Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 16;10(12):718.
doi: 10.3390/v10120718.

CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

Svenja Veit  1 , Sylvia Jany  2 , Robert Fux  3 , Gerd Sutter  4   5 , Asisa Volz  6   7
Affiliations

CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

Svenja Veit et al. Viruses. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection.

Keywords: MERS-CoV; MERS-CoV nucleocapsid protein; MVA vaccine; murine CD8+ T cell epitope.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Generation and characterization of recombinant Modified Vaccinia virus Ankara expressing the Middle East respiratory syndrome coronavirus N protein (MVA-MERS-N); (a) Schematic diagram of the MVA genome indicating the major deletion sites I-VI on the top. Flank-1 and flank-2 refer to MVA DNA sequences adjacent to corresponding insertion site. Deletion III was used to insert MERS-N encoding gene sequences under the transcriptional control of the vaccinia virus promoter PmH5. Repetitive sequences (FR) were designed to remove the mCherry marker by intragenomic homologous recombination (marker gene deletion); (b,c) PCR analyses of genomic viral DNA using oligonucleotide primers to confirm the correct insertion of recombinant MERS-N gene into deletion III (b), and the genetic integrity of the MVA genome for the C7L gene locus (c); (d) Multi-step growth analysis of recombinant MVA-MERS-N and non-recombinant MVA (MVA); Chicken embryo fibroblasts (CEF) and human HaCat or HeLa cells were infected at a multiplicity of infection (MOI) of 0.05 with MVA-MERS-N or MVA. Infected cells were collected at different time points after infection and titrated on CEF cells.
Figure 2
Figure 2
Analysis of recombinant MVA-MERS proteins; (a) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; (b–d) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody (b) or polyclonal sera from MERS-CoV infected rabbits (c) or cynomolgus macaques (d). Arrows indicate the N- or S-specific protein bands.
Figure 3
Figure 3
Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; (a–b) groups of BALB/c mice (n = 2 to 6) were vaccinated twice (21-day interval) by i.p. (a) or i.m. (b) application with 108 plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.
Figure 4
Figure 4
Mapping of H2-d restricted T cell epitopes in MERS-CoV N protein; (a–b) BALB/c mice (n = 2 to 4) were immunized twice (21-day interval) i.p. or i.m. with 108 PFU of recombinant MVA-MERS-N (MVA-N), non-recombinant MVA (MVA) or PBS. Splenocytes from vaccinated mice were incubated in the presence of subpools (V8.1, V8.2, H8.1, H8.2) from positive matrix pools (a) or individual 15-mers peptides #89 or #90 (b). IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were quantified by ELISPOT. The lines represent means.
Figure 5
Figure 5
Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; (a–d) Groups of BALB/c mice (n = 3 to 8) were vaccinated in a prime-boost regime with 108 PFU of MVA-MERS-N via i.p. (a) or i.m. (b–d) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. (a-b) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. (c–d) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide (c) or F2L26-34 peptide (d) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *< 0.05, **< 0.005.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zaki A.M., van Boheemen S., Bestebroer T.M., Osterhaus A.D., Fouchier R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012;367:1814–1820. doi: 10.1056/NEJMoa1211721. - DOI - PubMed
    1. WHO Middle East respiratory Syndrome Coronavirus (MERS-CoV) [(accessed on 22 November 2018)]; Available online: http://www.who.int/emergencies/mers-cov/en/
    1. Park J.E., Jung S., Kim A., Park J.E. MERS transmission and risk factors: A systematic review. BMC Public Health. 2018;18:574. doi: 10.1186/s12889-018-5484-8. - DOI - PMC - PubMed
    1. Alraddadi B.M., Watson J.T., Almarashi A., Abedi G.R., Turkistani A., Sadran M., Housa A., Almazroa M.A., Alraihan N., Banjar A., et al. Risk Factors for Primary Middle East Respiratory Syndrome Coronavirus Illness in Humans, Saudi Arabia, 2014. Emerg. Infect. Dis. 2016;22:49–55. doi: 10.3201/eid2201.151340. - DOI - PMC - PubMed
    1. Alshukairi A.N., Zheng J., Zhao J., Nehdi A., Baharoon S.A., Layqah L., Bokhari A., Al Johani S.M., Samman N., Boudjelal M., et al. High Prevalence of MERS-CoV Infection in Camel Workers in Saudi Arabia. mBio. 2018;9:e01985-18. doi: 10.1128/mBio.01985-18. - DOI - PMC - PubMed

Publication types

MeSH terms