Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region - PubMed (original) (raw)

. 2005 Mar;79(5):2678-88.

doi: 10.1128/JVI.79.5.2678-2688.2005.

Linqi Zhang, Chuan Qin, Lei Ba, Christopher E Yi, Fengwen Zhang, Qiang Wei, Tian He, Wenjie Yu, Jian Yu, Hong Gao, Xinming Tu, Agegnehu Gettie, Michael Farzan, Kwok-Yung Yuen, David D Ho

Affiliations

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region

Zhiwei Chen et al. J Virol. 2005 Mar.

Abstract

Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.

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Figures

FIG. 1.

FIG. 1.

Schematic representation of ADS-MVA construction (top and right panels) and the characterization of ADS-MVA by using avian cells (left bottom panel). The SARS-S gene was introduced, together with GFP gene, each under a separate promoter, into the Del III region of the MVA genome. (A) S glycoprotein was detected on CEF cells by using an anti-S polyclonal antibody (WH) in an immunofluorescence assay. (B) GFP coexpressed on the same cell population as seen in panel A.

FIG. 2.

FIG. 2.

Anti-SARS-CoV-specific neutralization antibody response in BALB/c mice after ADS-MVA vaccinations. Each mouse was designated with a number such as M260. The animals were immunized i.m. twice, 3 weeks apart, with 2 × 106 TCID50 (M260 and M262) or 2 × 107 TCID50 of ADS-MVA per mouse (M263, M264, and MM1 to MM4) or 2 × 107 TCID50 of ADC-MVA (M265 to M268). Two control mice (N1 and N2) only received saline under the same immunization schedule.

FIG. 3.

FIG. 3.

Anti-SARS-CoV specific neutralization antibody response in New Zealand White rabbits after ADS-MVA vaccinations. Each rabbit was designated with a number such as R524. The animals were immunized i.m. twice, 4 weeks apart, with 108 TCID50 of ADS-MVA per animal (R524 and R525) or 108 TCID50 of ADC-MVA (R520 and R521).

FIG. 4.

FIG. 4.

Anti-SARS-CoV-specific neutralization antibody response in Indian rhesus macaques after ADS-MVA vaccinations. Four animals (I624, N462, V876, and P283) were immunized i.m. twice, 4 weeks apart, with 1 × 108 and 3 × 108 TCID50 of ADS-MVA per animal, respectively. Another four macaques (AG11, AT14, I826, and V873) were treated the same but with ADC-MVA. (A) Neutralizing antibody responses after the first immunization. (B) Boosted neutralizing antibody responses after the second immunization.

FIG. 5.

FIG. 5.

Construction and expression of humanized fragmented S gene in the context of DNA vaccines. (A) Four S gene fragments (labeled A to D) were each constructed under cytomegalovirus (CMV) promoter in mammalian expression vector pcDNA3.1. (B) Fragmented S proteins of corresponding lengths were expressed in 293T cells in a Western blot analysis.

FIG. 6.

FIG. 6.

Anti-SARS-CoV-specific neutralization antibody response in New Zealand White rabbits after DNA vaccinations. Two animals per group were immunized twice, 4 weeks apart, using an in vivo electroporation technique with DNA vaccines S200 (R416 and R417), S400 (R418 and R419), S600 (R681 and R682), and S800 (R683 and R684). Another two rabbits were vaccinated with a DNA vaccine expressing HCVE1E2 gene (R518 and R519).

FIG. 7.

FIG. 7.

Absorption and removal of anti-SARS-CoV-specific neutralization antibodies of New Zealand White rabbits (A), BALB/c mice (B), and monkeys (C) by an ACE2 receptor-binding region and human Fc fusion protein (RBR-Fc). For rabbit and mouse sera, the neutralization antibody-RBR-Fc complex was removed by agarose beads that were precoated with specific anti-human Fc antibodies. The anti-human Fc antibodies do not cross-react with rabbit or mouse immunoglobulin but do cross-react with monkey samples. Therefore, for monkey sera, neutralization antibodies were removed by beads directly conjugated with RBR-Fc. In each experiment, the corresponding beads controls did not affect the activity of neutralization antibodies, as presented in groups of RBR-Fc(−). The experiment was carried out in triplicates, and the average values are presented. The error bars stand for the standard deviation.

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