- La variante Omicron del SARS-CoV-2 alberga 34 mutaciones en la espiga, más que otras variantes
- Dos dosis de vacunas basadas en ARNm provocan una pobre neutralización de Omicron
- Tres dosis de vacunas de ARNm provocan una potente neutralización cruzada de la variante, incluida Omicron
- El pseudovirus Omicron infecta las células con mayor eficacia que otras variantes del SARS-CoV-2
The SARS-CoV-2 Omicron variant harbors 34 mutations in the spike, more than other variants
Two doses of mRNA-based vaccines elicit poor neutralization of Omicron
Three mRNA vaccine doses elicit potent variant cross-neutralization, including Omicron
The Omicron pseudovirus infects cells more efficiently than other SARS-CoV-2 variants
Summary
Recent
surveillance has revealed the emergence of the SARS-CoV-2 Omicron
variant (BA.1/B.1.1.529) harboring up to 36 mutations in spike protein,
the target of neutralizing antibodies. Given its potential to escape
vaccine-induced humoral immunity, we measured the neutralization potency
of sera from 88 mRNA-1273, 111 BNT162b, and 40 Ad26.COV2.S vaccine
recipients against wild-type, Delta, and Omicron SARS-CoV-2
pseudoviruses. We included individuals that received their primary
series recently (<3 months), distantly (6–12 months), or an
additional “booster” dose, while accounting for prior SARS-CoV-2
infection. Remarkably, neutralization of Omicron was undetectable in
most vaccinees. However, individuals boosted with mRNA vaccines
exhibited potent neutralization of Omicron, only 4–6-fold lower than
wild type, suggesting enhanced cross-reactivity of neutralizing antibody
responses. In addition, we find that Omicron pseudovirus infects more
efficiently than other variants tested. Overall, this study highlights
the importance of additional mRNA doses to broaden neutralizing antibody
responses against highly divergent SARS-CoV-2 variants.
La
vigilancia reciente ha revelado la aparición de la variante Omicron del
SARS-CoV-2 (BA.1/B.1.1.529) que alberga hasta 36 mutaciones en la
proteína spike, el objetivo de los anticuerpos neutralizantes. Dado su
potencial para escapar a la inmunidad humoral inducida por la vacuna,
medimos la potencia de neutralización de los sueros de 88 receptores de
la vacuna mRNA-1273, 111 BNT162b y 40 Ad26.COV2.S contra los pseudovirus
SARS-CoV-2 de tipo salvaje, Delta y Omicron. Se incluyeron individuos
que recibieron su serie primaria recientemente (<3 meses), a
distancia (6-12 meses), o una dosis adicional de "refuerzo", teniendo en
cuenta la infección previa por SARS-CoV-2. Sorprendentemente, la
neutralización de Omicron fue indetectable en la mayoría de los
vacunados. Sin embargo, los individuos reforzados con vacunas de ARNm
mostraron una potente neutralización de Omicron, sólo 4-6 veces inferior
a la del tipo salvaje, lo que sugiere una mayor reactividad cruzada de
las respuestas de anticuerpos neutralizantes. Además, descubrimos que el
pseudovirus Omicron infecta más eficazmente que otras variantes
probadas. En general, este estudio subraya la importancia de las dosis
adicionales de ARNm para ampliar las respuestas de anticuerpos
neutralizantes contra las variantes altamente divergentes del
SARS-CoV-2.
The
SARS-CoV-2 Omicron variant (BA.1/B.1.1.529) was first detected in
Botswana and reported to the World Health Organization (WHO) in November
2021 as a novel variant with an unprecedented number of previously
described and novel mutations with immunevasive potential. A subsequent
and rapid increase in Omicron cases in South Africa resulted in its
designation as a novel variant of concern (VOC) by the WHO (www.who.int).
This variant harbors up to 59 mutations throughout its genome, with as
many as 36 of these occurring within the spike protein, the mediator of
host cell entry and the main target of neutralizing antibodies. Studies
of previous SARS-CoV-2 variants have demonstrated that mutations within
the receptor binding domain (RBD) mediate escape from vaccine-induced
neutralizing antibodies
La
variante Omicron del SARS-CoV-2 (BA.1/B.1.1.529) se detectó por primera
vez en Botsuana y se notificó a la Organización Mundial de la Salud
(OMS) en noviembre de 2021 como una nueva variante con un número sin
precedentes de mutaciones nuevas y descritas previamente con potencial
inmunoevasivo. Un posterior y rápido aumento de los casos de Omicron en
Sudáfrica dio lugar a su designación como nueva variante preocupante
(VOC) por parte de la OMS (www.who.int). Esta variante alberga hasta 59
mutaciones en todo su genoma, de las cuales hasta 36 se producen en la
proteína spike, el mediador de la entrada en la célula huésped y el
principal objetivo de los anticuerpos neutralizantes. Los estudios de
variantes anteriores del SARS-CoV-2 han demostrado que las mutaciones
dentro del dominio de unión al receptor (RBD) median el escape de los
anticuerpos neutralizantes inducidos por la vacuna
).
Omicron RBD contains 15 mutations, some of which overlap with
previously studied variants. For example, Beta (B.1.351) and Gamma (P.1)
harbor mutations in residues K417, E484, and N501 that potently
diminish vaccine-induced neutralization (
In
the United States, three vaccines have been approved by the FDA or are
under emergency use authorization (EUA), all of which use the original
wild-type SARS-CoV-2 spike protein first identified in Wuhan, China, as
the sole immunogen. These are formulated as spike-encoding mRNA in lipid
nanoparticles (BNT162b2 manufactured by Pfizer-BioNTech and mRNA-1273
manufactured by Moderna) or as an adenovirus vectored vaccine
(Ad26.COV2.S manufactured by Janssen/Johnson & Johnson) (
).
These SARS-CoV-2 vaccines have been remarkably successful in inducing
neutralizing humoral and cellular immunity and, more importantly,
reducing COVID-19 infections, hospitalizations, and deaths in clinical
trials (
).
However, it has now been shown that neutralizing antibody responses and
vaccine effectiveness vary by vaccine agent, decrease with increased
time post vaccination, and are negatively impacted by emerging variants (
).
In an effort to combat waning antibody responses and the emergence of
new variants, a third dose of mRNA vaccine (“boosters”) has been
approved for individuals vaccinated >6 months ago and has been shown
to be very effective at inducing high neutralizing antibody titers (
).
In the case of Ad26.COV2.S vaccinees, cross-over mRNA doses were
recommended >2 months from primary vaccination. However, while
neutralization of wild-type SARS-CoV-2 has been shown to predict the
effectiveness of vaccines against variants (
We
previously developed and validated a high-throughput pseudovirus
neutralization assay to understand differences in immunity by vaccine
and host characteristics against SARS-CoV-2 variants and other
coronaviruses (
).
Here, we used this assay to test sera from 239 individuals who had been
fully vaccinated with one of three vaccines approved in the United
States—mRNA-1273, BNT162b2, or Ad26.COV2.S—against wild-type, Delta, and
Omicron SARS-CoV-2 pseudoviruses. This included 70 individuals who had
received a third dose of mRNA vaccine after primary vaccination series
as either a cross-over or solely mRNA-based vaccination regimen.
Remarkably, we found that all three primary vaccine series resulted in
low-to-absent neutralization of SARS-CoV-2 Omicron. However, mRNA
vaccine recipients who received a third dose exhibited potent
neutralization against Omicron, despite exhibiting wild-type
neutralization titers similar to those in two-dose vaccinated
(“non-boosted”) individuals. In addition, in vitro infection
experiments demonstrated that the Omicron pseudovirus continues to rely
upon the human ACE2 receptor for target cell entry and infects target
cells 4-fold more efficiently than wild-type pseudovirus and 2-fold more
efficiently than Delta pseudovirus. Together, our results highlight
that the SARS-CoV-2 Omicron variant evades vaccine-induced neutralizing
immunity under current vaccine regimens and is more infectious than
previous variants. Nonetheless, our finding of potent cross-neutralizing
immunity against Omicron in individuals that received a third dose of
mRNA vaccine suggests that existing vaccines may overcome evasion of
humoral immunity by future variants of concern.
Results
Emergence of SARS-CoV-2 Omicron as a novel and highly mutated VOC
Over
the course of more than 270 million confirmed SARS-CoV-2 infections
worldwide, the virus has undergone remarkable diversification, producing
>1,500 uniquely identified Pango lineages (
) (coronavirus.jhu.edu).
Some of these have demonstrated evidence of increased transmissibility,
virulence, and/or immune evasion, prompting the WHO to classify five
lineages as current VOCs (www.who.int).
The Omicron variant, also known as PANGO lineage BA.1 or B.1.1.529, was
first reported in November 2021 and received its VOC designation within
days on account of its unique mutational profile and the dramatic rise
in cases observed in Gauteng, South Africa. While the Delta variant is
now the dominant SARS-CoV-2 variant worldwide after overtaking the Alpha
variant in July 2021, the rise of Omicron infections in regions where
Delta is circulating suggests that Omicron may overtake Delta to become
the next dominant strain. Despite the substantial recent expansion of
the Delta lineage, phylogenetic analysis suggests that the Omicron
variant was derived from the Alpha lineage and only recently detected by
genomic surveillance (Figure 1A).
In comparison to the nine mutations or deletions found in Delta, the
Omicron lineage we tested harbors 34 mutations (including three
deletions and one insertion) in the spike protein, including 15 within
the RBD region (Figure 1B).
These mutations are structurally focused at the top of the spike, in
regions accessible to antibodies, raising the likelihood of immune
evasion (Figure 1C).
Figure 1Emergence of SARS-CoV-2 Omicron among global variants of concern
Neutralizing antibody responses to SARS-CoV-2 variants demonstrate substantial escape by Omicron
We
accrued a diverse cohort of 239 COVID-19 vaccinees that were healthcare
workers and/or community dwellers from Boston or Chelsea, Massachusetts
(Table S1).
The entire cohort had a median age of 38 years (range: 18–78 years) and
was 63% female. Vaccinees had received a full series of mRNA-1273,
BNT162b, or Ad26.COV2.S and were subdivided into the following
subgroups: infection-naive individuals that received their primary
vaccination series within the last 3 months (“recent vax”),
infection-naive individuals that received their primary vaccination
series 6–12 months before (“distant vax”), individuals that received
their primary vaccination series 6–12 months before and had a history of
self-reported positive PCR and/or serologic evidence of SARS-CoV-2
infection as measured by anti-nucleocapsid antibodies (“distant vax +
infection”), and infection-naive individuals that received an additional
mRNA vaccine dose within the last 3 months (“booster vax”) (Figure 2A).
Figure 2Additional
mRNA vaccine dose (“booster”) induces potent neutralizing responses
against SARS-CoV-2 Omicron variant that are low-to-absent in primary
series (“non-boosted”) vaccinees
) to assess SARS-CoV-2 variant neutralization (Figure 2B).
In brief, pseudovirus encoding luciferase and bearing SARS-CoV-2
variant spike proteins were exposed to dilutions of vaccinee sera prior
to being added to ACE2-expressing target cells. After 48 h of
co-culture, luciferase activity of the dilution series was measured to
quantify infection rates and calculate the titer that achieved 50%
pseudovirus neutralization (pNT50). This pNT50 value was subsequently
converted to WHO IU/mL after correcting with a WHO reference standard
that was run in parallel (
),
individuals that were recently vaccinated with mRNA-1273 or BNT162b
achieved substantially higher wild-type neutralization titers than
Ad26.COV2.S vaccinees, with geometric mean neutralization titers (GMNTs)
of 1,362 IU/mL for mRNA-1273, 2,402 IU/mL for BNT162b, and 42 IU/mL for
Ad26.COV2.S (Figure 2C).
Individuals vaccinated >6 months prior exhibited substantially lower
but mostly detectable wild-type neutralization (GMNT 192 IU/mL for
mRNA-1273, 78 IU/mL for BNT162b, and 33 IU/mL for Ad26.COV2.S) (Figure 2C).
Prior history of infection was associated with high levels of wild-type
neutralization titers even in distantly vaccinated individuals,
particularly in Ad26.COV2.S vaccinees (GMNT 904 IU/mL for mRNA-1273, 947
IU/mL for BNT162b, and 603 IU/mL for Ad26.COV2.S) (Figure 2C).
However, recently boosted individuals exhibited among the highest
neutralization titers against wild-type SARS-CoV-2 pseudovirus (GMNT
3,862 IU/mL for mRNA-1273, 2,219 IU/mL for BNT162b, and 1,201 IU/mL for
Ad26.COV2.S) (Figure 2C).
Neutralization of the Delta variant pseudovirus was decreased relative to wild type for all subgroups (Figure 2C), as has been previously reported (
).
While Delta neutralization became undetectable in most individuals
vaccinated >6 months before blood draw, Delta neutralization was
detectable and only modestly decreased in recently vaccinated,
previously infected, and recently boosted vaccinees (Figure 2C).
In contrast, Omicron neutralization was dramatically decreased among
all subgroups, including recently vaccinated mRNA-1273 and BNT162b
recipients, which demonstrated a complete loss of neutralization in
>50% of individuals and GMNT decrease of 43-fold for mRNA-1273 and
122-fold for BNT162b (Figure 2C).
Previously infected vaccinees also had a substantial decrease in
Omicron neutralization titer (GMNT decrease of 9-fold for mRNA-1273,
12-fold for BNT162b, and 17-fold for Ad26.COV2.S), but most retained
detectable neutralization (Figure 2C).
Remarkably, however, recently boosted vaccinees exhibited potent
neutralization of Omicron variant pseudovirus that was only moderately
decreased relative to wild-type neutralization (GMNT decrease of 6-fold
for mRNA-1273, 4-fold for BNT162b, and 13-fold for Ad26.COV2.S) (Figure 2C).
Of note, among boosted vaccinees, all boosters were homologous (same
vaccine) except for 1 of 33 mRNA-1273 vaccinees that crossed-over to
BNT162b, 6 of 30 BNT162b vaccinees that crossed-over to mRNA-1273, and 7
of 8 Ad26.COV2.S vaccinees that crossed-over to mRNA-1273. Taken
together, our results indicate that two-dose mRNA-based vaccines are
effective at inducing neutralizing immunity to SARS-CoV-2 wild-type and
Delta variants but suboptimal for inducing neutralizing responses to the
Omicron variant.
Additional “booster” dose of mRNA vaccine increases breadth and cross-reactivity of neutralizing antibody response
Given
the drastic increase in cross-neutralization of the SARS-CoV-2 Omicron
pseudovirus in boosted versus non-boosted vaccinees, we directly
compared sera from individuals that recently received their primary
series to those that were boosted with an mRNA vaccine within the last
3 months. Wild-type pseudovirus neutralization was comparable between
individuals who received three versus two doses of either mRNA vaccine
(GMNT increase of 3-fold for mRNA-1273 and 1-fold for BNT162b). The
difference in neutralization of Delta pseudoviruses between three-dose
and two-dose vaccinees was similar for mRNA-1273 (GMNT increase of
3-fold) but increased in BNT162b vaccinees (GMNT increase of 9-fold).
Cross-neutralization of the Omicron variant was substantially higher in
individuals who received three doses of either mRNA vaccine (GMNT
increase of 19-fold for mRNA-1273 and 27-fold for BNT162b) (Figure 3A).
Interestingly, Ad26.COV2.S vaccinees boosted with mRNA-1273 showed
substantially higher wild-type, Delta, and Omicron pseudovirus
neutralization relative to those who received Ad26.COV2.S alone (Figure 3A).
Figure 3Cross-reactivity
of neutralizing antibody response is increased by an additional dose
(“booster”) of mRNA vaccine relative to primary vaccination series and
can be predicted by anti-spike antibody levels
To
better characterize the neutralization patterns observed between
individuals who were fully vaccinated with each of the three approved
vaccines and those who were boosted, we directly compared the wild-type
neutralization activity of these two groups of samples against the Delta
and Omicron pseudoviruses (Figure 3B).
Interestingly, we found that wild-type neutralization titers from
individuals who received their primary series correlated weakly to Delta
variant cross-neutralization (R2 = 0.35; slope = 0.44; p < 0.0001) and did not correlate with Omicron variant cross-neutralization (R2 = 0.03; slope = 0.05; p = 0.16). In contrast, wild-type neutralization of boosted individuals correlated strongly with Delta (R2 = 0.68; slope = 1.00; p < 0.0001) and Omicron (R2 =
0.56; slope = 0.94; p < 0.0001) variant cross-neutralization. This
indicates that, in addition to inducing higher neutralization titers
against wild-type SARS-CoV-2, boosting increases the breadth of humoral
immunity and cross-reactivity against highly mutated SARS-CoV-2 variants
such as Omicron.
Neutralization of SARS-CoV-2 variants can be predicted by anti-spike antibody levels
Given
the widespread accessibility of anti-SARS-CoV-2 spike serological
assays in the clinical setting, we correlated the neutralization signal
obtained by our high-throughput assay to measurements made by the Roche
Elecsys anti-SARS-CoV-2 spike semi-quantitative immunoassay in the
clinical laboratory. As expected, we observed higher neutralization of
wild-type, Delta, and Omicron pseudoviruses with higher levels of
anti-spike antibodies among all vaccinees (Figure 3C).
Receiver operating characteristic (ROC) analysis was performed to
assess how well anti-spike antibody levels performed at predicting
positive neutralization of wild-type, Delta, and Omicron pseudoviruses,
which was defined as neutralization titer >33 IU/mL (based on a
previously established cohort of 1,200 pre-pandemic samples (
)
and converted to WHO IU/mL). Area under curve (AUC) for wild type was
0.97, Delta was 0.91, and Omicron was 0.84. The optimal cut-offs that
maximized sensitivity (Se) and specificity (Sp) were determined using
the “Se + Sp” method, resulting in the following: for wild type, optimal
cut-off of 711 U/mL achieved 88.4% Se and 96.7% Sp; for Delta, optimal
cut-off of 1,591 U/mL achieved 88.4% Se and 83.8% Sp; and for the
Omicron variant, optimal cut-off of 10,300 achieved 67.2% Se and 90.6%
Sp (Figures 3C
and 3D). This highlights the potential use of this widely available
clinical diagnostic test in predicting SARS-CoV-2 variant
neutralization.
Omicron retains ACE2 usage and exhibits higher infection rates than other SARS-CoV-2 variants in vitro
Having
detected a substantial degree of evasion of vaccine-induced humoral
responses by Omicron, we next investigated how mutations in Omicron
might result in changes in infectivity compared with wild-type
SARS-CoV-2. To investigate whether the spike of circulating variants
mediate host cell entry via ACE2, we examined the ability of
pseudoviruses bearing wild-type, Delta, and Omicron spike to infect
293T-ACE2 cells or parental 293T cells devoid of ACE2 receptor (Figure 4A).
None of the pseudoviruses tested infected 293T cells in the absence of
ACE2 protein, confirming that, like other SARS-CoV-2 strains, Omicron
uses ACE2 for host cell entry (Figure 4B).
To comprehensively determine quantitative differences in the
infectivity of SARS-CoV-2 variants of concern, we compared the
efficiency of a panel of pseudovirus variants to infect 293T-ACE2 cells
over a range of viral concentrations. Remarkably, Omicron pseudovirus
exhibited greater infection of target cells regardless of concentration
when compared with all other tested variants (Figure 4C).
Comparison of the linear regressions of each pseudovirus to wild type
over the entire range revealed that, whereas the Gamma variant exhibited
similar infection rates to wild type, Beta showed less infection, and
Delta was nearly 2-fold more efficient at infecting target cells.
Strikingly, Omicron exhibited infection rates that were 4-fold higher
than wild type and 2-fold higher than Delta. Taken together, these data
strongly suggest distinct differences in infectivity according to spike
sequence, with Omicron exhibiting more efficient ACE2-mediated infection
than wild type or other variant strains (Figure 4D).
Figure 4SARS-CoV-2 Omicron pseudovirus demonstrates a substantial increase in infectivity of ACE2+ cells relative to other SARS-CoV-2 variants in vitro
Prior
characterization of vaccine-induced humoral immunity against SARS-CoV-2
variants of concern revealed significant loss of activity against the
Gamma and Beta variants, owing largely to three mutations in the RBD
region of the spike (
).
Given the 15 mutations present in the RBD of Omicron, which overlap the
three sites mutated in Gamma and Beta (K417, E484, and N501), as well
as prior work done to understand the potential for antibody escape (
),
it was anticipated that this variant would be significantly more
resistant to neutralization by vaccinee sera. To experimentally validate
this assumption, we generated a pseudovirus with the Omicron spike
incorporating 34 distinct mutations (including three deletions and one
insertion) relative to the Wuhan spike. Importantly, our spike did not
contain R346K, which has been observed at relatively low frequency
(<10%) within the Omicron lineage, but which is known to mediate
escape from class 3 neutralizing monoclonal antibodies such as
AZD1061/cilgavimab (
As
stated in our previous study, given the assumption of a polyclonal
response to vaccination, we would have initially anticipated that small
numbers of variations in spike protein would have only modest effects on
recognition by the immune system (
).
However, our earlier findings suggested that the response to primary
vaccination was insufficient to effectively neutralize spikes containing
a relatively small number of mutations. Consistent with this
observation, we found that the Omicron variant, harboring substantially
more mutations than prior variants, efficiently escapes humoral immunity
induced by primary vaccination. Strikingly, however, additional mRNA
vaccine doses appear to enable cross-neutralizing responses against
Omicron, either by further affinity maturation of existing antibodies or
targeting of new epitopes shared among variants.
Our
study included distantly vaccinated individuals (>6 months) with
evidence of prior infection. However, our approach could not distinguish
between infection prior to vaccination or asymptomatic breakthrough
infection. Irrespective of this distinction, these individuals exhibited
higher levels of neutralizing activity against the wild-type, Delta,
and Omicron pseudoviruses as compared with distantly vaccinated,
infection-naive individuals. These results suggest that additional
exposure to viral antigens through infection yields enhanced overall
neutralization activity. It is important to note, however, that an
additional dose of mRNA vaccine in infection-naive vaccinees yielded
substantially higher cross-neutralizing activity against Omicron as
compared with prior infection.
Interestingly,
vaccinated individuals who received an additional “booster” mRNA
vaccine dose generated the most potent and cross-reactive antibody
responses of any cohort tested, including individuals who recently
received their primary series. The effect was similar to that observed
following mRNA vaccine boosting in rhesus macaques (
).
This substantial difference in Omicron cross-neutralization was
surprising, given that two-dose and three-dose vaccine regimens elicited
comparable levels of wild-type neutralization and because the immunogen
was identical for all doses, which would be expected to elicit memory
responses from the existing, strain-specific antibody repertoire. This
difference in cross-reactivity between vaccinees that recently received
three versus two doses of vaccine could be the consequence of a third
dose amplifying existing, low-level neutralizing antibodies that target
conserved epitopes on spike protein or increasing the affinity of
existing neutralizing antibodies so as to render them less sensitive to
mutations in their target epitopes, or both. Although the emergence of
variants has catalyzed the development of variant-specific “booster”
shots to increase variant neutralization, our results suggest that
current wild-type-based mRNA vaccines are sufficient to stimulate
cross-reactive humoral responses greater than might have been
anticipated. Whether this is the result of third-dose timing or because
the primary series was simply insufficient to fully stimulate broadly
neutralizing B cell responses to spike antigen is unclear.
We
also found marked differences between approved vaccine regimens, with
mRNA-1273 and BNT162b2 vaccines offering significantly greater humoral
immune responses against all variants than Ad26.COV2.S, as has been
previously described (
).
Recipients of mRNA vaccines following Ad26.COV2.S exhibited markedly
improved neutralizing titers but did not achieve the breadth of response
seen with individuals that received three doses of mRNA vaccine. Our
results would suggest that these recipients of Ad26.COV2.S vaccines may
benefit from additional mRNA vaccine doses with the potential to further
raise titers and broaden their neutralizing activity.
Several
reports have measured the decay of neutralizing antibody responses
raised by mRNA COVID-19 vaccines with a half-life of 69–173 days (
).
Our results from distantly vaccinated samples are in line with these
reports, given that neutralization activity against wild-type
pseudovirus was more than 10-fold lower in distantly vaccinated
individuals. Given the activity generated against Omicron, it will be
important to determine the longevity of cross-neutralizing antibody
responses in individuals that received a third dose of mRNA vaccine, as
this could have a meaningful impact on vaccine efficacy as future
variants of concern continue to emerge.
In support of the rapid spread of Omicron globally (
), our in vitro
studies strongly suggest that Omicron spike mediates more efficient
target cell entry, resulting in increased infectivity. Interestingly,
while the Beta variant previously exhibited escape from vaccine-induced
humoral immunity, we found that infectivity of Beta was lower than wild
type, perhaps explaining the relatively lower epidemic spread of Beta.
In contrast, the now globally dominant Delta strain exhibited 2-fold
increased efficiency of pseudovirus entry relative to wild type,
consistent with prior studies reporting more efficient spike fusion that
may have contributed to its rapid spread (
).
Additional studies examining the interaction between Omicron spike and
ACE2, as well as other potential interaction partners, will be necessary
to discern whether more efficient infection of the SARS-CoV-2
pseudovirus is also observed in replication-competent viruses.
Taken
together, we demonstrate that Omicron drastically escapes
vaccine-induced immunity after primary vaccination series with mRNA-1273
(Moderna), BNT162b2 (Pfizer-BioNTech), or Ad26.COV2.S (Johnson &
Johnson/Janssen) and exhibits increased pseudovirus infection rates in vitro,
raising the potential for increased transmissibility. It is worth
noting that, despite escape from humoral immunity, Omicron breakthrough
infections may result in attenuated disease severity in vaccinees due to
pre-existing cellular and innate immunity. However, neutralization
remains the leading correlate of protection from infection, and this
study demonstrates that receiving a third dose of an mRNA-based vaccine
effectively yields a potent cross-neutralizing response against
SARS-CoV-2 Omicron, likely through increasing breadth and
cross-reactivity of neutralizing antibodies. These findings support the
need for rapid and synchronized widespread deployment of additional mRNA
vaccine doses as a public health measure to curtail the emergence and
spread of highly mutated SARS-CoV-2 variants.
Limitations of the study
Although
previous studies that used pseudovirus neutralization to model the
sensitivity of replicating SARS-CoV-2 to neutralizing antibodies have
shown excellent correlations (
),
it is possible that the mutations in the Omicron spike protein may
cause the Omicron pseudovirus to behave differently than previously
tested variants. However, recent reports have demonstrated similar loss
of neutralizing activity by vaccinee sera against intact Omicron
coronavirus (
).
In addition, while we confirmed that ACE2 expression is required for
infection of 293T cells, natural target cells in the respiratory tract
may express alternative receptors or attachment factors that facilitate
infection and are not adequately modeled in our system. In addition, our
cohort was cross-sectional and not longitudinal, which limits our
ability to estimate changes in neutralization titers over time across
single individuals. Furthermore, we did not assess other
antibody-mediated functions, such as complement deposition,
antibody-dependent cellular cytotoxicity, or antibody-dependent cellular
phagocytosis, which may contribute to protection even in the absence of
neutralizing antibodies. We did not assess the role of vaccine-elicited
cellular immune responses mediated by T cells and NK cells, which are
likely to play a key role in disease prevention for vaccine recipients.
Further
information and requests for resources and reagents should be directed
to, and will be fulfilled by, Alejandro Balazs ( abalazs@mgh.harvard.edu
).
Materials availability
All
unique plasmids generated in this study are available through Addgene.
Antibodies and other reagents are available from their respective
sources.
Experimental model and subject details
Human subjects
Use
of human samples was approved by Partners Institutional Review Board
(protocol 2020P002274). Serum samples from 239 COVID-19 vaccinees were
collected. For each individual, basic demographic information including
age and sex as well as any relevant COVID-19 history was obtained.
Cell lines
HEK293T
cells (ATCC) were cultured in DMEM (Corning) containing 10% fetal
bovine serum (VWR), and penicillin/streptomycin (Corning) at 37°C/5% CO2.
293T-ACE2 cells were a gift from Michael Farzan (Scripps Florida) and
Nir Hacohen (Broad Institute) and were cultured under the same
conditions. Confirmation of ACE2 expression in 293T-ACE2 cells was done
via flow cytometry.
Method details
Construction of variant spike expression plasmids
To
create Delta and Omicron variant spike expression plasmids, we
performed multiple PCR fragment amplifications utilizing
oligonucleotides containing each desired mutation (Azenta) and utilized
overlapping fragment assembly to generate the full complement of
mutations for each strain. Importantly, we generated these mutations in
the context of our previously described codon-optimized SARS-CoV-2 spike
expression plasmid harboring a deletion of the C-terminal 18 amino
acids that we previously demonstrated to result in higher pseudovirus
titers. Assembled fragments were inserted into NotI/XbaI digested
pTwist-CMV-BetaGlobin-WPRE-Neo vector utilizing the In-Fusion HD Cloning
Kit (Takara). All resulting plasmid DNA utilized in the study was
verified by whole-plasmid deep sequencing (Illumina) to confirm the
presence of only the intended mutations.
SARS-CoV-2 pseudovirus neutralization assay
To
compare the neutralizing activity of vaccinee sera against
coronaviruses, we produced lentiviral particles pseudotyped with
different spike proteins as previously described (
).
Briefly, pseudoviruses were produced in 293T cells by PEI transfection
of a lentiviral backbone encoding CMV-Luciferase-IRES-ZsGreen as well as
lentiviral helper plasmids and each spike variant expression plasmid.
Following collection and filtering, production was quantified by
titering via flow cytometry on 293T-ACE2 cells. Neutralization assays
and readouts were performed on a Fluent Automated Workstation (Tecan)
liquid handler using 384-well plates (Grenier). Three-fold serial
dilutions ranging from 1:12 to 1:8,748 were performed for each serum
sample before adding 50–250 infectious units of pseudovirus for 1 h.
Subsequently, 293T-ACE2 cells containing polybrene were added to each
well and incubated at 37°C/5% CO2 for 48 h. Following transduction, cells were lysed using a luciferin-containing buffer (
)
and shaken for 5 min prior to quantitation of luciferase expression
within 1 h of buffer addition using a Spectramax L luminometer
(Molecular Devices). Percent neutralization was determined by
subtracting background luminescence measured in cell control wells
(cells only) from sample wells, and dividing by virus control wells
(virus and cells only). Data were analyzed using Graphpad Prism, and
NT50 values were calculated by taking the inverse of the 50% inhibitory
concentration value for all samples with a neutralization value of 80%
or higher at the highest concentration of serum. NT50 values were
converted to WHO International Units using the Human SARS-CoV-2 Serology
Standard (Lot #COVID-NS01097) obtained from NCI-Frederick National
Laboratory for cancer research, which was calibrated to the WHO
SARS-CoV-2 Serology International Standard (20/136).
Titering and flow cytometry
To
determine the infectious units and infection rates of SARS-CoV-2
pseudovirus, we plated 400,000 293T-ACE2 cells per well of a 12-well
plate. SARS-CoV-2 pseudovirus supernatants that were produced in
parallel under identical conditions (as described above) were added 24 h
later in three ten-fold serial dilutions. Cells were incubated for 48 h
at 37°C/5% CO2 to allow for expression of ZsGreen reporter
gene and harvested with Trypsin-EDTA (Corning). Cells were resuspended
in PBS supplemented with 2% FBS (PBS+), and analyzed on a Stratedigm
S1300Exi Flow Cytometer to determine the percentage of ZsGreen+
cells. Infectious units were calculated by determining the percentage
of infected cells in wells exhibiting linear decreases in transduction
and multiplying by the average number of cells per well determined at
the initiation of the assay. At low MOI (infection rates <10%), each
transduced ZsGreen+ cell was assumed to represent a single
infectious unit. Surface expression of ACE2 on 293T and 293T-ACE2 cells
was confirmed by staining with 1 μL of anti-human ACE2, Alexa Fluor
647-conjugated antibody (R&D). Cells were incubated for 10 min at
25°C prior to running on a Stratedigm S1300Exi Flow Cytometer.
Quantitation of pseudovirus by RT-qPCR
To
determine the genome copy concentration of pseudotyped lentiviral
vectors, lentiviral RNA was extracted from pseudovirus supernatant using
the QIAamp viral RNA mini kit (Qiagen). Each sample was serially
diluted, and each dilution was treated with 1.2 U of Turbo DNase
(Invitrogen) at 37°C for 30 min followed by heat inactivation at 75°C
for 15 min. 10 μL of the treated RNA was used in a 20 μL qRT-PCR
reaction with the qScript XLT one-step RT-qPCR Tough Mix, low ROX mix
(Quanta Biosciences), a TaqMan probe containing locked nucleic acids
(/56-FAM/AGC+C/i5NitInd/GG+GA/ZEN/GCTCTCTGGC/3IABkFQ/) (IDT), and
primers designed for targeting the LTR gene of NL4-3 HIV genome, from
which the lentiviral vector was derived (5′-GGTCTCTCTIGITAGACCAG and
3′-TTTATTGAGGCTTAAGCAGTGGG). Each dilution was run in duplicate on a
QuantStudio 12K Flex (Applied Biosystems). The following cycling
conditions were used: 50°C for 10 min, 95°C for 3 min followed by 50
cycles of 95°C for 15 s and 60°C for 1 min. Virus titer was determined
by comparison with a standard curve generated using a plasmid standard
generated from serial dilution of CMV-Luciferase-IRES-ZsGreen lentiviral
backbone. DNase and No DNase controls were also included at 2.5 × 108 GC/mL of the same plasmid. The range of the assay was from 2.5 × 107 GC/mL to 1.5 × 103
GC/mL. Upon analysis, the average of the three most concentrated
dilutions within range of the standard were used to calculate genome
copies/mL.
Anti-SARS-CoV-2 spike and nucleocapsid antibody assays
The
EUA-approved electrochemiluminescence-based Roche Elecsys®
anti-SARS-CoV-2 spike antigen (semi-quantitative) and nucleocapsid
antigen (qualitative) immunoassays were used to detect total antibodies
(IgG, IgM, and/or IgA antibodies) to SARS-CoV-2 spike and nucleocapsid
in vaccinee sera. The upper limit of detection for the spike
semi-quantitative assay was 25,000 U/mL. The assay was run on a Roche
Cobas 8000 e801 Immunoassay Analyzer in the Massachusetts General
Hospital Core Laboratory.
Quantification and statistical analysis
Data
and statistical analyses were performed using GraphPad Prism 9.2.0, JMP
Pro 16.1.0 (SAS Institute), and R v4.0.2. Flow cytometry data were
analyzed using FlowJo 10.7.1. Student’s t tests were performed in the
indicated figures; all p values are unadjusted. Statistical significance was defined as p < 0.05. Error bars throughout all figures represent one standard deviation or standard error of the mean where indicated.
Data and code availability
This
study did not generate sequence data or code. Data generated in the
current study (including neutralization and antibody measurements) have
not been deposited in a public repository but are available from the
lead contact upon request.
Acknowledgments
We
thank Anand Dighe, MD; Andrea Nixon, BS; and the MGH Core Laboratory
for excellent assistance with clinical SARS-CoV-2 serology testing. We
wish to thank Michael Farzan, PhD, for providing ACE2-expressing 293T
cells. A.H. is supported by the DZIF (German Center for Infection
Research, TTU 01.709 ; 8009701709 ). B.M.H. is supported by award
number T32GM007753 from the National Institute of General Medical
Sciences and F30 AI160908 from the National Institute of Allergy and
Infectious Diseases . J.F. is supported by T32AI007245 . D.J.G. and
M.C.P. were supported by the VIC Innovation Fund . A.G.S. was supported
by NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis
Readiness (MassCPR) grant. A.H. is supported by the DZIF (German
Center for Infection Research, TTU 01.709 ). This work was supported by
the Peter and Ann Lambertus Family Foundation . V.N. received support
from a Medscape Young Investigators Lung Cancer Award . A.B.B. was
supported by the National Institute on Drug Abuse (NIDA) Avenir New
Innovator Award DP2DA040254 , the MGH Transformative Scholars Program
, and a Massachusetts Consortium on Pathogenesis Readiness (MassCPR)
grant.
Author contributions
W.F.G.B.,
K.J.S.D., E.C.L., V.N., and A.B.B. designed the experiments. W.F.G.B.,
K.J.S.D., E.C.L., M.L.S., A.D.N., and A.B.B. carried out experiments and
analyzed data. W.F.G.B., V.N., C.B., O.O., C.C.C., D.J.G., M.C.P., and
A.J.I. led and performed cohort recruitment and sampling. J.F., B.M.H.,
and A.G.S. provided key reagents and useful discussions and insights.
A.L.R. performed sequence validation of plasmids. A.B.B., V.N.,
W.F.G.B., A.D.N., and A.H. contributed to statistical and sequence
analyses. A.J.I. and V.N. provided key discussions and input into
experimental design. W.F.G.B., V.N., A.H., and A.B.B. wrote the paper
with contributions from all authors.
Declaration of interests
The authors declare no competing interests.
Inclusion and diversity
We
worked to ensure gender balance in the recruitment of human subjects.
We worked to ensure ethnic or other types of diversity in the
recruitment of human subjects. We worked to ensure that the study
questionnaires were prepared in an inclusive way. One or more of the
authors of this paper self-identifies as an underrepresented ethnic
minority in science. One or more of the authors of this paper
self-identifies as a member of the LGBTQ+ community.
Comparative
effectiveness and antibody responses to moderna and Pfizer-BioNTech
COVID-19 vaccines among hospitalized veterans—five Veterans Affairs
Medical Centers, United States, February 1–September 30, 2021.
Retroviruses
pseudotyped with the severe acute respiratory syndrome coronavirus
spike protein efficiently infect cells expressing angiotensin-converting
enzyme 2.
Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.
Cell.2021; 184: 2348-2361.e6
Traditional regimens of COVID-19 vaccines do not neutralize the fast-spreading variant
ByMGH News and Public Affairs
Date
An additional “booster” dose of Moderna or Pfizer mRNA-based
vaccine is needed to provide immunity against the Omicron variant of
SARS-CoV-2, the virus that causes COVID-19, according to a study by
researchers at the Ragon Institute of MGH, MIT and Harvard. The results
of this study, reported in the journal Cell,
indicate that traditional dosing regimens of COVID-19 vaccines
available in the United States do not produce antibodies capable of
recognizing and neutralizing the Omicron variant.
In late November, health officials in South Africa reported that a
previously unknown variant of SARS-CoV-2 was rapidly spreading
throughout the country. The variant, given the name Omicron by the World
Health Organization, would soon prove to be far more transmissible than
Delta, the variant that previously had caused the majority of COVID-19
infections. “People desperately wanted to know whether current vaccines
protect against Omicron,” says the senior author of the Cell paper,
Alejandro Balazs, whose laboratory at the Ragon Institute investigates
how to engineer immunity against infectious diseases.
To find answers, Balazs collaborated with a team that included the
lead author of the Cell paper, Wilfredo F. Garcia-Beltran, a clinical
pathology resident at MGH and a clinician-scientist fellow at the Ragon
Institute. The first step was to construct a harmless version of Omicron
known as a “pseudovirus” that could be used in the laboratory to
evaluate the effectiveness of the three COVID-19 vaccines available in
the United States, which include the two-dose Pfizer and Moderna
injections and the one-dose Johnson & Johnson vaccine. The
pseudovirus that Balazs and colleagues created mimicked the behavior of
Omicron, which has 34 mutations on its “spike” protein that are not
found on the original strain of SARS-Cov-2 first detected in Wuhan,
China, in December 2019. Scientists believe that these mutations may be
partially responsible for Omicron’s rapid spread throughout the world.
Next, Garcia-Beltran worked with colleagues at MGH, including
hematology-oncology fellow Vivek Naranbhai, to acquire blood samples
from 239 individuals who had been fully vaccinated with one of the three
COVID-19 vaccines. The study subjects included employees within the
Massachusetts General Brigham health care system and residents of
Chelsea, Massachusetts, a community with a high rate of COVID-19
infections. “It was important to us to have a diverse population
represented in the study,” says Garcia-Beltran. Included in this group
were 70 men and women who had received a third booster dose of either
the Pfizer or Moderna vaccine, according to recommendations by the
Centers for Disease Control and Prevention.
The blood samples were used to measure how effectively each vaccine
induces production of protective immunity in the form of antibodies
against the Omicron pseudovirus, as well as the Delta and wild type
viruses. The results were striking. “We detected very little
neutralization of the Omicron variant pseudovirus when we used samples
taken from people who were recently vaccinated with two doses of mRNA
vaccine or one dose of Johnson & Johnson,” says Balazs. “But
individuals who received three doses of mRNA vaccine had very
significant neutralization against the Omicron variant.”
It’s not yet clear why an mRNA booster dramatically improves immune
protection against Omicron, but Garcia-Beltran says one possibility is
that an additional dose creates antibodies that bind more tightly to the
spike protein, increasing their effectiveness. Also, a booster dose may
generate antibodies that target regions of the spike protein that are
common to all forms of SARS-CoV-2. Both theories may be true, says
Garcia-Beltran.
Balazs notes that the three-dose mRNA vaccine regimen — that is, the
traditional two doses and a booster of Pfizer or Moderna vaccines —
provides somewhat lower levels of neutralizing antibodies against
Omicron than it does against the COVID-19 wild type strain or Delta
variant. But the study’s results strongly support the CDC’s advice that
COVID-19 booster shots are appropriate for anyone 16 and older, and that
mRNA vaccines are preferred.
Balazs is a principal investigator at the Ragon Institute and an
assistant professor of Medicine at Harvard Medical School.
Garcia-Beltran recently established his own laboratory at the Ragon
Institute.
This work was supported by the Peter and Ann Lambertus Family
Foundation, the Massachusetts Consortium on Pathogenesis Readiness, the
National Institutes of Health, and the German Center for Infection
Research.
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