key: cord-0427859-ird9fgxr
authors: Eldi, Preethi; Cooper, Tamara H.; Prow, Natalie A.; Liu, Liang; Heinemann, Gary K.; Zhang, Voueleng J.; Trinidad, Abigail D.; Guzman-Genuino, Ruth Marian; Wulff, Peter; Hobbs, Leanne M.; Diener, Kerrilyn R.; Hayball, John D.
title: The vaccinia-based Sementis Copenhagen Vector COVID-19 vaccine induces broad and durable cellular and humoral immune responses
date: 2021-09-07
journal: bioRxiv
DOI: 10.1101/2021.09.06.459206
sha: f93b02b21efa8896704bd0ba820d8f3747d2d42b
doc_id: 427859
cord_uid: ird9fgxr

The ongoing COVID-19 pandemic perpetuated by SARS-CoV-2 variants, has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust Th1-biased, spike-specific neutralizing antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated neutralizing antibody activity was maintained up to 9 months post-vaccination in both young and aging mice, with durable immune memory evident even in the presence of pre-existing vector immunity. This immunogenicity profile suggests a potential to expand protection generated by current vaccines in a heterologous boost format, and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 . It was first detected in late 2019 1, 2 in Wuhan (Hubei Province, PRC) and has since spread globally with approximately 219 million confirmed cases and 4.5 million deaths as of September 2021 3 . Infection fatality rates are disproportionately high in aged individuals and individuals with co-morbidities such as diabetes, cardiovascular diseases, obesity, and immunosuppression [4] [5] [6] [7] [8] . The recent rapid spike in cases during the second and third waves of infection in several countries has been attributed to emerging variants of concern (VOC) with reported increased transmissibility of infection and/or disease susceptibility and severity associated with changes across the viral genome, in particular the spike protein [9] [10] [11] [12] [13] [14] .

Although initial counter measures such as social distancing, use of masks, and travel restrictions have played a major role in suppressing the spread of infection, vaccines currently continue to be the most effective means to control severe illness and fatality rates, and will play a major role in ending the enormous humanitarian and socioeconomic impact of the SARS-CoV-2 pandemic.

Currently 7 vaccines have been granted Emergency Use Listing by the World Health organization (WHO), with a further 112 in clinical development and 185 in pre-clinical development using a wide array of technologies including nucleic acid based vaccines (mRNA and DNA) 15, 16 , replicating and non-replicating viral vectored vaccines [17] [18] [19] [20] [21] , inactivated viruses 22, 23 , and protein subunit vaccines 24 using a range of new and repurposed adjuvants. Most of the vaccines in development, including all currently approved vaccines, exclusively target the SARS-CoV-2 spike glycoprotein to produce neutralizing antibodies to mediate protection. As a consequence of the unprecedented speed by which frontrunner vaccines were developed, there remains knowledge gaps in vaccine-mediated protection such as duration of protection, efficacy in specific clinically vulnerable populations, rare side effects affecting safety, induction and relative importance of cell-mediated immunity, cross-protection against other coronaviruses, and control of viral shedding and transmission. In addition, vaccines must meet the needs for rapid and successful deployment within the context of population-scale vaccination programs, with specific considerations given to large scale manufacturability and distribution logistics. This highlights the need for continued COVID-19 vaccine development strategies using novel technologies that are safe and build upon the successes of firstgeneration vaccines to provide robust, durable and broadly protective efficacy against variants as well as continue to address key logistical challenges.

The Sementis Copenhagen Vector (SCV) is a rationally designed, replication-defective viral vector technology based on the Copenhagen strain of vaccinia virus (VACV). Uniquely for a VACV-based vector, it has also been paired with a proprietary manufacturing cell line (MCL) to generate a platform system that can facilitate large scale and facile manufacturability of all SCVbased vaccines 25 . The SCV was generated by the targeted deletion of an essential viral assembly gene (D13L) 26, 27 to prevent viral replication whilst retaining the powerful immunogenicity and large payload capacity of the parental virus. With sites B7R/B8R, C3L, A39R and A41L designated as antigen insertion-sites, it is also an ideal vector technology for multi-antigen and multi-pathogen vaccines 28 . Systems vaccinology studies have demonstrated that SCV vaccination results in a localised Th1 signature and significant immunogen expression at the injection site 29 , with advanced preclinical studies showing a fully attenuated and safe vaccine capable of inducing robust and long-lived antigen-specific antibody and CD8 T cell responses equivalent to those elicited by replication competent VACV 25 . Vaccine efficacy of SCV vaccines has also been established in mouse models of disease and non-human primate immunogenicity and infectious disease challenge studies 25, 28, 30 .

In this study, a first-generation SCV vaccine encoding the full-length, native spike glycoprotein (SCV-S) was constructed, with cellular expression and cell surface anchorage of the spike protein in host cells confirmed. Detailed immunological analyses of vaccine-mediated immune responses demonstrated robust and long-lived spike-specific CD8 T cell and neutralizing antibody responses in young and aging mice, including inbred and outbred strains. Following a second booster dose, circulating neutralizing antibody levels were sustained without any discernible decay over a nine-month period. Assessment of long-lived CD8 T cell and antibodysecreting cell compartments confirmed induction of durable spike-specific immune memory following vaccination. Therefore, this study presents evidence that SCV-based COVID-19 vaccines can mediate robust and durable serological and cellular priming which supports progression towards efficacy studies of second-generation vaccine candidates incorporating additional SARS-CoV-2 antigens, to build synergistic layers of additional protection.

The proprietary SCV vaccine platform technology comprises (1) , a viral vector that is unable to produce infectious viral progeny through targeted deletion of an essential viral assembly protein (D13) from VACV, which maintains amplification of the viral genome and late gene expression of introduced vaccine antigens for immune stimulation. This is combined with (2), a manufacturing cell line based on Chinese hamster ovary (CHO) cells that constitutively express D13, for virion assembly, and CP77, an essential VACV host-range protein for CHO cells that provides replication capability for SCV vaccine production in suspension cultures using established commercial manufacturing technologies and facilities ( Figure 1a ).

To generate a spike encoding SCV vaccine (SCV-S), the full-length SARS-CoV-2 spike gene from the original Wuhan isolate-1 was placed under the control of a synthetic VACV early/late promoter 31 and introduced by homologous recombination into the A41L deletion locus of the SCV (Figure 1b ). PCR analysis of recombinant viral DNA confirmed site-specific insertion of the heterologous SARS-CoV-2 spike sequence into SCV and absence of D13L (Figure 1c ). Authentic expression of the spike protein was confirmed by infecting non-permissive human 143B cells 25 with SCV-S or parent control vector (SCV containing no vaccine antigen) and detection of S1 production by immunoblot analysis (Figure 1d ), as well as surface expression of the receptor binding domain (RBD) in infected cells by flow cytometric analysis (Figure 1e ).

The conversion of research grade vaccine stock to cGMP-compliant master and working virus seed (MVS and WVS) stocks requires several passages, followed by subsequent expansion during manufacturing for large scale SCV vaccine production. Therefore, the genetic stability of SCV-S was evaluated by serial passaging of the MVS up to 10 times (P10), which extends at least five-fold beyond the expected expansion requirement to reach commercial-scale vaccine batch production. Immunostaining of MVS and P10 infected cell monolayers using anti-RBD antibody confirmed spike protein expression in all foci of viral infection (Figure 1f) , with similar viral titer amplification ratios observed up to 72 hrs post-infection (Figure 1g ). Genetic integrity and sequence authenticity was also confirmed by next-generation sequencing of MVS and P10 stocks (data not shown), which together with characterised expression indicated the transgene stability and genetic integrity of the SCV-S vaccine candidate.

The capacity of SCV-S to induce an early spike-specific cellular immune response was evaluated in C57BL/6J mice. One week after vaccination, recovered splenocytes were stimulated with overlapping peptide pools spanning the S1 and S2 subunits of the SARS-CoV-2 spike protein and antigen-specific T cell responses evaluated by IFN-γ ELISPOT. Vaccination with SCV-S induced significant S1-and S2-specific IFN-γ spot forming units (SFU) compared to mice vaccinated with control vector (Figure 2a) . Consistent with the ELISPOT results, significantly elevated populations of S1-and S2-specific IFN-γ producing CD8+ T cells were detected in SCV-S vaccinated mice compared to controls by intracellular cytokine staining (Figure 2b ).

Effector CD8 T cells with cytotoxic potential produce granzyme B, a serine protease that is capable of mediating target cell lysis 32 , and a significant population of CD8 + T cells were shown to express granzyme B in response to SCV-S vaccination (Figure 2c ). To confirm the SCV-S vaccine induced a functional cytotoxic T lymphocyte response, day 7 splenocytes were incubated with radiolabelled target cells pulsed with either S1 N-terminal domain (S1-NTD)-, RBD-, or S2-specific spike subunit peptides (Supplementary Table 1 ). Significant cytolytic activity was detected against S1-NTD and RBD target cells (Figure 2d ). The absence of functional activity against the S2 subunit suggested that the SCV-S vaccine primarily triggers a S1-specific cytotoxic T cell profile. Together these results indicated that the SCV-S vaccine induced an early expansion of spike-specific effector CD8 T cells that display the potential to reduce the viral burden before the humoral arm of the immune response can be well established.

Previous studies have demonstrated CD8 T cells that produce multiple cytokines have enhanced effector functions and support maturation of an antigen-specific memory T cell population 33, 34 .

Therefore, S1-and S2-specific cytokine producing CD8 + T cells were profiled into seven distinct populations based on the production of IFN-γ, TNF, IL-2, and their combinations (Supplementary Figure 1) . Polyfunctional IFN-γ + TNF + cells dominated the responding S1-and S2-specific T cell population, followed by IFN-γ + TNF + IL-2 + (triple cytokine producing) and single IFN-γ + producing cell population ( Figure 2e ). Furthermore, significant numbers of S1-and S2specific triple cytokine producing CD8 + T cells (Figure 2f ), produced more cytokines on a per cell basis compared to double and single cytokine producing CD8 + T cells (Supplementary Figure 2) .

Immunogenicity of SCV-S was evaluated in C57BL/6J mice, with kinetics of spike binding antibody responses assessed up to day 28 post-vaccination. S1 and S2 IgM binding titers peaked at day 7 post-vaccination then returned to baseline levels by day 20 (Figure 3a ; top panels).

Class-switched S1 IgG binding titers were detected in all mice by day 9 post-vaccination, with the appearance of S2 IgG binding antibodies delayed to days 15-19 post-vaccination (Figure 3a , lower panels). The World Health Organisation (WHO) target product profile for COVID-19 vaccines 35 and current understandings in the field indicate that a Th1-biased humoral and cellular immune responses is required to prevent vaccine-associated enhanced respiratory disease [36] [37] [38] . Therefore, subclass profiling of S1 and S2 binding antibodies was assessed in day 28 post-vaccination serum samples. A strong IgG2c response with an associated IgG2c/IgG1 ratio of > 1 was observed confirming a Th1-biased spike-specific antibody response ( Outbred strains of mice are more representative of the genetic variability in the general human population and therefore the dominant S1 binding antibody responses in inbred and outbred strains of mice were compared at day 21 post-vaccination. S1 IgG binding titers were comparable between the two strains of mice, were significantly higher than the controls, and translated into significant levels of antibody-mediated neutralization capacity ( Figure 3c ).

To understand the long-term kinetics of the spike-specific antibody response, serum samples from outbred ARC(s) mice were evaluated up to 6 months post-vaccination. S1 binding titers remained significantly elevated compared to naïve mice at all time-points tested, although a trend in reduced titer was observed from 3 months. Significant neutralizing activity was also maintained, albeit with a small steady decline after 3 months (Figure 3d) . A similar trend was observed in C57BL/6J mice, with significant S1-specific antibody binding titers and neutralizing activity maintained up to 12 weeks post-vaccination, although neutralization activity had halved from 3 week levels by 12 weeks (Supplementary Figure 4) . Therefore, to stabilise the waning antibody responses, a 4-week homologous prime/boost vaccination strategy was assessed in C57BL/6J mice. At 3 weeks post-boost, a significant increase in both S1 IgG binding titers and neutralizing activity was detected in the prime-boost cohort compared to single dose and control vector vaccinated mice (Figure 3e) . A positive correlation between the S1-specific IgG binding titers and neutralization activity was confirmed (Supplementary Figure 5) .

Given the emergence of SARS-CoV-2 variants that can exhibit increased replicative and transmission fitness, the SCV-S vaccine-generated neutralization antibody response was assessed against the recognised Alpha and Beta VOCs. First, the capacity to block the interaction between viral RBD and the host angiotensin-converting enzyme 2 (ACE2) receptor was assayed using the RBD protein from the original Wuhan Isolate-1, N501Y Alpha VOC or the E484K, K417N and N501Y Beta VOC. All serum samples inhibited the binding between the ACE2 receptor and the RBD from original Wuhan isolate-1, with a 1.1-and 2.1-fold reduction in inhibition detected for the Alpha and Beta VOCs ( Figure 3f ). Next, using SARS-CoV-2 pseudotyped lentiviruses containing the spike sequence of either the Wuhan isolate-1, Alpha, or Beta VOCs, the effect of all the mutations in the spike protein on the serum neutralizing capacity was examined. A similar result, with a significant 3.2-and 4.1-fold reduction in the IC80 (80% inhibitory concentration) was noted for the Alpha and Beta VOCs compared to the original Wuhan isolate-1 (Figure 3g ).

The persistence of neutralizing antibody responses against SARS-CoV-2 is important in all populations, including the aging population. Therefore, the durability of the SCV-S induced neutralizing antibody activity was evaluated in both young (6-8 weeks old) and aging (9-10 months old) mice. Functional neutralizing antibodies were maintained without any significant decrease in titer up to the termination of the study at three months post-boost vaccination (a total of 4 months from first vaccination dose). Importantly, no significant differences were detected in the neutralizing capacity between the young and aging mice ( Figure 3h ). In summary, these results indicate that administration of SCV-S in a prime-boost vaccination strategy induces a robust and durable SARS-CoV-2 spike-specific antibody response with significant neutralizing capacity against the original Wuhan isolate-1, and both Alpha and Beta VOCs.

To examine SCV-S-induced cellular immune responses, C57BL/6J mice were immunised in a single dose (day 0) or prime-boost (day 0 and 28) vaccination protocol, with spike-specific memory CD8 + T cell populations studied three months later. Mice vaccinated with control vector in a prime-boost strategy were used as appropriate controls. Following a single dose of the vaccine, S1-NTD-specific IFN-γ + SFU were detected above the background determined by control vector vaccinated mice, however a significant 4.3-fold increase over controls was noted with a prime-boost regimen (Figure 4a ). This correlated with an increased frequency of IFN-γ + producing CD8 + T cells and absolute numbers of polyfunctional IFN-γ + TNF + IL-2 + -expressing CD8 + T cells, with the prime-boost regimen significantly elevating the response over that detected in single dose and control vaccinated mice (Figure 4b) . A similar significant increase in RBD-specific and S2-specific IFN-γ + SFU, and IFN-γ-expressing and polyfunctional CD8 + T cells was noted in the prime-boost vaccinated mice compared to the single dose and control vaccinated mice; however a single dose also induced a significant population of RBD-specific and S2-specific IFN-γ + CD8 + T cells and triple cytokine producing cells (Figures 4c-4f) .

Analysis of the total spike-specific CD8 + T cell IFN-γ + responses revealed an immunodominance of RBD-specific responses, with ~65% of the total spike-specific responses targeting the RBD region of the spike protein. Further cytokine profiling of this RBD-specific CD8 + T cell population revealed that the prime-boost vaccination regimen significantly increased the mean frequency of multifunctional T cells from 32% with a single dose, to 80% with a prime-boost. This Vaccine antigen-specific cellular immune responses were also examined in aging mice vaccinated once or in a prime-boost strategy. Similar trends to that seen in young mice was observed, with prime-boost vaccination inducing significantly higher S1-NTD-, RBD-, and S2specific IFN-γ producing cells compared to single dose and control vaccinated mice.

Additionally, the proportions of single TNF-secreting CD8 + T cells and numbers of triple cytokine producing CD8 + T cells were significantly increased over that induced by a single dose, as was the mean frequency and absolute number of multifunctional T cell proportions (Supplementary Figure 8) . Altogether, these results confirm that prime-boost vaccination with SCV-S enhances the spike-specific memory CD8 + T cell response in both young and aging populations of mice.

The presence of VACV-specific memory immune responses, particularly in a small cohort of the aged population that had previously received a smallpox vaccination, may impact on the induction of vaccine antigen-specific cellular and humoral immune responses from recombinant SCV vaccines. Therefore, a mouse model of robust pre-existing vector immunity was established to study its impact on spike-specific immune responses following vaccination with SCV-S. Mice were administered a single dose of replicative VACV, with presence of vector-specific antibody responses confirmed 6 weeks later (Figure 5a ). After eight weeks, mice were vaccinated with SCV-S vaccine in a 4-week prime-boost strategy, and the magnitude and quality of spike-specific antibody and T cell responses evaluated. Naïve mice were used as controls for vector-specific immune responses and mice vaccinated with control vector were used as controls for antigenspecific responses. Two weeks post SCV-S booster dose administration, no significant differences in S1-and S2-specific IgG binding titers between cohorts with (+) or without (-) preexisting immunity were observed ( Figure 5b ). Consistent with the binding titers, SARS-CoV-2 neutralization activity was comparable between the two groups of mice ( Figure 5c ). Importantly, S1 IgG binding titers and neutralizing activity were not impacted by pre-existing immunity even at three months post SCV-S vaccination ( Figure 5d ).

Next, the impact of pre-existing immunity on spike-specific memory CD8 T cell responses at three months post SCV-S vaccination was evaluated in the spleen using peptide pools specific to S1-NTD, RBD, and S2 regions of the spike-protein. As anticipated, irrespective of the preexisting immunity status, SCV-S vaccinated mice had significantly higher S1-NTD-, RBD-, and S2specific IFN-γ + SFU compared to control vaccinated mice ( Figure 5e ). However, approximately 10-15-fold lower S1-NTD-and RBD-specific T cell responses were noted in mice with preexisting VACV memory responses when compared to VACV naïve mice, suggesting the magnitude of the spike-specific T cell responses was impacted on by pre-existing vector immunity. Assessment of the IFN-γ + CD8 + T cell population confirmed a 20-fold increase in IFN-γ expression in response to SCV-S prime-boost vaccination in VACV naïve mice compared to VACV experienced cohorts (Figure 5f ).

Further analysis of the dominant RBD-specific CD8 + T cell responses revealed a higher proportion of RBD-specific multifunctional T cells in VACV naïve mice compared to VACV experienced mice, with a specific increase in IFN-γ + cells co-expressing TNF (Figure 5g ). Despite a lower proportion of multifunctional T cells within the cytokine producing CD8 + T cells, significant numbers of RBD-specific triple-cytokine positive cells in VACV experienced mice could still be detected compared to control vaccinated mice ( Figure 5h ). Together this indicated that pre-existing vector immunity had no significant effect on the humoral immune response, and whilst significantly reduced the magnitude of the spike-specific memory CD8 + T cell response, vaccination with SCV-S could still however induce significant spike-specific cellular responses.

Long-lived immunological memory forms the basis of robust prevention of disease following vaccination. Previous studies have demonstrated that impaired immune responses in older individuals can be associated with decreased immunological memory post-vaccination [39] [40] [41] .

Therefore, the ability of SCV-S to establish and retain a long-lived spike-specific immune response was investigated. Young and aging C57BL/6J mice were vaccinated in a 4-week primeboost regimen, and spike-specific immune responses were analysed nine months postvaccination. Significant S1 binding IgG titers were observed in both young and aging mice compared to control vaccinated mice, with no statistical difference observed between the groups (Figure 6a, left panel) . Surprisingly, S2 binding titers were detected only in 4 out of 5 aging mice, and while the mean S2 IgG binding titers in the aging mice were higher than the control vaccinated mice, it was not statistically different. The young mice on the other had significant S2 IgG binding titers compared to the control vaccinated mice (Figure 6a ; right panel). Retrospective analysis of the S2 binding titers in the young and aging mice during the vaccination time course revealed that the levels were comparable at day 21 post prime, however no further increase in the S2 binding titers in aging mice followed the booster dose. In comparison, an approximate 10-fold increase in the S2 binding titers were detected in young mice after boost vaccination (Supplementary Figure 9 ). Despite these differences in the S2 binding titers, the neutralisation activity was comparable between the young and aging mice at 9 months (Figure 6b ). Persistent antigen-specific antibody levels in the serum are maintained by long-lived antibody secreting cells (ASC) that reside in the bone-marrow [42] [43] [44] . Therefore, the capacity of SCV-S prime-boost vaccination to induce a S1-specific ASC population by B cell Figure 10) . Triple cytokine producing CD8 + T cells which have enhanced cytokine and memory potential were also detected in significant numbers in all groups except S2 targeting triple cytokine producing CD8 + T cells which suggested that S1specific polyfunctional effector cellular immune responses dominate the memory compartment following SCV-S vaccination. In summary, these results indicate that SCV-S vaccination induces a long-lived humoral and cellular immunity, with similar magnitude and quality of immune responses in both young and aging mice, demonstrating broad applicability of SCV-S.

Vaccine development programs that enhance existing vaccine technologies and advance development of novel vaccine platforms are critical in the current race to curb SARS-CoV-2 evolution and control the COVID-19 pandemic. Complementary and synergistic vaccination strategies and cohort-specific vaccination approaches are rapidly gaining acceptance, due to a lack of convenient and effective antiviral therapies, a broad range of vulnerable populations, and the intrinsic manufacturing challenges associated with global vaccination campaigns. In the current stage of the pandemic, a potential vaccine candidate should satisfy one or more of the following criteria: induce a broad and robust humoral and cellular immune response against the dominant circulating variants either as a stand-alone or effective booster to approved vaccines, enhance immunity in vulnerable populations, establish long-lived immune responses that can maintain herd immunity and prevent circulation of SARS-CoV-2 and variants, provide crossprotection against other coronaviruses, and have a stream-lined and facile manufacturing process.

The SCV vaccine platform technology builds on the favourable characteristics of the parental VACV vector, namely long-lasting cellular and humoral immune responses, significant antigen payload load capacity, cold-chain-independent vaccine distribution capability, and incorporates additional advantages primarily safety while maintaining immunogenicity. SCV-based vaccines have proven stability at 4°C for 6 months in simple salt buffered liquid formulations, and at least 1 year in dried formulations (unpublished data) which may address the logistical difficulties associated with vaccine deployment in hard to reach and vulnerable communities.

Furthermore, the SCV platform addresses the manufacturing challenges associated with the use of primary cells in empirically attenuated VACV-based vaccine production; industry-standard CHO cells were uniquely genetically engineered to allow high yield production of SCV vaccines.

This primary study describes the immunogenicity of a first-generation SCV-mediated COVID-19

vaccine. SCV-S incorporates a single antigen of the SARS-CoV-2, the spike glycoprotein, that mediates viral attachment and entry into host cells. The data presented here shows that SCV-S infected cells produce spike immunogen that is transported from the endoplasmic reticulum through the Golgi apparatus to the cell surface to stimulate antigen-specific immune responses.

Importantly, the spike insert was maintained in the viral genome without any loss or changes in transgene sequence or protein expression, thus confirming the genetic stability of the vaccine through several passages, which is an inherent characteristic to support large-scale vaccines manufacturing protocols.

Immunogenicity studies in mice confirmed that the vaccine induces spike-specific functional T cell responses in 1 week, and robust levels of circulating spike-specific antibodies in both inbred and outbred strains of mice, with significant neutralizing activity detected within two weeks of Analysis of the antibody repertoire in SARS-CoV-2 recovered individuals revealed the presence of neutralizing antibodies against the S1-NTD, and S2 region of the spike protein in addition to anti-RBD antibodies 51, 52 . Whilst the exact role and relative contributions of the non-RBD neutralizing antibodies in SARS-CoV-2 infection remains to be fully addressed, it is intuitive to deduce that broad spike-specific antibody responses spanning mutational hotspots (RBD and S1-NTD) and conserved regions (S2) of the spike region, via a combination of anti-S1 and anti-S2 neutralizing antibodies, will offer improved protective efficacy potential against emerging variants. T cell epitope analysis in naïve individuals revealed reactivity to SARS-CoV-2, with significant proportion of the epitopes mapped to the non-RBD (44%) region of the spike protein 53 , suggesting that T cell responses against the conserved S2 region can offer crossprotection against other betacoronaviruses. Our results demonstrate that SCV-S induces a broad-spike specific antibody and CD8 T cell response spanning both the S1 and S2 subunits, enhancing potential activity against other betacoronaviruses and emerging novel SARS-CoV-2 variants.

In summary, the data presented in this study demonstrate that the SCV-S vaccine induces a rapid, broad, robust, and durable spike-specific cellular and humoral response supporting the Binding antibody titers. S1-, S2-and vector-specific binding antibody titers were quantitated by enzyme linked immunosorbent assay (ELISA). High-binding 96-well plates (Nunc) were coated with the appropriate antigen (S1 protein 1.2 μg/ml; S2 0.8 μg/mL; SCV 10 5 PFU/well) in carbonate buffer (pH 9.8) and incubated at 4°C overnight. Plates were washed with PBS-T, blocked with 5% skim milk powder, and then incubated with 3 fold dilutions of relevant serum samples (starting incubated with the pseudoviruses for 30mins and plated onto ACE2-HEK293 cells. After 24hrs, the media was changed, and the following day cells were lysed using ONE-Step™ Luciferase kit and luciferase activity was measured. Data was normalised as percent neutralization using the readings from uninfected cell controls to set the baseline for 100% neutralization and the pseudovirus alone controls to set the baseline for 0% neutralization. IC80 titers were determined using a log (inhibitor) vs. normalized-response (variable slope) nonlinear regression model in Prism v9 (GraphPad).

Antibody secreting cell ELISPOT. S1-specific IgG producing antibody secreting cells in bonemarrow was examined by ELISPOT assay. ELISPOT plates (MSIPS4510; Millipore) were activated with 15% ethanol, washed, coated with S1 protein (10 μg/mL in PBS) and incubated overnight at 4°C. The next day, harvested bone-marrow cells were resuspended as 5x 10 6 cells/mL and 100µL added to washed and blocked ELISPOT plates for 24 hr culture at 37°C/5% CO2. Plates were subsequently washed, incubated with biotinylated anti-IgG detection antibody (Mabtech, 1 μg/mL), followed by streptavidin-alkaline phosphatase (Mabtech, 1:1,000 dilution). Spots were visualised using BCIP/NBT plus substrate and counted via ELISPOT plate reader.

Statistics. GraphPad Prism version 9.0 (GraphPad software) was used for data analysis and statistics. Data was log-transformed and where appropriate Brown-Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test for unpaired samples or repeated measures ANOVA with Tukey multiple comparison test for paired samples was used to determine statistical significance between three or more group. For statistical analysis between two groups, t-tests with Welch's correction was used. Correlation analysis was performed using Spearman rank test.

All datasets generated and analysed in the current research are available from the corresponding authors upon reasonable request. 

A Novel Coronavirus from Patients with Pneumonia in China

Novel coronavirus pneumonia (COVID-19) combined with Chinese and Western medicine based on "Internal and External Relieving -Truncated Torsion" strategy

COVID-19) dashboard

Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection?

Prevalence and impact of cardiovascular metabolic diseases on COVID-19 in China

Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease 2019 (COVID-19)

Obesity in Patients Younger Than 60 Years Is a Risk Factor for COVID-19 Hospital Admission

In-hospital mortality among immunosuppressed patients with COVID-19: Analysis from a national cohort in Spain

Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England

Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7

Detection of a SARS-CoV-2 variant of concern in South Africa

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus

Neutralization of variant under investigation B.1.617 with sera of BBV152 vaccinees

SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough. bioRxiv

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike

A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV152: interim results from a double-blind, randomised, multicentre, phase 2 trial, and 3-month follow-up of a double-blind, randomised phase 1 trial

Efficacy and safety of an inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac): interim results of a double-blind, randomised, placebo-controlled, phase 3 trial in Turkey

Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine

Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology

Immature viral envelope formation is interrupted at the same stage by lac operator-mediated repression of the vaccinia virus D13L gene and by the drug rifampicin

From crescent to mature virion: vaccinia virus assembly and maturation

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses

Injection site vaccinology of a recombinant vaccinia-based vector reveals diverse innate immune signatures

The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates

Compact, synthetic, vaccinia virus early/late promoter for protein expression

Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation

Pathogen-Specific T Cell Polyfunctionality Is a Correlate of T Cell Efficacy and Immune Protection

Generation of effector CD8+ T cells and their conversion to memory T cells

World Health organisation R&D Blue print. WHO Target Product Profiles for COVID-19 Vaccines

Rapid COVID-19 vaccine development

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals

Fading immune protection in old age: vaccination in the elderly

Age-related differences in humoral and cellular immune responses after primary immunisation: indications for stratified vaccination schedules

Bone marrow is a major site of long-term antibody production after acute viral infection

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans

A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection

Antibody responses to SARS-CoV-2 vaccines in 45,965 adults from the general population of the United Kingdom

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19

Mapping and role of T cell response in SARS-CoV-2-infected mice

Negligible impact of SARS-CoV-2 variants on CD4 (+) and CD8 (+) T cell reactivity in COVID-19 exposed donors and vaccinees

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans

Optimizing the immunogenicity of HIV prime-boost DNA-MVA-rgp140/GLA vaccines in a phase II randomized factorial trial design

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine

Safety and immunogenicity of a two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Europe (EBOVAC2): a randomised, observerblind, participant-blind, placebo-controlled, phase 2 trial

 32 The authors acknowledge the contributions of Paul M. Howley, past CEO/CSO of Sementis Ltd., and Robyn Kievit for technical support to this work, as well as The University of Queensland Protein Expression Facility and Genescript for providing reagents and additional support.