Emmanuel Margolin for the initial cloning of the SARS-CoV-2 gene used in this study. This work also allows for the direct comparison of the MVA platform with other platforms used by our group (DNA and plant-based subunit) as SARS-CoV-2 vaccines.
Emergence of SARS-CoV-2
- SARS-CoV-2 in Africa
The World Health Organization has declared it a pandemic, due to the alarming severity and rapid spread of the disease. With an initial reproduction number (R0) estimated to range between 1.5 – 3.5, the spread from China to the rest of the world has been unprecedented, with the WHO describing the global risk assessment in China in January as "very categorized high" and "high", finally declaring it a pandemic on March 11, 2020.
Coronavirus diversity and impact on human health
- Endemic human coronaviruses
- Highly pathogenic human coronaviruses
The most recent coronavirus to have been transmitted from animals to humans is the SARS-CoV-2 virus, so named because of the 79% genetic similarity to SARS, which was first discovered in 2002 [55]. Genetic sequencing analysis of the virus showed that it was in the same Betacoronavirus genus as SARS-CoV, lineage B [56], [57].
Diagnosis of SARS-CoV-2 infection
Different methods of diagnosis have had to be used to diagnose early viral infection, such as PCR and rapid antigen tests. For rapid diagnosis, a rapid antigen detection test is used, although the latter has been shown to be less reliable and thus used as a secondary test to PCR by reverse transcriptase [67].
Impact of the SARS-CoV-2 pandemic on Sub-Saharan Africa
Risk factors differ between countries, which significantly affects the impact of COVID-19 on the population. It was further described that delayed diagnosis is an additional strain due to the similarity of symptoms between COVID-19 and tuberculosis.
SARS-CoV-2 virus structure and genetic organization
The peak glycoprotein was identified as the protein showing the most diversity among coronaviruses, especially the S1 subunit of the protein. The peak glycoprotein is a trimeric class 1 fusion protein, like that of the influenza, HIV and Ebola viruses [100].
SARS-CoV-2 variants of concern and their impact on vaccines
An important role is played by the interaction of the SARS-CoV-2 S protein with the host cell angiotensin-converting enzyme 2 (ACE2) receptor as it serves as the entry point of SARS-CoV-2 into a cell. There are several regions of the SARS-CoV-2 peak that have been identified as areas of antibody vulnerability that are ideal for therapeutics.
Potential correlates of protective immunity against COVID-19
Additionally, it was found that there was a high proportion of CD4+ and CD8+ T cells in immunologically naïve SARS-CoV-2 individuals, suggesting cross-protection from endemics. The role of T cells as correlates of protection was further supported by a study where convalescent rhesus macaques with depleted CD8+ T cells were challenged with SARS-CoV-2 and had lower protection compared to the non-depleted group [136] .
Vaccine platforms for immunization against COVID-19
- Live-attenuated vaccines
- Inactivated vaccines
- Subunit vaccines
- Virus-like particles (VLPs)
- Nucleic acid-based vaccines
- DNA-based vaccines
- RNA-based vaccines
- Viral vectored vaccines
- Adenovirus-vectored vaccines
- Poxvirus vectored vaccines
- MVA as a vaccine vector
- The construction of recombinant MVA
- Project rationale
Chimpanzee adenoviruses (ChAd) are an alternative to Ad5 because they can evade preexisting immune challenge, as only about 1% of humans have antibodies against them [183], while Ad26 is a rarer virus in the human population and thus serves as an alternative to Ad5 vectors. [184]. A chimpanzee adenoviral vector vaccine, ChAdOx1-S nCoV-19, produced by Astra-Zeneca and the University of Oxford, has been shown to induce a strong and long-lasting immune response in both healthy adults and immunocompromised humans. Our group investigated the immunogenicity of an engineered version of the SARS-CoV-2 spike protein based on wild Wuhan virus on different platforms.
- Cloning
- Restriction enzyme digestion
- Separation of DNA fragments by agarose gel electrophoresis
- Excision and recovery of DNA fragments from agarose gels
- Ligation of DNA fragments
- Transformation of Escherichia coli
- Isolation of DNA
- Cell culture
- Cell lines
- Revival and expansion of frozen cells
- Growth and maintenance of cell lines
- Determination of cell count and cell viability
- Preparation of frozen cell lines for long-term storage in liquid nitrogen
- Transfection of mammalian cells
- Transfection of cells with plasmid DNA
- Generation of MVA-SARS2-SΔTM
- Infection of BHK21 cells with wild type MVA and transfection with pMVA-FNK1
- Validation and characterization of recombinant MVA-SARS2-SΔTM
- Polymerase Chain Reaction (PCR)
- DNA sequencing
- Western blot analysis
- Growth of MVA-SARS2-SΔTM in BHK21 and RK13 cells
- Large scale production and titration of recombinant MVA-SARS2-SΔTM vaccine stocks
- Titration of recombinant MVA-SARS2-SΔTM stocks
- Immunologic evaluation of MVA-SARS-2-SΔTM in BALB/c mice
- Mice vaccination regimen
A 20 µl aliquot of Proteinase K was added before 200 µl buffer AL was added and the cells were pipetted up and down. A 500 µl aliquot of 100% methanol (Sigma-Aldrich, France) was added and the plate was vortexed for 1 min. A 500 µl aliquot of SARS-CoV-2 spike rabbit primary antibody (1:500) was added to each well and the plate was incubated at room temperature for 90 min.
Cells were washed with 1x PBS to remove any unadsorbed virus before adding 2 ml of cDMEM to each well. Biotinylated detection antibody (1:250) was added and the plate was incubated for 2 hours at room temperature in the dark.
Construction of a mammalian expression plasmid encoding SARS-CoV-2 S∆TM
- Validation of SARS-CoV-2 S∆TM expression from the pMEx-FNK1 expression
- Construction of transfer vector pMVA-FNK1
- Generation of recombinant MVA by homologous recombination
The construction of pMEx-FNK1 that expressed the SARS-CoV-2-S∆TM antigen allows the plasmid to be used as a positive control for expression of the immunogen from the recombinant MVA-SARS2-S∆ TM vaccine. The resulting plasmid has the SARS-CoV-2 SΔTM gene downstream of the vaccinia virus mH5 promoter. The gene was ligated into the pSSPEx Du151 Gly V2.1 in place of the HIV Env gene, thus forming the transfer vector pMVA-FNK1.
BamHI and NcoI produced fragments of the expected size, indicating that the plasmid was indeed correct. Cells from A and B were used to isolate recombinant MVA, which is summarized in Figure 3.11.
Validation and characterization of recombinant MVA-SARS-2-S∆TM by PCR and
- PCR to confirm integrated gene cassette in the recombinant virus
- Sequencing to confirm the genetic integrity of the DNA in the recombinant virus
Confirmation of expression of SARS-CoV2-S∆TM by western blot analysis and
- Western blot analysis to confirm expression of S∆TM from the recombinant virus
- Scale up of MVA-SARS2-SΔTM
HEK293 cells were infected with MVA-SARS2-S∆TM and fixed as described in section 2.5.4, before observation under fluorescent light showing red Cy3 fluorescence (Figure 3.15). The presence of red fluorescence in the first and second rows of Figure 3.15 served as confirmation of SΔTM protein expression in cells infected with MVA-SARS2-SΔTM. The growth of recombinant MVA-SARS2-S∆TM was assessed in the two permissive cell lines, BHK21 and RK13 cells.
Both BHK21 and RK13 cells were found to be favorable for the growth of the recombinant MVA-SARS2-S∆TM, although the fluorescence was different in the two cell lines. In the experimental samples (lanes 1 and 2), cells were infected with MVA-SARS2-S∆TM and fixed with 4% paraformaldehyde and 100% methanol before being blocked with 2% BSA.
Immunogenicity of MVA-SARS-2-SΔTM in mice
- Quantification of serum binding antibodies in immunized mice
- Determination of the frequency of IFN-γ secreting cells following immunization 99
An ELISPOT test was used to determine the frequency of IFN-γ-secreting splenocytes as described in Section 2.7.1.2. A negative control of an irrelevant peptide produced negligible responses (mean 89.4 SFU/106 splenocytes), while the positive control, Con A, had a high response rate (mean 2610.3 SFU/106 splenocytes) as expected. Splenocytes were pooled from five mice and incubated in the absence of peptide (R10 medium only), stimulated with an irrelevant peptide, or stimulated with SARS-CoV-2 S1 and S2 peptides.
Serum neutralizing antibodies were tested in an assay where the serum of the vaccinated mice was tested to determine if it could block infection with a SARS-CoV-2 pseudovirus. Georgia Schafer (Full Member, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town), where the pseudovirus was created from the HIV backbone in HEK293TT cells, and then pseudotyped with the pcDNA3.3-SARS- CoV-2 peak Δ18.
SΔTM antigen design
Unique to the SARS-CoV-2 virus is the furin cleavage site in the spike glycoprotein, which has been documented to have enhanced infectivity [246]. The results of this current study showed that the SΔTM variant had the highest expression levels due to the deletion of the transmembrane domain. An additional modification used for our design was the replacement of the native leader sequence with a tissue plasminogen activator signal sequence, which has previously been shown to enhance secretion of the desired protein and has been shown to improve the immunogenicity of an MVA-based TB vaccine [248]. ].
A mammalian expression plasmid expressing the matching SARS-CoV-2 S∆TM protein was used as a positive control in this study. The plasmid was based on the pTHpCapR expression vector previously designed and used in our group [233].
Construction of a transfer vector pMVA-FNK2
A soluble antigen is better at producing recombinant proteins; therefore, the matched protein was used in this study. This strategy was previously used by Binley and colleagues [232], and was reported to improve processing of HIV gp140 trimers in mammalian cells. The antigen in our study was further modified by deleting the transmembrane and cytoplasmic domains, a strategy that has also been used by Jia et al., [247].
Smallpox promoters are important in that they uniquely direct poxviral transcription, due to the specific recognition of promoters by the poxviral transcriptional machinery. Thus, inclusion of the poxvirus p.75 promoter to drive expression of the green fluorescence reporter gene, eGFP, allowed visualization of the generated recombinant MVA-SARS2-SΔTM, as all three foreign genes (eGFP, K1L, and SARS- CoV- 2-SΔTM) were inserted between the flanking sequences I8R and G1L.
Generation of recombinant MVA-SARS2-SΔTM
During transcription, poxvirus RNA polymerase recognizes only unique poxvirus promoters controlled by early, intermediate, and late genes. In this study, the SΔTM gene was cloned into the MVA pSSPEx backbone, putative clones were confirmed by restriction enzyme digestion, and plasmid DNA was purified for use in transfections. bp) could contribute to this, as large fragments are generally difficult to amplify [256]. The use of "long fragment polymerases" designed to specifically amplify fragments larger than 6 kbp could give a positive result.
The GC-rich sequence of the recombinant (Appendix B) could also have contributed to the inability to amplify the entire foreign gene cassette in the recombinant. In this project, neither the use of Taq polymerase nor dimethyl sulfoxide (DMSO) was successful.
Confirmation of expression of protein SΔTM
104 . bp) could have contributed to this as it is generally difficult to amplify large fragments [256]. The difficulty of amplifying templates that have a high percentage of GC runs is a well-documented challenge and as such there are many protocols developed to circumvent it.
Immunogenicity assessments in mice
A possible explanation for the failure of MVA-SARS2-SATM to induce neutralizing antibodies in this study could be the low expression of the spike protein seen in the Western blot results, where the assay yielded a faint band of the protein. In addition, the vaccine titer used in this study to inoculate the mice was relatively low at 105. Interestingly, in this study there was a Th1 bias, with the vaccine coupled with adjuvant Advax-2 producing stronger T-cell responses in contrast for antibody reactions.
In our study, MVA-SARS2-SΔTM followed a pattern similar to the vaccine coupled with Advax-2, whereby there was a Th1 bias likely explained by the low dose of the MVA-based vaccine used for inoculation. This may likely be due to the length of S2, which was shorter in our SΔTM gene.
Future work
