Dear Editor,
An outbreak of unusual pneumonia in Wuhan, China recently was caused by infection
of a novel type of corona-virus. The virus and disease were denoted as 2019-nCoV and
COVID-19, respectively, by the World Health Organization (WHO). Most recently, 2019-nCoV
was renamed SARS-CoV-2 by Coronaviridae Study Group (CSG) of the International Committee
on Taxonomy of Viruses (ICTV) (Gorbalenya et al.
2020), or HCoV-19, as a common name for the consistence with COVID-19, by a group
of virologists in China (Jiang et al.
2020a, b). As of 7 March 2020, a total of 80,651 confirmed cases, including 3070 deaths,
were reported in China (China CDC 2020). Global spread is undeniable with serious
implications for public health, thus calling for rapid development of effective therapeutics
and prophylatics (Jiang et al.
2020a, b).
Very recently, it was found that horse anti-SARS-CoV and convalescent SARS patient
sera had neutralizing effects against 2019-nCoV at a low dilution of 1:80 and 1:25,
respectively (Zhou et al.
2020; Hoffmann et al.
2020). Although several SARS-CoV receptor-binding domain (RBD)-specific human monoclonal
antibodies (mAbs), such as S230, m396 and 80R, exhibited no cross-reactivity with
2019-nCoV RBD (Wrapp et al.
2020). CR3022, a SARS-CoV RBD-specific human mAb, was reported to bind with the RBD
of spike (S) protein in 2019-nCoV with potential cross-neutralizing activity (Tian
et al.
2020). These studies suggest that the anti-SARS-CoV antibodies might be useful for
the treatment of 2019-nCoV infection and people with history of SARS-CoV infection
many years ago might be resistant to 2019-nCoV infection.
The S protein in coronavirus plays an essential role in virus entry, and it is also
the main target of neutralizing antibodies (Du et al.
2009). Sequence alignment indicated a 75.9% amino acid sequence identity between the
S protein of SARS-CoV and that of 2019-nCoV (Supplementary Figure S1, Supplementary
Table S1). SARS-related coronavirus (SARSr-CoV) WIV1 and Rs3367 have 92.3% amino acid
sequence identity to SARS-CoV in S protein, while those between SARS-CoV and MERS-CoV
is 28.7% (Supplementary Figure S1, Supplementary Table S1). It is worth noting that
the RBDs of SARS-CoV and 2019-nCoV exhibited a significant difference, although they
bind the same receptor (angiotensin-converting enzyme 2, ACE2) during virus infection
(Zhou et al.
2020). Therefore, despite sequence and receptor similarities, it is unclear whether
the anti-SARS-CoV serum produced by immunization with S protein-based vaccine could
cross-neutralize 2019-nCoV infection.
We previously developed SARS pseudovirus (SARS-PsV) and MERS pseudovirus (MERS-PsV),
which were prepared by cotransfecting the plasmids of pNL4-3.luc.RE and pcDNA3.1-SARS,
or MERS-CoV-S protein, to HEK293T cells (Fig. 1A) (Xia et al.
2019). These pseudoviruses (PsVs) bear the S protein and a defective HIV-1 genome,
including a luciferase reporter gene, and could simulate the virus to infect target
cells, thus having native conformations (Fig. 1A) (Xia et al.
2019). Consequently, we believed these PsVs to be potential immunogens to produce
neutralizing antibodies. Female Balb/C mice (n = 5) were subcutaneously injected with
SARS-PsV, MERS-PsV or PBS as a control using Freund’s complete adjuvant for the prime
immunization and vaccinated at 28 days after the first immunization using Freund’s
incomplete adjuvant for the boost immunization (Fig. 1B). Sera samples were collected
from day 7 after the final vaccination.
Fig. 1
Neutralizing effects of sera from mice treated with SARS-PsV or MERS-PsV against SARS-PsV,
MERS-PsV, 2019-nCoV-PsV, WIV1-PsV and Rs3367-PsV infection. A Schematic representation
of the production of SARS-PsV and MERS-PsV. B The immunization procedure. C Detection
of sera from SARS-PsV-, MERS-PsV- and PBS-treated mice for neutralization activity
against SARS-PsV infection. D Assessment of sera from SARS-PsV-treated mice for neutralizing
antibody titers against SARS-PsV infection. E Detection of sera from SARS-PsV-, MERS-PsV-
and PBS-treated mice for neutralizing antibody titers against MERS-PsV infection.
F Assessment of sera from MERS-PsV-treated mice for the neutralizing antibody titers
against MERS-PsV infection. G Detection of sera from SARS-PsV-, MERS-PsV- and PBS-treated
mice for neutralization activity against 2019-nCoV-PsV infection. H–I Detection of
sera from SARS-PsV-, MERS-PsV- and PBS-treated mice for neutralization activity against
WIV1-PsV and Rs3367-PsV, respectively. J–K Detection of sera from SARS-PsV-treated
mice for neutralization activity against WIV1-PsV and Rs3367-PsV infection. I NT50
value of mouse sera agaisnt SARS-PsV, MERS-PsV, WIV1-PsV, Rs3367-PsV and 2019-nCoV-PsV
infection. GraphPad Prism version 5.0 was used to perform all statistical analyses.
ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
We first tested day-35 sera for neutralization of SARS-PsV infection using ACE2-expressing
293T cells. As shown in Fig. 1C, compared with the PBS-treated group, significant
neutralization (P < 0.0001) was observed in the sera from SARS-PsV-treated mice at
dilution of 1:100, 1:200, 1:400 and 1:800. The percent of inhibition of SARS-PsV infection
in sera from the SARS-PsV-vaccinated group could reach ~ 97%, even at a dilution of
1:800. Then, the NT50s (50% neutralizing antibody titers) of these sera were further
tested. As shown in Fig. 1D, the mean NT50 of the sera was 41,425. The sera from MERS-PsV-treated
mice, as negative control, did not reach our NT50 threshold at a dilution of 1:100
and did not show a significant difference (P > 0.5) compared with the PBS-treated
control group. Then, we tested the neutralization effects of the sera against MERS-PsV
infection using the Huh-7 cell line. We found significant MERS-PSV neutralization
effects in the sera from MERS-PsV-treated mice (Fig. 1E). The NT50 of these sera was
also detected as 5945 (Fig. 1F). However, the sera from SARS-PsV-treated mice could
not significantly inhibit MERS-PsV infection, even at a dilution of 1:100 (Fig. 1E).
In spite of the high neutralization titer in both groups, we did not observe a significant
cross-neutralization effect.
To assess whether the SARS-PsV-treated mouse sera could show cross-neutralization
effects for 2019-nCoV, we tested the inhibition effects of all sera using a 2019-nCoV
pseudovirus system and Huh-7 cells as the target cell. No neutralization was detected,
either for sera from SARS-PsV-treated mice or for MERS-PsV-treated mice, even at a
dilution of 1: 100, compared with the sera from PBS-treated mice (Fig. 1G).
To investigate the ability to neutralize SARSr-CoVs, we further tested the inhibitory
activities against two SARSr-CoVs, WIV1 and Rs3367. Interestingly, sera from SARS-treated
mice could significantly neutralize WIVI-PsV and Rs3367-PsV with ~ 92% and ~ 87% inhibitory
activities at a dilution of 1:800 (Fig. 1H and 1I). However, no neutralization was
observed for MERS-PSV-treated mice sera, even at a dilution of 1:100, compared with
the sera from PBS-treated control (Fig. 1H and 1I). Then, we further investigated
whether the sera from SARS-CoV spike protein-treated mice could also cross-neutralize
bat SARSr-CoV, WIV1-PsV and Rs3367-PsV with high titers. The NT50s for WIV1-PsV and
Rs3367-PsV were 8787 and 7345, respectively (Fig. 1J and 1K), demonstrating that sera
from mice treated with SARS-CoV S protein could potently cross-neutralize infection
by SARS-CoV and SARSr-CoVs, but weakly for 2019-nCoV infection, although its S protein
sequence is highly similar to that of SARS-CoV spike protein (Supplementary Figure
S1, Supplementary Table S1).
Taken together, our results indicated that the sera of mice treated with pseudotyped
SARS-CoV exhibited low titer of 2019-nCoV neutralization activity (< 1:100), implying
that it may not be practical to treat 2019-nCoV infection with anti-SARS-CoV antibodies
and that people with history of SARS-CoV infection many years ago may not be resistant
to 2019-nCoV infection. However, we cannot exclude the possibility that the low titer
of the cross-neutralizing antibody is due to the low density of S protein on the surface
of pseudovirus particles. Furthermore, modifying or reconstructing the SARS-CoV S
protein may enhance the exposure of conformational neutralizing epitopes in RBD, in
order to increase its immunogenicity for eliciting higher cross-neutralizing antibody
responses against 2019-nCoV and emerging/reemerging coronaviruses in the future.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Supplementary material 1 (PDF 844 kb)