the wells of a 96-well plate. The plates were sealed with a plastic sheet. All samples were tested in triplicate.

For control curve preparation, samples of the control RNA were prepared to contain 10 6 to 10 7 copies per 3 µL. Eight (8) 10-fold serial dilutions of control RNA were prepared using RNAse-free water by adding 5 µl of the control to 45 µl of water and repeating this for 7 dilutions resulting in a standard curve with a range of 1 to 10 7 copies/reaction. Duplicate samples of each dilution were prepared as described above.

If the copy number exceeded the upper detection limit, the sample was diluted as needed. For amplification, the plate was placed in an Applied Biosystems 7500 Sequence detector and amplified using the following program: 48ºC for 30 minutes, 95ºC for 10 min followed by 40 sheet. All samples were tested in triplicate. For control curve preparation, the control viral RNA was prepared to contain 10 6 to 10 7 copies per 3 µl. Seven 10-fold serial dilutions of control RNA were prepared using RNAse-free water by adding 5 µl of the control to 45 µl of water and repeating this for 7 dilutions.

This gives a standard curve with a range of 1 to 10 7 copies/reaction. The sub-genomic-N uses a known plasmid for its curve. Duplicate samples of each dilution were prepared as described above. If the copy number exceeded the upper detection limit, the sample was diluted as needed. For amplification, the plate was placed in an Applied Biosystems 7500 Sequence detector and amplified using the following program: 

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Linear B-cell epitopes in the spike and nucleocapsid proteins as markers of SARS-CoV-2 exposure and disease severity

Effect of COVID-19 vaccination on transmission of Alpha and Delta variants

Association of a Third Dose of BNT162b2 Vaccine With Incidence of SARS-CoV-2 Infection Among Health Care Workers in Israel

Effectiveness of a third dose of mRNA vaccines against COVID-19-associated emergency department and urgent care encounters and hospitalizations among adults during periods of delta and omicron variant predominance -VISION Network, 10 States

Survival of long-lived plasma cells (LLPC): piecing together the puzzle

Germinal centre-driven maturation of B cell response to mRNA vaccination

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses

mRNA COVID-19 vaccines and long-lived plasma cells: a complicated relationship

Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Hyperimmune Immunoglobulin Demonstrates Potent Neutralization and Antibody-Dependent Cellular Cytotoxicity and Phagocytosis Through N and S Proteins

T cell responses to adenoviral vectors expressing the SARS-CoV-2 nucleoprotein

CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer

CD8 + T cells specific for conserved coronavirus epitopes correlate with milder disease in patients with COVID-19

Adenovirus-based vaccines: Comparison of vectors from three species of adenoviridae

Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues

Adenovirus Serotype 5 Neutralizing Antibodies Target both Hexon and Fiber following Vaccination and Natural Infection

Novel, chimpanzee serotype 68-based adenoviral vaccine carrier for induction of antibodies to a transgene product

Pre-clinical testing of two serologically distinct chimpanzee-origin adenovirus vectors expressing spike of SARS-CoV-2. Immunology

Broad anti-SARS-CoV-2 antibody immunity induced by heterologous ChAdOx1/mRNA-1273 vaccination

Distinct homologous and variant-specific memory B-cell and antibody response over time after severe acute respiratory syndrome coronavirus 2 messenger RNA vaccination

Cutting Edge: Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity

Human T cell memory: A dynamic view

An efficient method of directly cloning chimpanzee adenovirus as a vaccine vector

Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

This work was funded by grants from The G. Harold and Leila Y. Mathers Charitable Foundation, the Commonwealth of Pennsylvania, and the Wistar Science Discovery Fund. MH was the recipient of fellowship from Fellowship from Janssen Scientific Affairs. Support for Shared Resources utilized in this study was provided by Cancer Center Support Grant (CCSG) P30CA010815 to The Wistar Institute. We wish to thank Dr. Elledge (Massachusetts General Hospital, Boston, MA) for providing the cDNA sequences for N and S of SARS-CoV-2.

HCJE holds equity in Virion Therapeutics. She serves as a Consultant to several Gene Therapy companies.