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. 2024 Dec 31;20(1):2351664.
doi: 10.1080/21645515.2024.2351664. Epub 2024 May 17.

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen

Rahul Ahuja  1 , Preeti Vishwakarma  1 , Sneha Raj  1 , Varun Kumar  1 , Ritika Khatri  1 , Bharat Lohiya  1 , Shikha Saxena  1 , Gurleen Kaur  1 , Gagandeep Singh  1   2 , Shailendra Asthana  1   2 , Shubbir Ahmed  1   3 , Sweety Samal  1
Affiliations

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen

Rahul Ahuja et al. Hum Vaccin Immunother. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Keywords: MERS-CoV vaccine; immunogenicity; prefusion spike; thermostability, antibody response.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Expi293F based mammalian expression system produced soluble trimeric prefusion MERS-CoV Spike. (a) schematic representation of color-coded expression cassette used in mammalian (pcDNA3.1 (1)) expression vector; blue box represents signal peptide sequence at N-terminal domain for mammalian expression; light-green box represents human-codon-optimized gene encoding M-CoV-S sequences from 16–1291 amino acids taken from England 1 strain; and at C-terminal domain dark-green box represents T4 fibritin trimerization domain and pink box for 8×His-tag. (b)12% reducing SDS-PAGE of purified M-CoV-S (Lane1: Marker, Lane 2: M-CoV-S). (c) Western blot image of inhouse M-CoV-S and commercial MERS-RBD (#10621-CV, R&D systems) probed with anti-M-CoV-S mouse polyclonal serum (Lane 1, 4: Marker, Lane 2: In-house M-CoV-S, Lane 3: recombinant MERS-RBD). (d) Blue native-PAGE image of purified M-CoV-S protein. Lane 1: native Marker, Lane 2: native M-CoV-S. (e) Purified M-CoV-S protein was subjected to de-glycosylation by using PNGase F enzyme (Lane 1: Marker, Lane 2: PNGase treated M-CoV-S, Lane 3: untreated M-CoV-S).
Figure 2.
Figure 2.
Biophysical characterization of purified M-CoV-S protein. (a) Size exclusion chromatography using superdex 200 increase. The purified M-CoV-S was injected manually using a 500 µl capillary loop. The column pre-equilibrated with 10 mM PBS was eluted with 1.5 CV of at flow rate of 0.750 ml/minute. The protein eluted at 9.0 ml. The inset graph shows the blue-native PAGE image of M-CoV-S protein after purification eluted at an size of ~ 1045 KDa. (b) Hydrodynamic size and apparent zeta potential measurement of M-CoV-S using Malvern Zetasizer. (c) Intrinsic tryptophan fluorescence of M-CoV-S. 1 mg/ml M-CoV-S was excited at wavelength of 280 nm and emission spectra acquired (300–400 nm). An emission maximum was recorded at 336 nm. (d) Far UV circular dichroism spectroscopy of M-CoV-S (250 µg/ml) in the range 195–250 nm range.
Figure 3.
Figure 3.
Thermal stability analysis of purified M-CoV-S. (a) differential scanning colorimetry for estimation Tm. M-CoV-S (0.5 mg/ml) was loaded onto nano-DSC with 10 mM PBS as a reference. Thermal melting at scanning rate of 1 °C/min and 3.0 atmospheres of pressure. NanoAnalyze software showed a Tm of 58°C. (b) SDS-PAGE analysis of M-CoV-S stored at different temperatures. Lane 1: Marker, lane 2: 4°C day 3, lane 3: 4°C day 7, lane 4: 25°C day 3, lane 5: 25°C day 7, lane 6: 37°C day 3, lane 7: 37°C day 7. (c) Intrinsic tryptophan fluorescence of M-CoV-S at different temperatures. (d) Far UV circular dichroism spectroscopy of M-CoV-S at different temperatures.
Figure 4.
Figure 4.
Evaluation of Immunogenic potential of MERS-CoV prefusion spike given with either AddaVaxTM or imject adjuvants. (a) 6–8 weeks old BALB/c mice were immunized with M-CoV-S prefusion protein either with addavax or imject in a prime-boost regimen at 28 days apart. Serum sample was harvested on 14 days after prime and day 14 and day 28 after boosting. Serum was separated, heat inactivated and stored at − 20°C and was used for IgG and its subtypes endpoint titer estimation. (b) Table describing different groups, dose of M-CoV-S immunogen and route of immunization. 29 female BALB/c mice were divided in to six groups having 6 mice each except PBS control group had 5 mice. (c) Total IgG end point titer estimation of prime (day 14) serum by homologous protein. (d) IgG estimation at day 14 post boost. (e) IgG estimation day 21 post boost. (f) IgG subtyping using anti-mouse HRP conjugated secondary antibodies (IgG1, IgG 2a, IgG 2b, and IgG3). (g) Th1/Th2 cytokines level was estimated in boost serum using the cytometric bead array. The values shown in the graphs are the geometric mean titers ± S.E. of triplicate wells. The statistical significance of the experiment was calculated between two groups by students t-test and between more than two groups with the one-way ANOVA test (Tukey-Kramer test). Where, *p < .05, **p < .01, ***p < .001, and ****p < .0001 were considered significant.
Figure 5.
Figure 5.
Molecular modeling indicates the possible binding site of antibody: (a) modelled protein (chain A: in green and rendered in cartoon) and antibody (chain B and C: multi-colored and rendered in cartoon). (b) Residues of MCA1 antibody involved in interaction with modeled MERS spike protein. Pink residues represent the MCA1 antibody, and green residues represent the MERS spike protein. All the residues are shown in atom-wise (C: light red/green, N: blue, O: red and H: white). (c) 2D representation of interacting residues. (d) Quantitative interaction map of interacting residues. The color of bubbles indicates the residues basic: blue, acidic: red, aromatic: purple, nonpolar: gray and polar: green. (e) Binding affinity comparison of MCA1antibody with M-CoV-S protein. The modeled in blue and co-crystal in red. All the values are in kcal/mol.
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Grants and funding

In this study the MERS antigen design and characterization was partially supported by the grant from the Coalition for Epidemic Preparedness Innovations (“CEPI”) towards a Consortium involving THSTI and Panacea Biotec ltd., to develop a Multi-epitope, Nanoparticle based broadly protective Beta coronavirus candidate vaccine (“Project”). The other works were supported by Translational Health Science and Technology Institute (THSTI) core funding and INDCEPI project, an institute functioning under the Biotechnology Research and Innovation Council (BRIC) of the Department of Biotechnology, Ministry of Science and Technology, Government of India.

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