Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein

Abstract

The recent identification of a novel human coronavirus responsible of a SARS-like illness in the Middle-East a decade after the SARS pandemic, demonstrates that reemergence of a SARS-like coronavirus from an animal reservoir remains a credible threat. Because SARS is contracted by aerosolized contamination of the respiratory tract, a vaccine inducing mucosal long-term protection would be an asset to control new epidemics. To this aim, we generated live attenuated recombinant measles vaccine (MV) candidates expressing either the membrane-anchored SARS-CoV spike (S) protein or its secreted soluble ectodomain (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S induced the highest titers of neutralizing antibodies and fully protected immunized animals from intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted recombinant Ssol protein, recombinant MV induced stronger and Th1-biased responses, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine.

Keywords: Coronavirus; Measles vaccine; Severe acute respiratory syndrome; Spike glycoprotein.

Fig. 1

Characterization of MVSchw-SARS recombinant viruses expressing the full-length transmembrane spike glycoprotein (S) or its secreted ectodomain (Ssol). (A) Schematic representation of the pTM-MVSchw-ATU2 vector containing the Schwarz MV cDNA with a green fluorescent protein (eGFP) gene as an additional transcription unit (ATU). Codon-optimized synthetic genes coding for the full-length S protein or secreted ectodomain (Ssol) were inserted into pTM-MVSchw between the BsiWI and BssHII sites of the ATU, in place of the eGFP gene. MV genes are indicated: N (nucleoprotein), PVC (phoshoprotein and V/C accessory proteins), M (matrix), F (fusion), H (hemaglutinin), L (polymerase). T7: T7 RNA polymerase promoter. hhR: hammerhead ribozyme. T7t: T7 RNA polymerase terminator. h∂vR: hepatitis delta virus (HDV) ribozyme. (B) Growth kinetics of recombinant MVSchw-SARS viruses. Vero cells were infected with the parental MVSchw (open circles) or recombinant MV-S (filled squares) or MV-Ssol (filled triangles) viruses at an M.O.I. of 0.01. At the indicated time points, cells were collected and cell-associated virus titers were determined as described in Materials and methods. (C) Immunostaining of spike polypeptides in syncytia of MVSchw-SARS-infected Vero cells. Cells were fixed 24–48 h after infection with the indicated viruses and permeabilized with triton X-100 (upper panels) or non-permeabilized (lower panels), then labeled as described in Materials and methods with anti-S mouse polyclonal antibodies and Cy3-conjugated anti-mouse IgG antibodies. (D) Lysates and supernatants of Vero-NK cells infected with MVSchw-SARS viruses were analyzed by western blot as described in Materials and methods using rabbit polyclonal antibodies specific for the S protein. As controls, whole cell lysates prepared from uninfected or SARS-CoV infected VeroE6 cells were analyzed. The positions of full-length S and Ssol polypeptides as well as molecular weight markers (kDa) are shown.

Fig. 2

Antibody response in IFN-α⧸βR^−/− CD46^+/− mice immunized with MVSchw-SARS recombinant viruses. Groups of 6 IFN-α⧸βR^−/− CD46^+/− mice were injected twice intraperitoneally at four-week interval with 10⁵ TCID₅₀ of the indicated recombinant MVSchw-SARS measles viruses or with parental MVSchw, as control. Another group of mice was immunized with two intramuscular injections of 2 µg of purified Ssol polypeptide adjuvanted with 50 µg Alum. Sera were collected before immunization (PI) or 3 weeks after each injection (IS1 and IS2, respectively). SARS-CoV-specific (A) or MV-specific (B) IgG antibody titers were determined by indirect ELISA, as described in Materials and methods. (C) SARS-CoV-specific, IgG1 (filled circles) and IgG2a (open circles) isotype titers were determined for each IS2 serum by indirect ELISA. (D) SARS-CoV-specific IgA antibody titers were determined for each IS2 serum by indirect ELISA. Values obtained for each individual mouse are represented with circles, with means for each group of mice shown by horizontal bars. Detection limits of the assays are indicated by dotted lines.

Fig. 3

Neutralizing antibody response in IFN-α⧸βR^−/− CD46^+/− mice immunized with MVSchw-SARS recombinant viruses. SARS-CoV neutralizing antibody titers were determined for each IS2 serum as the reciprocal of the highest dilution of serum which completely prevented SARS-CoV cytopathic effect in 50% of the wells. Values obtained for each individual mouse are represented with circles, with means for each group of mice shown by horizontal bars. Detection limits of the assays are indicated by dotted lines.

Fig. 4

Protection of MVSchw-SARS-immunized mice from intranasal challenge with SARS-CoV. Five weeks after the second injection of MVSchw-SARS recombinant viruses or Ssol polypeptide, mice were subjected to intranasal challenge with 10⁵ pfu of SARS-CoV. SARS-CoV infectious titers (expressed as log₁₀ pfu/lungs for individual mice) were dermined in lung homogenates collected two days after challenge, using plaque assay on Vero cells. Values for each individual mouse are represented with filled circles, and means for each group by horizontal bars. The detection limits of the assays are indicated by dotted lines. Data shown are from one experiment representative of two.