A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

Abstract

First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

Conflict of interest statement

Competing interests: D.B.W. has grant funding, participates in industry collaborations, has received speaking honoraria, and fees for consulting. This service includes serving on scientific review committees and advisory boards. Remuneration includes direct payments and/or stock or stock options and in the interest of disclosure therefore he notes potential conflicts associated with this work with Inovio where he serves on the SAB, Merck, VGXI, OncoSec, Roche, Aldevron, and possibly others. Licensing of technology from his laboratory has created over 150 jobs in the biotech/pharma industry. The other authors declare no competing interests.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. Construction and characterization of the MERS vaccine plasmid construct

(A) Schematic diagram of MERS S protein gene inserts used to generate the codon-optimized DNA vaccines, designated as MERS vaccine. Different S protein domains (TmD, transmembrane domains; CD, cytoplasmic domain) are indicated. (B) Expression of the MERS S protein detected by SDS–polyacrylamide gel electrophoresis and Western blot. The expression of S protein from the indicated amount of MERS vaccine in 293T cells was analyzed. The arrows indicate theSprotein and β-actin control. (C) Immunofluorescence assay of Vero cells transfected with the MERS vaccine. S protein expression is indicated by Alexa Fluor 488 (AF488) staining, and 4′,6-diamidino-2-phenylindole (DAPI) staining shows cell nuclei. MW, molecular weight; FITC, fluorescein isothiocyanate.

Fig. 2. Functional profile of cellular immune responses elicited by MERS vaccine in mice

(A) The S protein-specific cellular immune response in mice 1 week after the final immunization with the MERS vaccine. IFN-γ responses were assessed by ELISpot assays using six peptide pools encompassing the entire S protein. Values (that is, SFU per 106 cells) represent mean responses in each group (n = 3) ± SEM. (B) Characterization of MERS-CoV S protein-specific dominant epitopes in C57BL/6 mice. IFN-γ responses were assessed by ELISpot assays with matrix pools of peptides, indicating the presence of immunodominant epitopes. Values represent mean responses in each group (n = 3) ± SEM. Similar results were obtained in two separate experiments. (C) The functional profile of CD4+ and CD8+ T cell responses elicited by MERS vaccine. Mouse splenocytes (n = 3) were isolated 1 week after the final DNA immunization and were stimulated with pooled MERS S protein peptides ex vivo. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2, and then analyzed by fluorescence-activated cell sorting (FACS). The bar graph shows subpopulations of mono-, double-, and triple-positive CD4+ and CD8+ T cells releasing the cytokines IFN-γ, TNF-α, and IL-2. The pie charts show the proportion of each cytokine subpopulation. Values represent mean responses in each group (n = 3) ± SEM.

Fig. 3. Humoral immune responses elicited by MERS vaccine in mice

(A) Serum IgG responses specific for MERS S protein. Serum from individual mice (1 week after the third immunization) was serially diluted, and anti-MERS S protein–specific total IgG was measured by ELISA. Values represent mean responses in each group (n = 9) ± SEM. (B) Endpoint binding titers for the MERS vaccine– immunized mouse sera were calculated at the indicated time points. Values for individual mice are shown (n = 9) and lines represent the geometric mean ± SEM. (C) Western blot analysis of the presence of IgG specific for recombinant full-length MERS S protein (or recombinant HIV gp120 as a negative control) in immune sera. Pooled sera were used as the primary antibody at a 1:250 dilution. (D) NAb responses detected by the viral infection assay in sera collected 1 week after the final immunization. NAb titers are presented as the sera dilution that mediates 50% inhibition (IC50) of virus infection of the target cells. Values of individual mice are shown (n = 9) and lines indicate the mean of each group ± SEM. (E) Neutralization with MERS and related CoV pseudoviruses by MERS vaccine–immunized mouse sera. Serially diluted pooled sera from four mice 1 week after the third immunization were analyzed in duplicate. These assays were performed twice for consistency, with one of these shown. VSV-G–pseudotyped virus was used as the control for neutralization specificity. OD, optical density.

Fig. 4. Humoral immune responses elicited by MERS vaccine in camels

(A) Three dromedary camels were immunized three times at 4-week intervals with the MERS vaccine delivered by EP. Blood was taken at week 0 (prebleed) and week 11 (3 weeks after the third immunization), and sera were isolated for the assessment of the humoral immune response. (B) Western blot analysis of the presence of IgG specific for recombinant full-length MERS S protein (or recombinant HIV gp120 as a negative control) in immune sera. Sera from individual animals were used as the primary antibody at a 1:250 dilution. (C) NAb responses detected by the viral neutralization assay in sera collected 3 weeks after the final immunization. NAb titers are presented as the sera dilution that mediates IC50 of virus infection of the target cells. Each sample was run in duplicate. The data shown are the mean titers for each animal ± SEM.

Fig. 5. Potent T cell responses elicited by MERS vaccine in rhesus macaques

(A) Time course of MERS vaccine immunization, viral challenge, and immune analysis. (B) The S protein–specific cellular immune response in PBMCs isolated from NHP 2 weeks after the final immunization with MERS vaccine. IFN-γ responses were assessed by ELISpot assays using six peptide pools encompassing the entire S protein. Values represent mean responses in each group (n = 4) ± SEM. (C) The functional profile of CD4+ and CD8+ T cell responses elicited by low and high dose MERS vaccine. PBMCs (n = 4) were isolated 2 weeks after the final MERS vaccine immunization and were stimulated with pooled MERS S protein peptides ex vivo. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. The bar graph shows the mean total percentage ± SEM of CD4+ and CD8+ T cells in the blood expressing the indicated cytokine. RhM, rhesus macaque.

Fig. 6. Humoral immune responses elicited by MERS vaccine in rhesus macaques

(A) Endpoint antibody titers were determined for all rhesus macaques before and after each immunization with MERS vaccine. Values for individual NHP are shown (n = 4) and lines represent the group mean ± SEM. (B) NAb responses detected by the viral infection assay in sera collected 2 weeks after the final immunization. NAb titers are presented as the sera dilution that mediates IC50 of virus infection of the target cells. Values of individual NHP are shown (n = 4) and lines indicate the mean of each group ± SEM. (C) Western blot analysis of the presence of IgG specific for recombinant full-length MERS S protein in immune sera. Pooled immune sera were used as the primary antibody at a 1:250 dilution. (D) Percent neutralization of S protein–pseudotyped viruses by sera from MERS vaccine–immunized NHP. The values are expressed as percent neutralization of the average of duplicate wells. The assay was performed two times. Gray bar represents immunized sera, and green bar represents prebleed sera. VSV-G pseudotyped virus was used as the control for neutralization specificity.

Fig. 7. Protection from live MERS-CoV viral challenge by MERS vaccine in rhesus macaques: Evaluation of clinical signs and viral loads

(A) Radiographic changes. Ventrodorsal thoracic x-rays from pVax1- and MERS vaccine–immunized rhesus macaques imaged on day 6 after MERS-CoV infection. Infiltration is highlighted by the white circles. (B) Histology of lung sections. Lung from a pVax1-vaccinated animal (4× and 20×) indicating coalescing subacute bronchointerstitial pneumonia with abundant alveolar edema and fibrin and type II pneumocyte hyperplasia. Lungs from rhesus macaques immunized with high or low doses of the MERS vaccine demonstrating minimal focal interstitial pneumonia with mild subacute perivasculitis and minimal focal interstitial pneumonia. Histology pictures are all taken of tissue from the left middle lobe. (C) Viremia in the indicated tissues from rhesus macaques immunized with MERS vaccine and challenged with MERS-CoV (n = 4 per group). RNA was extracted from control and vaccinated NHPs, and viral load was determined as TCID50 equivalents (TCID eq/g) by qRT-PCR. TCID50 eq/g were extrapolated from standard curves generated by adding dilutions of RNA extracted from a MERS-CoV EMC/2012 stock with known virus titer in parallel to each run. All values are mean ± SEM. (D) Cumulative viremia in all tissues from rhesus macaques in each vaccination group (n = 4 per group). All values are mean ± SEM. P values determined by an unpaired t test are indicated as comparison between different groups.