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Evaluation of candidate vaccine approaches for MERS-CoV.

Wang L, Shi W, Joyce MG, Modjarrad K, Zhang Y, Leung K, Lees CR, Zhou T, Yassine HM, Kanekiyo M, Yang ZY, Chen X, Becker MM, Freeman M, Vogel L, Johnson JC, Olinger G, Todd JP, Bagci U, Solomon J, Mollura DJ, Hensley L, Jahrling P, Denison MR, Rao SS, Subbarao K, Kwong PD, Mascola JR, Kong WP, Graham BS - Nat Commun (2015)

Bottom Line: Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses.Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates.Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.

No MeSH data available.


Related in: MedlinePlus

Varied vaccination regimens elicit neutralizing antibodies that target different regions of the MERS-CoV Spike glycoprotein.Immunization with different vaccine regimens elicited neutralizing antibodies that target the Spike glycoprotein within and outside the RBD. (a) Cell adsorption assay. Sera from mice immunized with MERS-CoV S DNA-only, S DNA prime and S1 protein plus Ribi adjuvant, or S1 protein plus Ribi adjuvant prime and boost were evaluated for neutralization activity against pseudotyped MERS-CoV England1 after adsorption with HEK 293T cell-surface-expressed MERS-CoV Spike proteins: S, RBD, S1, S2. Serum neutralization was tested at a single dilution. Sera adsorbed with untransfected HEK 293T cells served as controls and retained 95% of neutralization activity. Each bar represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown. (b) Protein competition neutralization assay. Sera at a single dilution from the immunized mice were also assayed for neutralization of MERS-CoV England1 pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteins at concentrations of 0.016–50 μg ml−1. Each data point represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown.
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f2: Varied vaccination regimens elicit neutralizing antibodies that target different regions of the MERS-CoV Spike glycoprotein.Immunization with different vaccine regimens elicited neutralizing antibodies that target the Spike glycoprotein within and outside the RBD. (a) Cell adsorption assay. Sera from mice immunized with MERS-CoV S DNA-only, S DNA prime and S1 protein plus Ribi adjuvant, or S1 protein plus Ribi adjuvant prime and boost were evaluated for neutralization activity against pseudotyped MERS-CoV England1 after adsorption with HEK 293T cell-surface-expressed MERS-CoV Spike proteins: S, RBD, S1, S2. Serum neutralization was tested at a single dilution. Sera adsorbed with untransfected HEK 293T cells served as controls and retained 95% of neutralization activity. Each bar represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown. (b) Protein competition neutralization assay. Sera at a single dilution from the immunized mice were also assayed for neutralization of MERS-CoV England1 pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteins at concentrations of 0.016–50 μg ml−1. Each data point represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown.

Mentions: We analysed the specificity of NAb responses against the different subunits of the MERS-CoV Spike glycoprotein using several methods. First, sera were pre-adsorbed with HEK 293T cells expressing transmembrane-anchored versions of S, S1, S2 and RBD (Fig. 1a and Supplementary Fig. 4a) and were screened for neutralization (Fig. 2a). Sera pre-adsorbed with untransfected cells retained 95% of their original neutralization activity, while negative control pre-immune sera showed no capacity for virus neutralization. The full-length Spike-transfected cells adsorbed virtually all neutralization activity from the immune sera of all three vaccine groups, while the RBD expressed by itself on the cell membrane only adsorbed about half of the neutralizing activity, and only in the S1 protein group, suggesting the possibility that the conformation of RBD presented in the full-length Spike and in the truncated versions may differ. Adsorption with S1 also removed all neutralization activities in the S1 protein group and ∼60% of neutralizing capacity in the two DNA-primed groups. In contrast, S2 did not deplete neutralization activity in any of the groups, except slightly in mice vaccinated with DNA alone. It is likely that expression of S2 by itself on cells resulted in rearrangement to the post-fusion conformation, and therefore neutralizing epitopes present on the prefusion structure would not have been available for adsorption. Flow cytometric analysis of serum bound to cells that expressed different Spike subunits gave consistent results (Supplementary Fig. 4c).


Evaluation of candidate vaccine approaches for MERS-CoV.

Wang L, Shi W, Joyce MG, Modjarrad K, Zhang Y, Leung K, Lees CR, Zhou T, Yassine HM, Kanekiyo M, Yang ZY, Chen X, Becker MM, Freeman M, Vogel L, Johnson JC, Olinger G, Todd JP, Bagci U, Solomon J, Mollura DJ, Hensley L, Jahrling P, Denison MR, Rao SS, Subbarao K, Kwong PD, Mascola JR, Kong WP, Graham BS - Nat Commun (2015)

Varied vaccination regimens elicit neutralizing antibodies that target different regions of the MERS-CoV Spike glycoprotein.Immunization with different vaccine regimens elicited neutralizing antibodies that target the Spike glycoprotein within and outside the RBD. (a) Cell adsorption assay. Sera from mice immunized with MERS-CoV S DNA-only, S DNA prime and S1 protein plus Ribi adjuvant, or S1 protein plus Ribi adjuvant prime and boost were evaluated for neutralization activity against pseudotyped MERS-CoV England1 after adsorption with HEK 293T cell-surface-expressed MERS-CoV Spike proteins: S, RBD, S1, S2. Serum neutralization was tested at a single dilution. Sera adsorbed with untransfected HEK 293T cells served as controls and retained 95% of neutralization activity. Each bar represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown. (b) Protein competition neutralization assay. Sera at a single dilution from the immunized mice were also assayed for neutralization of MERS-CoV England1 pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteins at concentrations of 0.016–50 μg ml−1. Each data point represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525294&req=5

f2: Varied vaccination regimens elicit neutralizing antibodies that target different regions of the MERS-CoV Spike glycoprotein.Immunization with different vaccine regimens elicited neutralizing antibodies that target the Spike glycoprotein within and outside the RBD. (a) Cell adsorption assay. Sera from mice immunized with MERS-CoV S DNA-only, S DNA prime and S1 protein plus Ribi adjuvant, or S1 protein plus Ribi adjuvant prime and boost were evaluated for neutralization activity against pseudotyped MERS-CoV England1 after adsorption with HEK 293T cell-surface-expressed MERS-CoV Spike proteins: S, RBD, S1, S2. Serum neutralization was tested at a single dilution. Sera adsorbed with untransfected HEK 293T cells served as controls and retained 95% of neutralization activity. Each bar represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown. (b) Protein competition neutralization assay. Sera at a single dilution from the immunized mice were also assayed for neutralization of MERS-CoV England1 pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteins at concentrations of 0.016–50 μg ml−1. Each data point represents the mean of triplicate assays with s.e.'s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown.
Mentions: We analysed the specificity of NAb responses against the different subunits of the MERS-CoV Spike glycoprotein using several methods. First, sera were pre-adsorbed with HEK 293T cells expressing transmembrane-anchored versions of S, S1, S2 and RBD (Fig. 1a and Supplementary Fig. 4a) and were screened for neutralization (Fig. 2a). Sera pre-adsorbed with untransfected cells retained 95% of their original neutralization activity, while negative control pre-immune sera showed no capacity for virus neutralization. The full-length Spike-transfected cells adsorbed virtually all neutralization activity from the immune sera of all three vaccine groups, while the RBD expressed by itself on the cell membrane only adsorbed about half of the neutralizing activity, and only in the S1 protein group, suggesting the possibility that the conformation of RBD presented in the full-length Spike and in the truncated versions may differ. Adsorption with S1 also removed all neutralization activities in the S1 protein group and ∼60% of neutralizing capacity in the two DNA-primed groups. In contrast, S2 did not deplete neutralization activity in any of the groups, except slightly in mice vaccinated with DNA alone. It is likely that expression of S2 by itself on cells resulted in rearrangement to the post-fusion conformation, and therefore neutralizing epitopes present on the prefusion structure would not have been available for adsorption. Flow cytometric analysis of serum bound to cells that expressed different Spike subunits gave consistent results (Supplementary Fig. 4c).

Bottom Line: Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses.Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates.Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.

No MeSH data available.


Related in: MedlinePlus