Miljø- og Fødevareudvalget 2020-21
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Preliminary rapport on SARS-CoV-2 spike mutations arising in Danish mink,
their spread to humans and neutralization data.
SARS-CoV-2 spike mutations arising in Danish mink and their spread to
humans
Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, DENMARK
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 MOF, Alm.del - 2020-21 - Endeligt svar på spørgsmål 226: MFU spm. om dokumentation, der konkluderer, at mutationen cluster 5 er en reel trussel for udviklingen af den danske og internationale COVID-19 vaccineudvikling, til sundheds- og ældreministeren
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Background
Despite control measures, SARS-CoV-2 continued to spread among mink farms across northern
Denmark, with more than 200 farms infected by November 2020. SARS-CoV-2 genome sequences
obtained from infected mink and humans living on the farms provided evidence of SARS-CoV-2 spread
between mink and human in zoonotic events. This study investigates the amino acid changes in the
spike surface glycoprotein that appeared during this outbreak and their effect on the antigenicity of
the SARS-CoV-2 virus.
Spike mutations
Within the infected mink, the SARS-CoV-2 virus mutated, giving rise to several amino acid changes in
the spike protein. The first was a tyrosine to phenylalanine at amino acid 453 (Y453F), a mutation that
also appeared during the Dutch mink farm outbreaks. It is a conservative amino acid substitution in
the receptor binding domain that directly contacts the host ACE2 receptor at amino acid 34 (Wang et
al). This ACE2 contact position differs between human and mink (histidine [34H] in humans and
tyrosine [34Y] in mink and other mustelids (Damas et al)), which suggests that Y453F is an adaptation
mutation to mink ACE2. Importantly, 453F increases affinity for human ACE2, which may explain its
successful introduction and establishment in humans.
Following the appearance of 453F, additional spike mutations were observed in minks and the humans
epidemiologically linked to the infected mink farms (Fig. 1). These include: i) 69-70deltaHV - a deletion
of a histidine and valine at amino acid positions 69 and 70 in the N-terminal domain of the S1 subunit;
ii) I692V
–
a conservative substitution at position 692 that is located seven amino acids downstream
of the furin cleavage site; iii) S1147L
–
a non-conservative substitution at position 1147 in the S2
subunit; and iv) M1229I
–
a conservative substitution located within the transmembrane domain.
Clinical isolates
Efforts are underway to isolate each mink-associated SARS-CoV-2 spike mutant strain that occurs in
people residing in Denmark. To date, Statens Serum Institut in Denmark has isolated two strains of
mink-associated SARS-CoV-2 viruses. These include an isolate with the 453F spike mutation (F-spike)
from cluster 1 and an isolate with a 69-70deltaHV, 453F, 692V, and 1229I mutation combination from
Cluster 5 (hereafter referred to as
ΔFVI-spike).
To ensure that subculturing of SARS-CoV-2 clinical
isolates on VeroE6 cells did not induce additional spike mutations, each isolate was sequenced. The
spike protein of the cultured virus was identical to that of the SARS-CoV-2 virus in the original clinical
sample.
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 MOF, Alm.del - 2020-21 - Endeligt svar på spørgsmål 226: MFU spm. om dokumentation, der konkluderer, at mutationen cluster 5 er en reel trussel for udviklingen af den danske og internationale COVID-19 vaccineudvikling, til sundheds- og ældreministeren
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The clinical isolates bearing the Y453F spike mutation replicated as efficiently as the
unmutated/wildtype SARS-CoV-2 virus that predominates in Denmark (data not shown). Conversely,
the SARS-CoV-2 virus with four mutations grew slower than both the wildtype virus and other SARS-
CoV-2 virus isolates (Fig. 2). The cytopathic effect (CPE) induced by the
ΔFVI-spike
mutant virus
appeared later and was less pronounced and had an approximate 10-fold lower titer 24 hours post-
inoculation compared to human SARS-CoV-2 isolates prepared under the same conditions (Fig. 2A). At
96 hours post-inoculation the
ΔFVI-spike
mutant virus titer was comparable to that of the wildtype
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Figure 1. The mink-associated mutations in the SARS-CoV-2 spike protein.
A) The combination and frequency
of mink-associated spike mutations detected in SARS-CoV-2 infected humans B) The crystal structure of a closed
prefusion spike trimer [PDB: 6ZGE] with the position of the Y453F variant in the receptor binding motif, the
position of two amino acids deleted in the N-terminal domain, and the position of the I692V variant. The regions
encompassing the S1147L and M1229I mutations are not within the crystal structure; however, their relative
positions are indicated. C) The position the Y453F variant in a receptor binding domain complexed with a host
ACE2 receptor [PBD: 6LZG].
 MOF, Alm.del - 2020-21 - Endeligt svar på spørgsmål 226: MFU spm. om dokumentation, der konkluderer, at mutationen cluster 5 er en reel trussel for udviklingen af den danske og internationale COVID-19 vaccineudvikling, til sundheds- og ældreministeren
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virus and exceeded other SARS-CoV-2 viruses isolated and subcultured under the same conditions (Fig.
2B). The
ΔFVI-spike
mutant virus titer increased 54.7-fold from 24 to 96 hours post-inoculation,
compared to an average of 4-fold (range: 2.6 to 5.7-fold) over the same time for other SARS-CoV-2
isolates. The ability to replicate to high viral titers is consistent with high levels of the
ΔFVI-spike
mutant virus detected in throat swab samples of infected persons, as indicated by an average qPCR
assay (E-Sarbeco) cycle threshold of 24.7 (range: 20-35). Further evaluation of the SARS-CoV-2
ΔFVI-
spike strain growth kinetics in other cells systems are warranted.
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Virus neutralization
The introduction of SARS-CoV-2 spike mutant viruses raises concerns about a potential reduced
recognition of the protein by antibodies induced after SARS-CoV-2 infection or vaccination that may
have implications for re-infections and vaccine efficacy, respectively. To evaluate the effect of the
mink-associated SARS-CoV-2 spike mutant viruses on antigenicity, neutralizing activity of convalescent
plasma from persons who recovered from a SARS-CoV-2 infection and sera from immunized rabbits
were compared between the
ΔFVI-spike
mutant virus and an unmutated wildtype virus.
The neutralization activity was tested using a micro-neutralization assay that was adapted from the
World Health Organization protocol for influenza virus neutralization. The assay was developed at
Statens Serum Institut and validated on >300 convalescent plasma/serum samples as well as sera from
vaccinated mice and rabbits. In brief, 2-fold serial dilutions of plasma/sera were pre-incubated with
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Figure 2. Growth kinetics of the SARS-CoV-2
ΔFVI-spike
mutant virus.
A) Virus titers 24h post-inoculation for
SARS-CoV-2 viruses isolated from clinical samples under the exact same conditions. Isolate 1-3 each have
different spike mutations unrelated to mink outbreaks, these include N439K (isolate 1), N439K+69-70delHV
(isolate 2), and S477N (isolate 3). B) The growth kinetics of the
ΔFVI-spike
mutant virus relative to other clinical
isolates, including the nonmutated virus (wildtype) that predominates in Denmark and spike mutant viruses
(isolate 1 and 2 as for [A]).
 MOF, Alm.del - 2020-21 - Endeligt svar på spørgsmål 226: MFU spm. om dokumentation, der konkluderer, at mutationen cluster 5 er en reel trussel for udviklingen af den danske og internationale COVID-19 vaccineudvikling, til sundheds- og ældreministeren
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SARS-CoV-2 virus for 1 hour before addition to a monolayer of VeroE6 cells prepared in 96-well plates.
After a 24 hour incubation, the cells were fixed to the plates and the level of virus determined using a
standard ELISA targeting the SARS-CoV-2 nucleocapsid protein. To determine the amount of virus to
add to the assay, clinical isolates are usually titrated at 24 hours and from these titers 100× TCID
50
virus used in the neutralization assay. This equates to approximately 300× TCID
50
from titers calculated
96 hours post-inoculation. Due to the difference in growth kinetics of the
ΔFVI-spike
mutant virus, the
TCID
50
titer calculated at 96 hours was deemed to reflect the amount of infectious particles in the virus
stock more accurately than that measured at 24 hours post-inoculation. Thus, each serum samples
were tested in duplicated with 300× TCID
50
as calculated from 96 hours post-inoculation titers.
The convalescent plasma was selected from persons living in the South of Denmark, geographically
separated from the mink outbreaks in the North of Denmark, and had a documented SARS-CoV-2
infection at the beginning of the Danish epidemic before the mink outbreaks occurred. Since the effect
of the spike mutations on different levels of neutralizing antibodies is unknown, sera with known low
(N=4), intermediate (N=3) and high (N=2) neutralization titers were tested. Each plasma sample
represents a different donor and was tested in duplicate.
The different convalescent plasma were not equally affected by the
ΔFVI-spike
mutant virus. The two
plasma samples with high neutralization titers were largely unaffected, while plasma with low and
intermediate titers were more likely to experience a loss in neutralization activity (Fig. 3a). In these
preliminary data from 9 convalescent plasma, an average 3.58-fold (range: 0 to 13.5) reduction was
observed. Only two plasma samples had a greater than 4-fold reduction, a threshold set for
neutralization resistance by Li et al. who evaluated other spike mutants presented on pseudovirus
particles. It is important to note that the findings are preliminary and warrant further investigation in
other SARS-CoV-2 neutralization assays.
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Figure 3. Neutralization of the SARS-CoV-2
ΔFVI-spike
mutant virus relative to an unmutated SARS-CoV-2
virus.
A) Convalescent plasma from nine individuals with known low, intermediate, or high neutralizing titers
were used to assess the effect of the spike mutations on neutralization activity of antibodies induced following
infection with an unmutated SARS-CoV-2 virus. The neutralization titer was determined as follows: a 50% cut-
off value was calculated using quadruplicate virus controls (prepared for each virus) and cell controls included
on each plate. The titer was calculated as the interpolation of a 5-parameter titration curve with the 50% cut-
off value. The reciprocal serum dilution is reported as the 50% neutralization antibody titre. B) The fold-change
in neutralization titer for the SARS-CoV-2
ΔFVI-spike
mutant virus relative to an unmutated SARS-CoV-2 virus.
The horizontal dotted line indicates a 4-fold reduction. The bars represent the mean of duplicate measurements
with the standard deviation.
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PRELIMINARY References
Wang et al (2020) Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2
Damas et al (2020) Broad host range of SARS-CoV-2 predicted by comparative and structural analysis
of ACE2 in vertebrates
Li et al (2020) The impact of mutations in SARS-CoV-2 spike on Viral Infectivity and Antigenicity. Cell
182, 1284-1294
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