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Assessment of hybrid population
immunity to SARS‑CoV‑2
following breakthrough infections
of distinct SARS‑CoV‑2 variants
by the detection of antibodies
to nucleoprotein
Gerco den Hartog
1,2,5, Stijn P. Andeweg
3,5, Christina E. Hoeve
3, Gaby Smits
1,
Bettie Voordouw
4, Dirk Eggink
4, Mirjam J. Knol
3 & Robert S. van Binnendijk
1*
Immunity induced by vaccination and infection, referred to as hybrid immunity, provides better
protection against SARS‑CoV‑2 infections compared to immunity induced by vaccinations alone. To
assess the development of hybrid immunity we investigated the induction of Nucleoprotein‑specic
antibodies in PCR‑conrmed infections by Delta or Omicron in vaccinated individuals (n = 520).
Eighty‑two percent of the participants with a breakthrough infection reached N‑seropositivity.
N‑seropositivity was accompanied by Spike S1 antibody boosting, and independent of vaccination
status or virus variant. Following the infection relatively more antibodies to the infecting virus variant
were detected. In conclusion, these data show that hybrid immunity through breakthrough infections
is hallmarked by Nucleoprotein antibodies and broadening of the Spike antibody repertoire. Exposure
to future SARS‑CoV‑2 variants may therefore continue to maintain and broaden vaccine‑induced
population immunity.
A large part of the global population has acquired immunity through vaccination, infection or a combination
of both i.e. hybrid immunity against SARS-CoV-2 in late 20221. Especially Omicron variants have shown their
potential to escape vaccine-induced humoral immunity, resulting in many vaccine breakthrough infections and
the development of hybrid immunity2–7. Previous infection with Omicron protects against subsequent infections
by other Omicron variants, and this protection may be better than hybrid immunity induced by SARS-CoV-2
variants preceding Omicron8,9. How Omicron-induced hybrid immunity protects against future variants remains
to be seen.
Knowledge about immunological protection induced by vaccines, previous infection, or hybrid immunity is of
great importance for COVID-19 intervention policies and further understanding of immunological mechanisms
protecting against infectious diseases. Besides that SARS-CoV-2 infection is expected to broaden the immune
response as it taps into new antigenic epitopes presented to the immune system, another mechanism explaining
increased protection by hybrid immunity is believed to be enhanced mucosal immunity resulting in better local
protection against the virus10.
Assessment of the development of hybrid immunity in the population requires a clear identication of
a passed SARS-CoV-2 infection. Such information has oen been obtained from testing registries based on
OPEN
1Centre for Immunology of Infectious Diseases and Vaccines, Centre for Infectious Disease Control, National
Institute for Public Health and the Environment, Bilthoven, The Netherlands. 2Laboratory of Medical Immunology,
Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The Netherlands. 3Centre for Infectious
Diseases, Epidemiology and Surveillance, Centre for Infectious Disease Control, National Institute for Public
Health and the Environment, Bilthoven, The Netherlands. 4Centre for Infectious Diseases Research, Diagnostics
and Laboratory Surveillance, Centre for Infectious Disease Control, National Institute for Public Health and the
Environment, Bilthoven, The Netherlands. 5These authors contributed equally to the study: Gerco den Hartog and
Stijn P. Andeweg. *email: rob.van.binnendijk@rivm.nl
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diagnostic SARS-CoV-2 RT-PCR and rapid antigen testing. However, testing behavior and policy varies over
time and since April 2022 community testing has been scaled down. Serological testing for virus-induced anti-
bodies could be an alternative to detect SARS-CoV-2 infections and can usually be detected many months aer
virus exposure.
Immunogenic SARS-CoV-2 proteins that are absent in most vaccines, such as Nucleoprotein (N), can be
regarded as a potential tool to identify the development of hybrid immunity through breakthrough infections
in a vaccinated population11. Both the development of Spike-antibody mediated hybrid immunity and induction
of detectable N-specic antibodies require immune activation through replication of SARS-CoV-2 aer break-
through infection. Sucient immune activation aer breakthrough infection may be limited in a proportion of
the vaccinated population, due to e.g. presence of vaccine-induced S-specic antibodies that may reduce viral
replication, thereby also reducing de novo induced N-specic antibodies following breakthrough infection12,13.
Identication of breakthrough infections by antibodies to non-vaccine viral antigens would allow for research
further elucidating of characteristics of the development of hybrid immunity and also shed light on risk factors for
breakthrough infection, e.g. pre-infection antibody levels, virus variants, comorbidities or vaccination status14,15.
Next to the induction of N-specic responses, breakthrough infections resulting in immune activation will
most likely also boost antibodies towards the spike protein, a mechanism probably casually related to the devel-
opment of hybrid immunity. Quantifying the additional immune boosting of vaccine targets by an infection
in primary and booster vaccination recipients with and without previous infection provides insight into how
breakthrough infections support better immunity. Epidemiological studies show a benet of hybrid immunity
over vaccine- or infection-induced immunity9,16,17. ese ndings are substantiated by immunological data
showing high levels of antibodies and neutralization in vaccinated persons with a history of infection prior to
vaccination15. e level and duration of boosting by infections in vaccinated persons is largely unknown, as well
as the factors inuencing the response.
A major factor for breakthrough infections involves the genetic dri of virus strains, resulting in their poten-
tial to escape humoral immunity18. Protection against infection from various existing and novel SARS-CoV-2
strains is likely dependent on the ability to acquire B cells recognizing new epitopes. Increased antibody reactivity
to a new variant causing relative to antibody reactivity to previous variants could demonstrate the acquisition
of such B-cells recognizing new epitopes. Hybrid immunity therefore, may not only boost pre-existing antibody
responses but also gain new reactivity to new variants.
Here we assess serological immune response aer SARS-CoV-2 breakthrough infection in persons with
primary and booster vaccination with and without previous infection. First, we determine the sensitivity of
N-antibodies as a tool to identify breakthrough infection. Subsequently, we investigate the boosting of Spike
S1-specic responses aer breakthrough infection and the inuence of time since vaccination and N-serocon-
version on the S1-specic antibody levels. e study was performed during the transition period of the Delta
variant to Omicron9, which provided a unique basis to relate immune activation to the virus strain involved.
erefore, lastly, we investigate the change in the response towards the variant of infection as an indication for
the development of broader hybrid immunity.
Results
Study population
520 vaccinated persons from the prospective VASCO study with a SARS-CoV-2 infection between October 1st
2021 and February 13th 2022 were enrolled in the study (Table1). e median age was 55 (IQR 43–64) years and
67% were female. Among the 444 participants of the rst round of inclusion in the transitioning period from the
Delta to the Omicron variant, 165 (37.2%) had a swab sample which could be retrieved and typed. In addition, 76
cases with an infection between October 1st 2021 and November 15th 2021 were included in the second round
of inclusions, categorized as Delta infections based on calendar time. From the rst inclusion round 35 (21.2%)
were typed by variant-PCR and 130 (78.8%) by whole genome sequencing. Of the breakthrough infections 135
(26.0%) were Delta, 99 (60.0%) were Omicron BA.1 and 7 (4.2%) were BA.2 infections. Age and sex distributions
were largely similar between the Delta and Omicron infected individuals. However, dierent distributions of
vaccination status were observed among the Delta and Omicron infected individuals, with individuals experienc-
ing Omicron breakthrough infection more frequently having received a booster vaccination (Table1). In total,
26 participants (5.0%) were partially vaccinated and 494 (95.0%) completed their primary schedule, of which
236 (47.8%) participants also received one (n = 230, 97.5%) or more (n = 6, 2.5%) booster doses. Samples prior
to infection were available with a collection time of 60days (median, with minimal 3 and maximal 233days)
before the reported positive SARS-CoV-2 test date. Post-infection measurements were taken 22days (median, at
minimal 5 and maximal 75days) aer the reported positive SARS-CoV-2 test date. Of the included individuals,
32 out of 479 (7.5%) had evidence of a previous infection.
Serological response post‑infection
Participants were enrolled in the study following reporting an infection provided the individual was vaccinated
at least once. Of the enrolled participants, blood samples available prior to the infection were analyzed for
antibodies to Spike S1, RBD and Nucleoprotein. Pre-infection samples were collected irrespective of the date of
vaccination so between dierent samples non or several vaccination doses could have been administered (le
panels Fig.1). Aer vaccination an antibody response is observed to spike S1 and its subdomain RBD (Fig.1A,
D). As expected, N-specic antibody responses were not induced by vaccination (Fig.1G). Of the previously
infected participants 26 (81.2%) had N-specic IgG antibodies in their most recent pre-infection serological
measurement and 6 (18.8%) participants reported a previous infection without showing N-specic antibodies in
the pre-infection serum (Fig.1G). Aer conrmed breakthrough infection, parental N-, S1- and RBD-specic
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antibody concentrations increased with time since positive test and saturated aer 4–5weeks (Fig.1B and C, E
and F, H and I). In previously uninfected individuals the geometric mean antibody concentration (GMC) for N
reached 35.7 BAU/mL at 4weeks aer infection (Fig.S1). In persons with a history of infection prior to break-
through infection the concentration of N-specic antibodies at 4weeks was 246.7 BAU/mL (Fig.S1). Following
breakthrough infection the levels of S1 were boosted reaching a GMC of 10,829.9 BAU/mL aer 5weeks, and
in the second week aer infection concentrations of 4000–6000 BAU/mL were already observed (Figs.1B, S1).
Individuals with a positive N-specic IgG pre-measurement had an equal geometric mean S1 IgG concentration
aer infection compared with individuals without previous infection (Figs.1B, S1).
N seropositivity following breakthrough infection
In individuals without a history of previous infection, the probability of N-seropositivity was 54% (95% CI 46–62)
and 82% (95% CI 75–86) at two weeks and four weeks aer positive test, respectively (Fig.2A, le panel). In
persons with a previous infection 30 out of 32 (94%) were N-seropositive aer the current infection (Fig.2A,
right panel. In these persons N seropositivity was mostly already achieved in the second week aer breakthrough
infection (8 out of 9). Persons reporting symptoms did not show a signicant higher proportion of N positivity
Table 1. Characteristics of study participants (n = 520).
Delta Omicron Unknown
N (%) 135 (26.0) 106 (20.4) 279 (53.7)
Age group
18–29 3 (2.2) 9 (8.5) 16 ( 5.7)
30–44 22 (16.3) 23 (21.7) 70 (25.1)
45–59 34 (25.2) 34 (32.1) 89 (31.9)
60–74 71 (52.6) 40 (37.7) 101 (36.2)
75 + 5 (3.7) 0 ( 0.0) 2 ( 0.7)
Unknown 0 (0.0) 0 ( 0.0) 1 ( 0.4)
Sex
Female 83 (61.5) 65 (61.3) 201 (72.0)
Male 52 (38.5) 41 (38.7) 78 (28.0)
COVID-19 symptom status
Asymptomatic 19 (14.1) 21 (19.8) 74 (26.5)
Mild symptomatic 35 (25.9) 27 (25.5) 73 (26.2)
Symptomatic 78 (57.8) 56 (52.8) 129 (46.2)
Unknown 3 (2.2) 2 ( 1.9) 3 (1.1)
Number of pre infection samples
0 7 (5.2) 10 ( 9.4) 31 (11.1)
1 106 (78.5) 82 (77.4) 182 (65.2)
2 13 (9.6) 10 (9.4) 55 (19.7)
3 9 (6.7) 4 (3.8) 11 ( 3.9)
Interval between pre infection sample and SARS-CoV-2 positive test in days mean (SD) 109.93 (48.60) 62.44 (39.28) 77.02 (52.99)
Interval between post infection sample and SARS-CoV-2 positive test in days mean (SD) 26.22 (12.46) 24.90 (9.73) 20.83 (8.64)
Evidence of previous infection
Yes 6 (4.2) 2 (1.9) 24 ( 8.6)
No 125 (92.6) 95 (89.6) 227 (81.4)
Unknown 4 (3.0) 9 (8.5) 28 (10.0)
Vaccination status (at post infection measurement)
Partially 15 (11.1) 3 ( 2.8) 8 ( 2.9)
Full 117 (86.7) 29 (27.4) 112 (40.1)
Booster 3 (2.2) 74 (69.8) 159 (57.0)
Vaccine type primary series (at post infection measurement)
AstraZeneca (Vaxzevria) 39 (32.5) 33 (32.0) 75 (27.8)
BioNTech/ Pzer (Comirnaty) 71 (59.2) 39 (37.9) 115 (42.6)
Janssen 5 (4.2) 11 (10.7) 27 (10.0)
Moderna (Spikevax) 5 (4.2) 20 (19.4) 53 (19.6)
Vaccine type booster series (at post infection measurement)
BioNTech/ Pzer (Comirnaty) 0 (0.0) 36 (48.6) 87 (54.7)
Moderna (Spikevax) 3 (100.0) 38 (51.4) 65 (40.9)
Unknown 0 (0.0) 0 ( 0.0) 7 ( 4.4)
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compared to persons considered mild symptomatic or asymptomatic (p = 0.12, Fig.2B). Neither did seropositivity
dier between Delta and Omicron infections (p = 0.3) nor vaccination history (vaccination status: partially, full
and booster, p = 0.2, last vaccine brand used, p = 0.9, time since vaccination, p = 0.09).
S1 antibody levels as a function of time since infection and vaccination
We found a decrease in Spike S1 levels with increasing time since vaccination. Upon breakthrough infection,
increasing time between the last vaccination and breakthrough infection was associated with a faster increase
and higher levels of Spike S1 antibodies (p < 0.001, Fig.3). Individuals with measurable N-specic antibodies
post-breakthrough infection showed higher levels of antibodies to Spike S1 (p < 0.001, Fig.3) compared to indi-
viduals who failed to seroconvert for N. Similar results were found for antibodies to RBD compared to Spike S1
(Fig.S2). As expected, the N-specic antibody response develops independent from vaccination (p = 0.22) and is
only aected by time since infection (p < 0.001, Fig.1H). Individuals with N seropositivity prior to breakthrough
infection had lower levels of Spike S1 antibodies aer infection (Fig.S1B, p < 0.001).
e level of N and S1-specic IgG antibodies and the duration to reach peak levels was also independent of
the virus variant (p = 0.88 and p = 0.10, respectively).
Figure1. Antibody levels following vaccination and breakthrough infection. (A, D, G) pre-infection antibody
concentrations by time since rst vaccination for N-, S1, and RBD-specic IgG, respectively (n = 598). Colors
indicate the vaccination status at the time of blood collection. Measurements from the same individual are
connected (gray line). (B, E, H) Post-breakthrough infection antibody concentrations by time since positive test
for N-, S1-, and RBD-specic IgG, respectively (n = 520). Circle colors indicate the history of previous infection
(see methods) and circles are lled by pre-infection N, S1, or RBD concentration. Absent pre-infection sample
is indicated in grey. Black line shows the estimated mean serological response in not previously infected. Shaded
areas represent 95% condence envelopes. Red horizontal line indicates the seropositivity threshold for N (14.3
BAU/mL) and S1 (10.1 BAU/mL). (C, F, I) Histograms of the pre-infection and post-infection concentrations
for N-, S1-, and RBD-specic IgG, respectively.
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Response towards the SARS‑CoV‑2 variant of infection
To investigate whether novel type-specic antibodies were induced by Omicron infections we compared the ratio
of antibodies to Delta and Omicron BA.1 RBD for persons with a Delta and Omicron breakthrough infection.
Ratios were used instead of comparing antibody levels, as both RBD variants will bind a signicant propor-
tion of cross-reactive antibodies aer breakthrough infection, which cannot be directly compared, as both are
scaled according to their own allocated arbitrary unitage. If no variant-specic antibodies are induced following
breakthrough infection the ratio between Omicron BA.1 and Delta antibodies should be the same between the
groups infected with the Delta and the Omicron variant, with dierences in the ratios indicating variant-specic
antibodies being induced. Compared with Delta breakthrough infections, Omicron BA.1 breakthrough infections
resulted in a higher ratio of Omicron BA.1 over Delta antibodies, indicating the generation of Omicron-specic
antibodies (Fig.4). A similar pattern in ratio towards variant of infection is observed for RBD Omicron BA.1
over parental and RBD parental over Delta (Fig.S3A,B). However, S1 Omicron BA.1 over WT did not dier by
variant (Fig.S3C).
Discussion
e aim of this study was to investigate the humoral immune response following infection in vaccinated persons
and how this relates to two dierent SARS-CoV-2 virus variants responsible for the breakthrough infections,
i.e. Delta and Omicron. We show that SARS-CoV-2 breakthrough infections can be identied by N-specic IgG
antibodies and boosting of vaccine-induced immunity by infection, leading to hybrid immunity. We show that
up to 82% of the individuals that experienced their rst infection with SARS-CoV-2 aer vaccination developed
antibodies to N of SARS-CoV-2, regardless of the virus variant and independent from COVID-19 vaccina-
tion. Following breakthrough infection, N-seroconversion was associated with increased S1 antibody levels. N
Figure2. Estimated probability of N-seropositivity by time since positive test. (A) Estimates of the probability
of N-seropositivity as a function of time since positive test and history of previous infection (n = 479). Shaded
areas represent 95% condence intervals/envelopes. (B) Estimates of the probability of N-seropositivity as a
function of time since positive test, history of previous infection and COVID-19 symptom status (n = 474).
Shaded areas represent 95% condence intervals/envelopes.
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seroconversion might therefore be a more reliable proxy for the development of hybrid immunity rather than a
positive PCR or antigen test only conrming breakthrough infection.
To date, very few studies have systematically investigated N-specic seroconversion as a useful marker for
breakthrough infection, let alone to relate this to the induction of hybrid immunity19. In a recent population
serosurvey among individuals without a history of COVID-19 vaccination, 79% of the participants that had
reported a PCR-conrmed infection and clinical symptoms were N seropositive between 2 and 6weeks aer
infection. For PCR-positive individuals that did not report symptoms, this was 67%11. Despite vaccine-induced
immunity, the sensitivity to N in the current study was found to be similar to what we and others have found in
unvaccinated populations11,20. Our estimates of N seropositivity aer breakthrough infection were a little lower
compared to a recent study by Mizoue etal.19 that reported N seropositivity ranging from 78% up to 97% for
those infected 4–5months aer vaccination, but not as low as 26% (95% CI 11–49) as reported by Allen etal.,
the latter of which concerned a small number of investigated persons and with no clear documented timeline of
infection13. e dierences between these two studies and ours could be related to the timing between infection
and antibody measurement. e sensitivity of the detection of breakthrough infections by N-specic antibodies
is dependent on a minimum time since infection estimated to be about 3weeks. On the other hand, waning of
N-specic antibodies in individuals has been noticed to occur in infected (nonvaccinated) persons, resulting in
partial loss in N seropositivity within 5–6 months11. Whether a similar waning occurs aer breakthrough infec-
tion needs to be determined, whilst such an assessment may be hampered by new and consecutive breakthrough
infection events. Still, our data support the identication of vaccine-breakthrough infection by the detection
of N-specic antibodies applicable within a timeframe of at least 6months aer breakthrough infection. at
Nucleoprotein antibodies are induced at similar rates for Delta and Omicron SARS-CoV-2 is likely because the
Nucleoprotein is conserved and doesn’t show the many mutations observed for the Spike protein. In addition, S1
antibodies are reported to persist longer than N antibodies, resulting in extended duration of hybrid immunity.
It is interesting to note that a small subset of participants with RT-PCR/rapid antigen-conrmed breakthrough
infection (6.7%) had most likely experienced earlier infection prior to this study. is group of participants
was characterized by a rapid onset of N antibodies, almost complete (93.8%) N-seropositivity following the
Figure3. Estimates of the mean S1 antibody levels as a function of time since infection and vaccination in not
previously infected (n = 447). Panels show the dierent time since vaccination (30days intervals) and x-axis
the time since breakthrough infection. Orange and green indicate the persons with and without N-specic
antibodies following breakthrough infection. Shaded areas represent 95% condence intervals/envelopes. RBD
estimates are shown in Fig.S2.
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breakthrough infection and also reaching much higher levels, indicative for a secondary response to the N
protein.
In vitro assays have shown largely reduced neutralization of Omicron variants by pre-Omicron convalescent
sera and by sera of individuals vaccinated by monovalent vaccines3,4,6,18. In addition, epidemiological studies
have shown immune escape by the Delta and Omicron variants2,21. Apart from immune escape, a few reports
suggest that the vaccines used against SARS-CoV-2 provide a limited degree of mucosal immunity22,23. Limited
mucosal immunity aer primary and booster vaccination may allow a more replication of the virus aer expo-
sure, leading to immune activation and the generation of antibodies to the internal Nucleoprotein of the virus.
Following such initial replication, the pre-existing immunity by B cell-derived antibodies or memory T cells may
also enhance the activation of the immune system resulting in not only boosting of Spike-specic antibodies
(hybrid immunity), but also de novo antibodies to other viral targets, while in parallel also improving mucosal
immunity because of a rst contact with infectious virus at the mucosal site23,24. If that indeed happens, hybrid
immunity is not only characterized by increased antibody levels and enhanced mucosal immunity, but also by
broadened immune responses25,26. at this broadening of immunity may occur is indicated by our nding of
relatively higher Omicron-specic RBD antibodies in individuals experiencing Omicron breakthrough infec-
tions compared to persons with a Delta breakthrough infection. is broadening may continue to occur with
subsequent exposures to other variants of the virus. To better assess the development of de novo B cell reactivity,
future studies with longer follow-up periods and type-specic virus neutralization assays are needed.
ere are some limitations to our study. First, infections not directly adjacent to a retrospective antibody
measurement to determine the infection, may be missed, e.g. due to antibody waning as described for N11.
erefore, this leaves the possibility for an earlier infection to have occurred unnoticed. Secondly, variant of
infection changes with calendar time like vaccinations were administered at given time periods resulting in a
correlation between protection by vaccination and the virus variant causing the breakthrough infection. Also,
previous infection diers between the Delta and Omicron variants. is leads to dierences in vaccination and
previous infection status between Delta and Omicron infections, where Omicron cases more oen had received
their COVID-19 booster vaccination.
In conclusion, protection against future variants by antibodies as determined by antibody concentrations,
overlap in antibody-binding epitopes and anity to the targets. Here we showed that breakthrough infection
results in de novo responses to non-vaccine targets, resulting in detectable N-specic IgG antibodies in 82% of
the cases. We propose that the observed association of N seroconversion with stronger boosting of S1 antibodies
makes the N response a better predictor for the development of hybrid immunity than a positive PCR test, since
positive tests in some cases are accompanied with a weak humoral immune response. Although breakthrough
infections by distinct virus variants are equally detected by the induction of N antibodies, the breakthrough infec-
tions also result in variant-specic antibody levels during the development of hybrid immunity. e generation
Figure4. Ratio of the RBD Omicron BA.1 over RBD Delta serological response for Delta and Omicron BA.1
infections in not previously infected individuals. Dierent subplots indicate time since infection in days. See
Fig.S3 for additional antigenic target ratio results. P-value is indicated with ***for < 0.001, **for < 0.01, *for < 0.05
and not signicant (NS).
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of de novo responses, the boosting of vaccine-target antibody levels, and broadening of humoral immunity by
breakthrough infections likely enhances immunity to current Omicron and future variants.
Materials and methods
Study design and population
e VAccine Study COvid-19 (VASCO) is an ongoing prospective cohort study into eectiveness of COVID-19
vaccination in the Netherlands, in which information is collected through regular questionnaires and ngerpick
samples for serology were taken every six months14. From this study, vaccinated participants with a reported
SARS-CoV-2 infection between December 1st 2021 and February 13th 2022 (circulating Delta or Omicron
BA.1/2 variant infections) were asked to donate an additional ngerpick blood sample in 1–8weeks aer infec-
tion, with outliers up to 11weeks. Aer the rst round of inclusions, the study was extended with vaccinated
individuals with an infection between 1 October up to 15 November 2021 (assumed to be Delta infections, as this
was the only variant circulating in the Netherlands at that time27) and from which a serum sample was available
between 3 and 7weeks aer infection, as Delta infections with a longer interval between infection and blood
sample appeared underrepresented in the primary selection.
Data on symptoms were collected directly aer a positive test and one month aer this positive test. Partici-
pants reporting fever, dyspnea, muscle ache, extreme tiredness, general malaise, painful respiration, joint pain,
diarrhea, or stomach ache were regarded as COVID-19 symptomatic as these symptoms relate to a systemic
infection. Participants with a runny nose, sore throat, anosmia/ageusia, headache, coughing or without symptoms
were considered mild symptomatic or asymptomatic as an indication of non-systemic infection.
e VASCO study is conducted in accordance with all relevant guidelines and regulations. e study protocol
was approved by the independent Medical Ethics Committee of the Stichting Beoordeling Ethiek Biomedisch
Onderzoek (BEBO), Assen, the Netherlands (NL76815.056.21). All participants provided written informed
consent.
Variant detection
Positive national community testing SARS-CoV-2 specimens from participants were collected and variant
detection was performed by whole genome sequencing and variant-PCR (S gene target failure), as previously
described9,21. Sequences obtained in this study are available on GISAID.org (accession IDs provided in Tabel S1).
Antibody measurements
Nucleoprotein (N)-, Spike S1 (S1)-, and Receptor binding domain (RBD)-specic IgG was measured aer break-
through infection, referred to as post-infection measurement, and in all samples available prior to the break-
through infection, referred to as pre-infection measurement. Antibodies to antigenic targets N, S1 and RBD of
the parental strain, RBD of the Delta variant and RBD and S1 of the Omicron BA.1 variant were detected using
a uorescent bead-based assay, as described previously18,28,29. Briey, samples were diluted (1:400 and 1:10,000)
in SM01 (Surmodics, USA) supplemented with 2% FCS and added to the bead mixture. Sample bead mixtures
were incubated while shaking in the dark at room temperature. Following washing (3 × PBS) PE-conjugated goat
anti human IgG (1:400) was added and incubated for 30min as above. Aer washing, samples were acquired
on a FlexMap 3D (Luminex) and interpolated using pooled sera calibrated against the international reference
(NIBSC, 20/136) and expressed in international units BAU/mL.
e threshold for seropositivity to N (14.3 BAU/mL, reference11) and Spike S1 (10.1 BAU/mL, reference29)
were determined by receiver operator characteristics analysis and mixed modeling using pre-pandemic negative
control samples and a heterogeneous mix of samples of PCR-conrmed cases with varying severity (asympto-
matic individuals, moderately-ill cases and hospitalized patients)28,29.
Vaccination status and evidence of previous infection
Vaccination status was determined at the date of blood collection. If the self-reported date of blood collection
was missing, the received date of the blood sample minus two days (median dierence between date of blood
collection and date received in non-missing) was used. Partial primary vaccination was dened as having received
one dose of Comirnaty, Spikevax or Vaxzevria before date of blood collection, or two doses of Comirnaty, Spik-
evax or Vaxzevria less than 14days before this date. Full vaccination is dened as having received two doses of
Comirnaty, Spikevax or Vaxzevria at least 14days before date of blood collection, one dose of Jcovden at least
28days before this date, or a booster dose less than 7days before this date. Booster vaccination is dened as
one or more doses aer a complete primary vaccination schedule, where the rst booster dose is at least 7days
before date of blood collection.
Evidence of an infection before the studied breakthrough infection was based on self-report of a positive
SARS-CoV-2 test or the presence of N-specic antibodies prior to breakthrough infection.
Statistical analyses
Log-transformed N-, S1- and RBD-specic responses (BAU/mL values) were modelled using a Gaussian general-
ized additive model, with the R package mgcv30. We modeled the N-, S1- and RBD-specic serological response
in individuals without a previous infection as a function of time since positive test in days (in Fig.1). In addition,
for the RBD and S1 responses we expanded this model with an interaction term for time since vaccination and
N-seropositivity of the post-infection sample (in Fig.3). Time since positive test and time since vaccination were
included as a tensor product of penalized cubic splines (15 knots), using second order penalties.
Probability of N-seropositivity is modelled using a logistic regression in a generalized additive model as a
function of time since positive test (penalized cubic spline, 15 knots, in Fig.2A). We expand this model separately
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for categorical variables COVID-19 symptom status (in Fig.2B, absent/present), variant of infection (Delta/
Omicron), vaccination status (partial, full, or booster vaccination), last used vaccine type (Comirnaty, Vaxzevria,
Spikevax, or Jcovden), and time since vaccination. Participants only reporting the answer option ‘other symptoms’
were excluded from the analysis into the eect of symptoms. e output of the logistic regression (log-odds) is
transformed into a probability of N seropositivity using the inverse logit function,
log
(
p
n+
1−p
n+
)
.
We tested dierences in ratios in Delta and Omicron-specic IgG levels between individuals with an Omicron
or Delta infection stratied by time since infection with a Wilcoxon test.
Data availability
All antibody data obtained are presented in the manuscript. SARS-CoV-2 variant sequencing data acquired for
this study (accession numbers provided in TableS1) are available on https:// gisaid. org/.
Received: 8 June 2023; Accepted: 23 October 2023
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Acknowledgements
We thank the laboratory team of the National Institute for Public Health and the Environment (RIVM), Ae Vog-
elzang, Annemarie van den Brandt, Bas van der Veer, Jeroen Cremer, Jil Kocken, Jordy de Bakker, Kim Freriks,
Lisa Wijsman, Lynn Aarts, Ryanne Jaarsma, Sanne Bos and Sharon van den Brink. We thank Teun Guichelaar
and Joanna Kaczorowska for critically reviewing the manuscript.
Author contributions
Conceptualization: G.d.H., R.S.v.B. Methodology: G.d.H., R.S.v.B., S.P.A., M.J.K. Investigation: G.d.H., S.P.A.,
G.S., D.E., R.v.B., M.J.K. Visualization: S.P.A. Supervision: R.S.v.B., M.J.K. Writing—original dra: G.d.H., S.P.A.
Writing—review & editing: R.v.B., G.S., C.E.H., B.V., M.J.K.
Funding
e study was funded by the Dutch Ministry of Health, Welfare and Sports.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 45718-8.
Correspondence and requests for materials should be addressed to R.S.B.
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