Abstract
SARS-CoV-2 causes COVID-19 pandemic and continues to pose a threat to
global public health through genetic mutation. In this study, we have
found that an ACE2-specific monoclonal antibody at low concentration was
able to greatly enhance SARS-CoV-2 infection and growth in cell culture.
Strikingly, it promotes SARS-CoV-2 plaque formation, resulting in
accurate titration of different SARS-CoV-2 variants, particularly the
newly emerged Omicron variants, which otherwise cannot be determined by
standard plaque assays. Quantification of infectious titers of the newly
emerged variants will facilitate the development and evaluation of
vaccines and antiviral drugs against SARS-CoV-2.
Emerging viral infections continue to pose a major threat to global
public health. The coronavirus disease 2019 (COVID-19) pandemic caused
by a newly emerged severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) has unprecedently resulted in hundreds of millions of
infections and millions of deaths worldwide since its outbreak in
December 2019 (1). The virus belongs to the family ofCoronaviridae , consisting of 4 genera: Alpha, Beta, Gamma, and
Delta coronaviruses. Seven of them were found to infect humans,
including CoV-229E, CoV-NL63, CoV-HKU1, CoV-OC43, SARS-CoV, MERS-CoV,
and SARS-CoV-2 (2). Coronaviruses are enveloped RNA viruses with a
single-stranded RNA genome in positive polarity. Like other enveloped
viruses, SARS-CoV-2 enters the cell through receptor(s)-mediated
endocytosis (3, 4), which depends on both angiotensin-converting enzyme
2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) (5). Upon
internalization, viral RNA genomes replicate in the cytoplasm. The
genomic RNA acts as an mRNA for translation of polyprotein 1a/1ab, which
encodes nonstructural proteins to form the replication‐transcription
complex. It also serves as the template for synthesis of a nested set of
subgenomic RNAs (sgRNAs) by discontinuous transcription. The
minus‐strand sgRNAs serve as the templates to produce subgenomic mRNAs
for production of different viral proteins. Nascent RNA genomes are
synthesized from the full-length minus-strand RNA and are assembled with
viral structural proteins to form progeny virions egressing from
infected cells (6).
Various methods have been developed for rapid diagnosis of SARS-CoV-2
infection by detecting viral proteins and genomic RNAs (7). Serological
methods have also been used for detection and titration of viral
proteins-specific antibodies produced among people infected with
SARS-CoV-2. In general, accurate quantification of infectious SARS-CoV-2
is more complex, requiring amplification and titration of viruses in
cell culture (8). Both plaque-forming units and the 50% tissue culture
infectious dose (TCID50), are routinely used for
measuring infectious SARS-CoV-2 based on its induction of cytopathic
effect (CPE) . However, TCID50 is more qualitative rather than
quantitative as a measure of infectious SARS-CoV-2 (9). Most of the
recently emerged variants of SARS-CoV-2 are attenuated and do not form
clear plaques even using the most sensitive Vero E6 cells that
overexpress the SARS-CoV-2 receptors ACE2 and TMPRSS2 (Fig. 2). A
reliable method to quantify infectious SARS-CoV-2 is urgently needed for
development and evaluation of COVID19 vaccines and antiviral drugs.
Over the course of our studies on SARS-CoV-2 infection, we have found
that the growth of SARS-CoV-2 was significantly enhanced by an
ACE2-blocking monoclonal antibody(10108-MM37, Sino Biological). This
observation is exemplified by a dose-dependent growth enhancement of
both SARS-CoV-2 wild type and Omicron BA.2 variant in Vero E6 cells that
overexpress both ACE2 and TMPRSS2 (Fig. 1). The levels of viral NP
protein of SARS-CoV-2 wild type and BA2 variant were increased to 34 and
78 folds, respectively, by the anti-ACE2 antibody at a concentration of
0.25 μg/mL (Fig. 1A). The infectious titers of wild type SARS-CoV-2 and
BA2 variant were also enhanced by 10,000 and 100,000 folds,
respectively, at 0.25 μg/mL of the antibody (Fig 1B). Subsequently, we
tested the antibody for its enhancement of SARS-CoV-2-induced plaque
formation. Strikingly, the anti-ACE2 antibody promoted plaque formation
of SARS-CoV-2 wild type and various variants when 0.2 μg/mL of the
antibody was added to the agarose overlay, as shown by large plaques
with uniform sizes at the bottom half of each 6-well plate (Fig. 2).
Both plaque size and numbers were significantly increased, particularly
for Omicron variants (BA1, BA2, BA4, BA5, and BF5) that otherwise did
not form clear plaques in the absence of the anti-ACE2 antibody (top
half of each 6-well plate). Both alpha and delta variants formed smaller
and highly heterogeneous plaques in the absence of anti-ACE2 antibody.
More significantly, the addition of the ACE2-blocking antibody resulted
in a more accurate titration with uniform plaque sizes like that of wild
type SARS-CoV-2. The numbers of plaques formed by SARS-CoV-2 variants
were much less compared to that formed in the presence of anti-ACE2
antibody. Additionally, the ACE2-blocking antibody induced a more rapid
plaque formation, requiring only 2 days instead of routinely 3 days to
form large plaques. However, the concentration of anti-ACE2 monoclonal
antibody is critical as it inhibits virus growth and plaque formation at
higher (> 0.5 μg/mL) concentrations. In general, 0.2 μg/mL
of anti-ACE2 antibody resulted in large and uniform plaques of all
SARS-CoV-2 variants tested in this study.
It was previously reported that ACE2 autoantibody could be detected
among some COVID-19 patients (10). The question arose whether ACE2
autoantibody developed in patients is associated with COVID-19 disease
severity by enhancing SARS-CoV-2 infection. This possibility is
warranted for futureP investigation. Nevertheless, our in vitrostudy demonstrated that anti-ACE2 antibody can promote SARS-CoV-2 growth
and formation of plaques with large, clear, and uniform sizes, resulting
in accurate titration of all infectious SARS-CoV-2 isolates, which
otherwise cannot be accomplished by standard plaque assays.