Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

Abstract

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Expression and purification of SARS S and S/M1 chimeric VLP. (A) pFastBac1 baculovirus transfer vector for SARS S/Flu M1 chimeric VLP vaccine: SARS S protein with the influenza A/Indonesia/5/2005 hemagglutinin (HA) transmembrane and carboxyl terminus (TM/CT) and the M1 influenza matrix protein 1. Each gene is under the control of its own polyhedrin promoter; (B) pFastBac1 baculovirus transfer vector for wild type full length SARS S protein with its native TM/CT; (C) purified SARS VLP vaccine, left panel: coomassie blue stain, right panel: Western blot using anti-S and anti-M1 antibodies. Positions of the 160 kDa SARS S protein and the 25 kDa M1 protein are labeled. Bands labeled # and * were identified as baculovirus gp64 protein and insect cell β tubulin protein, respectively; (D) purified native SARS S protein, left panel: coomassie blue stain, right panel: Western blot using anti-SARS S antibody. Lane M contains precision plus protein molecular weight marker (Bio-Rad, Hercules, CA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Transmission electron microscopy (EM) analysis of SARS VLPs. (A) Phosphotungstic acid negative stain of EM image of SARS VLPs; (B) immuno EM image of SARS VLPs using anti-SARS S protein primary antibody and 6 nm colloidal gold labeled secondary antibody. Black dots indicated gold labeled antibody binding to the SARS S protein. Bar: 100 nm.

Fig. 3

Survival rate and lung virus titer of mice intramuscularly (IM) immunized with SARS S protein vaccine or SARS VLP vaccine after SARS-CoV lethal challenge. Mice were immunized (IM) with vehicle, 0.8 μg or 4 μg of SARS S protein or SARS VLPs, with or without aluminum hydroxide, on days 0 and 21 (n = 14 or 15). Mice were challenged with 2 LD50 of SARS-CoV strain v2163 on day 42. Five mice per group were sacrificed on day 45. Lung tissues were collected, homogenized, and assayed for virus titer. The remaining mice were monitored for survival and body weight from day 42 to day 63. (A) Survival percentage curve; (B) survival table with the mean day of death; (C) animal weight change for 21 days post challenge; (D) lung SARS-CoV titer at day 45, 3 days post challenge. Virus titer was calculated as log10 CCID50/gram lung tissue with limit of detection as 0.75 (dashed line). Results were expressed as mean titer ± SEM. Statistical significance between the vehicle and vaccine groups was determined by the student t test. **p < 0.01, ***p < 0.001.

Fig. 4

Survival rate and lung virus titer of mice intranasally (IN) immunized with SARS S protein vaccine or SARS VLP vaccine after SARS-CoV lethal challenge. Mice were immunized (IN) with vehicle, 0.8 μg or 4 μg of SARS S protein or SARS VLPs, without adjuvant, on day 0 and 21 (n = 14 or 15). Mice were challenged with 2 LD50 of SARS-CoV strain v2163 on day 42. Five mice per group were sacrificed on day 45. Lung tissues were collected and assayed for virus titer. The remaining mice were monitored for survival and body weight from day 42 to day 63. (A) Survival percentage curve; (B) survival table with the mean day of death; (C) animal weight change for 21 days after challenge; (D) lung SARS-CoV titer at day 45, 3 days post challenge. Virus titer was calculated as log10 CCID50/gram lung tissue. Results were expressed as mean titer ± SEM. Statistical significance between the vehicle and vaccine groups was determined by the student t test. ***p < 0.001.

Fig. 5

Neutralizing antibody titer of mice intramuscularly (IM) immunized with SARS S protein vaccine or SARS VLP vaccine. Mice were immunized (IM) with vehicle, 0.8 μg or 4 μg of SARS S protein or SARS VLPs, with or without aluminum hydroxide, on days 0 and 21. Mice were bled on day 21 (grey bar) and 42 (black bar). Sera samples were 2 fold serially diluted and assayed for neutralizing antibodies. SARS-CoV neutralizing antibody titer was defined as the inverse of the greatest dilution of serum that neutralized 100% and had no visible virus CPE. Results are expressed as the geometric mean titer ± SEM. A 1:20 dilution was the lowest dilution of serum tested. Statistical significance between vehicle and vaccine groups for day 42 sera samples was determined by the student t test. *p < 0.05.