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Group 1 and group 2
hemagglutinin stalk antibody
response according to age
Laura Sa
´nchez-de Prada
1,2
, Iva
´n Sanz-Muñoz
1
, Weina Sun
3
,
Peter Palese
3
, Rau
´l Ortiz de Lejarazu
1
, Jose
´Marı
´a Eiros
1,2,4
,
Adolfo Garcı
´a-Sastre
3,5,6,7,8
*
†
and Teresa Aydillo
3,5
*
†
1
National Influenza Center of Valladolid, Valladolid, Spain,
2
Department of Microbiology and
Immunology, Hospital Clı
´nico Universitario de Valladolid, Valladolid, Spain,
3
Department of
Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States,
4
Centro de
Investigacio
´n Biome
´dica en Red de Enfermedades Infecciosas, Instituto de Salud Carlos III,
Madrid, Spain,
5
Global Health and Emerging Pathogens Institute, Icahn School of Medicine at
Mount Sinai, New York, NY, United States,
6
Department of Pathology, Molecular and Cell-Based
Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States,
7
Department of
Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York,
NY, United States,
8
The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York,
NY, United States
Objective: Antibodies elicited by seasonal influenza vaccines mainly target the
head of hemagglutinin (HA). However, antibodies against the stalk domain are
cross-reactive and have been proven to play a role in reducing influenza disease
severity. We investigated the induction of HA stalk-specific antibodies after
seasonal influenza vaccination, considering the age of the cohorts.
Methods: A total of 166 individuals were recruited during the 2018 influenza
vaccine campaign (IVC) and divided into groups: <50 (n = 14), 50–64 (n = 34), 65–
79 (n = 61), and ≥80 (n = 57) years old. Stalk-specific antibodies were quantified by
ELISA at day 0 and day 28 using recombinant viruses (cH6/1 and cH14/3)
containing an HA head domain (H6 or H14) from wild bird origin with a stalk
domain from human H1 or H3, respectively. The geometric mean titer (GMT) and
the fold rise (GMFR) were calculated, and differences were assessed using ANOVA
adjusted by the false discovery rate (FDR) and the Wilcoxon tests (p <0.05).
Results: All age groups elicited some level of increase in anti-stalk antibodies
after receiving the influenza vaccine, except for the ≥80-year-old cohort.
Additionally, <65-year-old vaccinees had higher group 1 antibody titers versus
group 2 before and after vaccination. Similarly, vaccinees within the <50-year-
old group showed a higher increase in anti-stalk antibody titers when compared
to older individuals (≥80 years old), especially for group 1 anti-stalk antibodies.
Conclusion: Seasonal influenza vaccines can the induction of cross-reactive
anti-stalk antibodies against group 1 and group 2 HAs. However, low responses
were observed in older groups, highlighting the impact of immunosenescence in
adequate humoral immune responses.
KEYWORDS
influenza, stalk antibodies, influenza vaccines, age, elderly
Frontiers in Immunology frontiersin.org01
OPEN ACCESS
EDITED BY
Simon Evan Hufton,
National Institute for Biological Standards
and Control, United Kingdom
REVIEWED BY
Othmar Engelhardt,
Medicines and Healthcare Products
Regulatory Agency, United Kingdom
Anke Huckriede,
University Medical Center Groningen,
Netherlands
*CORRESPONDENCE
Teresa Aydillo
teresa.aydillo-gomez@mssm.edu
Adolfo Garcı
´a-Sastre
adolfo.garcia-sastre@mssm.edu
†
These authors have contributed
equally to this work and share
last authorship
RECEIVED 26 March 2023
ACCEPTED 12 May 2023
PUBLISHED 29 May 2023
CITATION
Sa
´nchez-de Prada L, Sanz-Muñoz I, Sun W,
Palese P, Ortiz de Lejarazu R, Eiros JM,
Garcı
´a-Sastre A and Aydillo T (2023)
Group 1 and group 2 hemagglutinin
stalk antibody response according to age.
Front. Immunol. 14:1194073.
doi: 10.3389/fimmu.2023.1194073
COPYRIGHT
© 2023 Sánchez-de Prada, Sanz-Muñoz,
Sun,Palese,OrtizdeLejarazu,Eiros,Garcı
´a-
Sastre and Aydillo. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 29 May 2023
DOI 10.3389/fimmu.2023.1194073
1 Introduction
The influenza virus, with three to five million severe cases and
between 290,000 and 650,000 annual respiratory deaths (1),
represents a major socioeconomic burden (2). Currently, the best
approach to preventing infection and reducing disease severity is
annual vaccination. However, influenza vaccine effectiveness is
moderate, varying from 20% to 70% depending on the season.
Additionally, influenza vaccines provide short-lasting and strain-
specific protection (3). Most neutralizing antibodies induced by
vaccination target hemagglutinin (HA), particularly the
immunodominant head domain, which constantly undergoes
antigenic drift by accumulating amino acid substitutions and
additional glycosylation sites (4). Additionally, during some
seasons, the strains contained in the vaccine do not match the
circulating strain(s) due to viral evolution. Such low effectiveness
makes it necessary for vaccines to be reformulated and re-
administered annually (5).
The HA is the most abundant surface glycoprotein and has two
major domains: the globular head (HA1) and the stalk region
(HA2). The HA stalk domain is highly conserved between
influenza virus strains due to functional restraints and low
immune pressure (6). There a re currently 18 hemagglutinin
subtypes for influenza A virus, which are classified into two
phylogenetic groups based on their antigenic properties: group 1
consists of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and
H18; while group 2 contains H3, H4, H7, H10, H14, and H15 (7).
Antibodies against the stalk are more cross-reactive and can bind
different strains of the same phylogenetic group, providing broad
protection. Mechanisms of these antibodies may include
impairment of viral and endosomal membrane fusion, inhibition
of viral release, and interruption of HA maturation. In addition,
these antibodies are functionally involved in antibody-dependent
cell cytotoxicity and phagocytosis (ADCC and ADCP) and
complement-dependent cytotoxicity (CDC) (8). Novel influenza
vaccine designs are focused on the development of influenza
vaccines that would increase the breadth and duration of
protection. Some of the most advanced vaccine candidates target
conserved epitopes of the HA protein, such as the subdominant
stalk domain, with the aim of providing long-lasting protection
against different strains and subtypes of the virus (8,9).
The aim of our study is to investigate the level of pre-
existing anti-stalk antibodies against phylogenetic groups 1 and 2,
and after seasonal influenza vaccination according to age.
Immunodominance profiles and antibody titers against different
antigenic sites in the HA head of A(H1) that matched influenza
vaccine strains were previously studied in this cohort. Classically,
five antigenic sites in the head of the HA have been defined as Sb, Sa,
Cb, Ca1, and Ca2 and are the main targets of the humoral response
upon vaccination or infection. The first two are placed at the distal
tip of each monomer, while Cb, Ca1, and Ca2 are placed proximally,
near the stalk domain. The receptor binding site (RBS), where the
attachment to sialic acids occurs, is located between Sb, Ca2, and Sa
(10,11). We found that the immune response was mainly directed
at Sb, followed by Ca2, and that adjuvants can broaden responses to
subdominant antigenic sites (12). Here we expand on our previous
study and now investigate the antibody response to the stalk
domain according to age.
2 Materials and methods
2.1 Patient recruitment
A total of 166 individuals were recruited from vaccination
programs during the Influenza Vaccine Campaign (IVC) 2018
conducted by the Influenza Sentinel Surveillance Network of
Castile and Leon (Spain) (ISSNCyL). All serum samples obtained
were shipped to Mount Sinai Hospital in New York (USA) and were
used to determine HA stalk-specific antibodies. Serum samples
were obtained before and 28 days after vaccination and stored
at −20°C in the National Influenza Centre of Valladolid (Spain)
before being sent. Two seasonal influenza vaccines were used
following the recommendations of the World Health
Organization (WHO) for the northern hemisphere: A/Michigan/
45/2015 (H1N1)pdm09-like virus, A/Singapore/INFIMH-16-0019/
2016 (H3N2)-like virus, and B/Colorado/06/2017-like virus (B/
Victoria/2/87 lineage) for the trivalent vaccine, and also B/
Phuket/3073/2013-like virus (B/Yamagata/16/88 lineage) for the
quadrivalent one. Following the recommendations for vaccination
in Spain, subjects ≥65 years old received an adjuvanted trivalent
influenza vaccine (ATIV) and subjects <65 years old received a
quadrivalent influenza vaccine (QIV). Two patients from each
group received the other group’s vaccine due to a lack of vaccine
availability. Written informed consent was obtained from the
participants. This research was performed according to the
Declaration of Helsinki and was approved by the Ethics
Committee of the East-Valladolid Health Area under the code PI
21-2314.
2.2 Stalk-specific antibodies
To quantify the levels of the stalk-specific antibodies, two
reassortant viruses were used: a cH6/1N5 and a cH14/3N5. The
first one had an HA stalk derived from the pandemic H1N1 virus
(A/California/04/09) containing an exotic H6 head domain (H6N1
virus A/mallard/Sweden/81/02) and an exotic N5 (H12N5 virus A/
mallard/Sweden/86/03). HA head domains were of wild bird origin,
and hence no specific antibodies should be present in the patients’
serum samples. The methods and description of the generation of
this virus in cell culture by using reverse genetics have been
previously published (13–15). The second virus had an HA stalk
derived from an H3N2 virus A/Hong Kong/4801/2014 combined
with an exotic H14 head domain A/mallard/Gurjev/263/1982
and an exotic N5 from the H12N5 virusA/mallard/Sweden/86/03
(for virus generation, see the Supplementary Appendix).
Reassortant viruses were cultured in 10-day-old embryonic
chicken eggs and titered to confirm the growth and ensure they
had similar hemagglutination units. Then, a purification
by ultracentrifugation in a sucrose gradient was performed
(Supplementary Appendix). Antibodies in human serum were
Sa
´nchez-de Prada et al. 10.3389/fimmu.2023.1194073
Frontiers in Immunology frontiersin.org02
measured using an enzyme-linked immunosorbent assay (ELISA)
as described before (16) (for the ELISA protocol, see the
Supplementary Appendix). The optical density (OD) for each well
was calculated by subtracting the average background plus three
standard deviations. The area under the curve (AUC) was
computed using GraphPad Prism v.10 software.
2.3 Statistical analysis
All ELISA values were log10-transformed to improve linearity.
The GMT and 95% confidence intervals (CI 95%) were computed
by taking the exponent (log10) of the mean and the lower and upper
limits of the 95% CI of the log10‐transformed titers. Fold rise was
calculated as the ratio between days 0 and 28. GMFR was computed
by taking the exponent (log10) of the mean fold rise and the lower
and upper limits of the CI 95% of the log10‐transformed titers.
Statistical significance was established at p <0.05. All reported
p values are based on two‐tailed tests. For antibody levels, the
Brown–Forsythe and Welch ANOVA test was adjusted by
controlling the false discovery rate (FDR) with the two-stage
linear procedure of Benjamini, Krieger, and Yekutieli for multiple
comparisons, and the Wilcoxon matched pairs signed rank test was
used when appropriate. All tests were performed using IBM SPSS
Statistics (version 26) and GraphPad Prism (version 10).
3 Results
3.1 Human cohorts
A total of 166 individuals were recruited during the Influenza
Vaccine Campaign (IVC) 2018. Two different inactivated influenza
vaccines were applied according to age following Spanish
recommendations: a quadrivalent influenza vaccine (QIV) in 46
subjects of 28–64 years and two subjects of 73 and 74 years old
(28.9%), and an MF-59 adjuvanted trivalent influenza vaccine
(ATIV) in 116 subjects ≥65 years old and two subjects of 57 years
old (71.1%). To assess the presence of HA stalk-specific antibodies,
vaccinees were divided according to age into four groups: <50, 50–
64, 65–79, and ≥80 years old. Epidemiological and clinical
characteristics are described in Table 1.
3.2 Anti-stalk antibodies according to age
To better understand the baseline antibody landscape, we first
profiled the pre-existing immunity before vaccination. For this, we
investigated the levels of anti-stalk antibodies against HA groups 1
and 2 using reassortant viruses containing an exotic HA head domain
and an exotic NA to whom humans should not have specific
antibodies and a conserved stalk from human pandemic H1N1
virus A/California/04/09 and H3N2 virus A/HongKong/4801/2014
(groups 1 and 2, respectively). Purified viruses were then used to
perform ELISA assays. To improve visualization, the levels of anti-
stalk antibodies of each individual together with the geometric mean
titer (GMT, CI95%) at day 0 are shown in Figure 1A and
Supplementary Table 1. All vaccinees presented anti-stalk
antibodies against both phylogenetic groups. The stalk antibody
levels against HA group 1 in the 50–64-year-old group were
significantly higher compared to <50-year-old, ≥80-year-old, and
65–79-year-old groups. Additionally, antibody levels in the <50-year-
old group were also significantly higher than those in the 65–79-year-
old group. In contrast, stalk antibodies against group 2 were lower in
the <50-year-old cohort compared to the 50–64-year-old and the
≥80-year-old groups. Since different years of birth could influence
previous exposure to different influenza viruses and therefore pre-
existing immunity to influenza A viruses (IAVs), we next investigated
the levels of immunity in the context of historical IAV circulation. In
order to understand whether first exposure to influenza A viruses
could have had an impact on preexisting immunity of anti-stalk HA
group 1 versus HA group 2 antibodies, we analyzed antibody levels in
the context of birth year. To do so, anti-stalk antibody levels based on
birth year against each HA group were plotted, and Lowess curves
were generated (Figure 1B). The timeline and emergence of different
influenza A viruses and their circulation over the years are indicated
TABLE 1 Cohort description and epidemiological and clinical characteristics.
<50 years old 50–64 years old 65–79 years old ≥80 years old
No. 14 34 61 57
Age (Median, IQR) 37.5
(29.75–45.5)
59.5
(55.0–62.25)
70.0
(68.0–74.0)
85.0
(83.0–90.0)
Men (%) 14.3 55.9 60.7 49.1
Type of vaccine Vaxigrip Vaxigrip Chiromas Chiromas
Comorbidities (n, %) 1 (7.1) 4 (11.8) 10 (16.4) 4 (7.0)
Diabetes mellitus 0 1 3 1
Heart disease 0 1 4 2
COPD 0 0 0 1
Immunocompromised 1 2 3 0
No, number; IQR, interquartile range; COPD, chronic obstructive pulmonary disease.
Sa
´nchez-de Prada et al. 10.3389/fimmu.2023.1194073
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to represent the likelihood of group 1 or 2 HA influenza primary
infection. There is not a clear pattern, indicating that the likelihood of
first exposure to either HA group virus could have an influence on
pre-existing immunity. However, higher antibody levels against HA
group 1 were found in younger adults when compared to older age
groups, while an increasing tendency with age was found for group 2
anti-stalk antibodies (Figure 1A).
To characterize the antibody response to both groups after
influenza vaccination, we next investigated anti-stalk antibody
levels at day 28. A modest but significant increase compared with
baseline levels was observed in all age groups, except for anti-group
1 stalk antibodies in the ≥80-year-old group (Figure 2A). Post-
vaccination stalk antibody titers against group 1 were significantly
higher in the 50–64-year-old group compared to the other groups.
Again, the titers in the <50-year-old group were significantly higher
than those in the 65–79-year-old group and the ≥80-year-old group.
Stalk antibodies against group 2 showed the same profile as before
vaccination and were lower in the <50-year-old group compared to
the 50–64-year-old and ≥80-year-old groups (Figure 2B)
(Supplementary Table 1).
To quantify the induction of an antibody response to
vaccination, we next calculated the geometric mean fold rise
(GMFR). Despite only a few patients displaying a higher than
4-fold increase, more than 70% of the individuals in all age
groups showed some level of increase in anti-stalk antibodies,
except for the ≥80-year-old group, where 59.65% of them showed
an increase in group 1 anti-stalk antibodies (Figure 2C). The
distribution of fold-rise levels is also detailed in Supplementary
Figure 2. Adjusted two-tailed p-values for multiple comparisons
after Brown–Forsythe and Welch ANOVA were used to compare
B
A
FIGURE 1
(A) Individual anti-stalk antibodies and geometric mean titer (GMT, 95% CI) before vaccination against HA groups 1 and 2 in all groups. To compute
differences between age cohorts: The two-tailed p-values were calculated with the Brown–Forsythe and Welch ANOVA test adjusted by controlling
the false discovery rate (FDR) with the two-stage linear procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons. *P <0.05, ****P
<0.0001. (B) Stalk antibody pre-immunity trend based on birth year. To represent anti-stalk antibodies based on theoretical first exposures to A
viruses, individual antibody levels of patients based on their birth year were represented against both HA groups, and Lowess curves were designed
with medium smoothing, taking 10 points in the smoothing window.
Sa
´nchez-de Prada et al. 10.3389/fimmu.2023.1194073
Frontiers in Immunology frontiersin.org04
GMFR against each group (Figure 2D). Overall responses were
similar for both HA groups 1 and 2. However, there was a
significantly higher response to group 1 in the youngest patients
compared to the oldest (p = 0.0382). No differences were found in
fold induction levels in group 2 HAs between different age groups or
when comparing the responses of groups 1 and 2 within age groups.
4 Discussion
The results of our study indicate that (a) pre-existing HA stalk
immunity against phylogenetic group 1 is higher in younger
populations; (b) seasonal influenza vaccines can moderately (on
average less than two times) boost cross-reactive antibody responses
against the stalk domain of both group 1 and group 2 HA viruses;
and (c) age and previous exposures could impact responses to
conserved epitopes, such as those against the stalk.
Responses to the influenza virus in adults are variable and
complex as they are influenced by many factors (17). Humoral
responses to the influenza virus rely on individual histories of
exposure to the virus and are mainly targeted at the HA head
(18). However, rapid evolution and antigenic drift make them of
lesser importance when we talk about lifelong protection. In
contrast, anti-stalk antibodies target more conserved epitopes and
provide cross-reactive protection against different strains of the
same phylogenetic group, resulting in an attractive approach to new
vaccine development (19). Additionally, they have been recently
associated as an independent correlate of protection in the case of
group 1 HAs (20). Those antibodies are elicited most effectively
after natural infection or vaccination with antigenically diverse
strains. Our results showed higher baseline antibody levels against
group 1 HAs in individuals <65 years old, in particular those 50–64
years old. This is in contrast with previous findings that suggest that
they tend to increase with age (21–24). In the present study, only in
group 2 HAs, anti-stalk antibodies seemed to increase with age.
Although those studies included different age groups and vaccines,
their results agree with ours in finding better responses in young
adults <50 years old against group 1 HAs (21,22) and no differences
in responses in group 2 HAs (22).
On the other hand, unlike group 1 Has, of which several different
antigenic strains have circulated in humans (H1N1, H2N2, and
H1N1pdm09), antigenically similar group 2 HA viruses have
circulated in humans since 1968 (25)(Supplementary Figure 3).
It has been shown that divergent strains are more likely to drive the
expansion of cross-reactive antibodies against more conserved
epitopes, such as the HA stalk, than similar ones (24). It is possible
that the lack of stimulus from substantially divergent strains is
responsible for the lower magnitude of antibodies against group 2
A
B
DC
FIGURE 2
(A) Individual antibody levels and geometric mean titer (GMT, 95% CI) before and after vaccination against groups 1 and 2 of HAs in each group. The
two-tailed p-values were calculated with the Wilcoxon matched pairs signed rank test. **P <0.01, ****P <0.0001. (B) Individual anti-stalk antibodies
and geometric mean titer (GMT, 95% CI) after vaccination against HA groups 1 and 2 in all groups. To compute differences between cohorts, the
two-tailed p-values were calculated with the Brown–Forsythe and Welch ANOVA test adjusted by controlling the false discovery rate (FDR) with the
two-stage linear procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons. *P <0.05, ***P <0.001, ****P <0.0001. (C) Percentage of
responders and non-responders to seasonal influenza vaccination against groups 1 and 2 of HAs in each group. Responders are considered to have
a fold rise of anti-st alk antibodies >1. (D) Geometric mean fold rise (GMFR, 95% CI) of stalk antibody levels. To compute differences between cohorts:
The two-tailed p-values were calculated with the Brown–Forsythe and Welch ANOVA test adjusted by controlling the false discovery rate (FDR) with the
two-stage linear procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons. To compute differences in antibody levels within the same
cohort: The two-tailed p-values were calculated with the Wilcoxon matched pairs signed rank test. *P <0.05.
Sa
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Frontiers in Immunology frontiersin.org05
HAs in younger individuals. These results align with a previous
longitudinal study where the highest levels of group 1 HAs were
found in individuals exposed to the most diverse group 1 viruses (26).
Additionally, responses against the head domain have also been
described as being lower against phylogenetic group 2 (27).
Therefore, the lower magnitude in these age groups could also
suggest that group 2 HA viruses are less immunogenic.
The term antigenic seniority or antigen imprinting describes
how influenza antibody responses in humans are shaped by the first
encounters in life, usually at an early stage, and upon repeated
exposure, either by infection or vaccination. This concept is
commonly known as the original antigenic sin. Humoral
responses after natural infection induce broader and longer-
lasting responses than after vaccination (28). However, responses
to vaccination are not equal and depend on the immunodominance
of different epitopes as well as the age of individuals (12). In fact, it
has been shown that antibody responses against the stalk domain
are suppressed in favor of the head domain with currently licensed
influenza vaccines (29,30). Not many studies attribute an increase
in stalk antibodies to seasonal vaccination (31). Although our
previous results confirmed that most responses are directed
against the HA head (12), here we show that a modest but
significant rise in stalk titers can be found in most individuals
after influenza vaccination. Also, these responses were higher in
younger populations despite receiving a non-adjuvanted influenza
vaccine, in contrast to the adjuvanted vaccine received by older
individuals. This reduction inimmuneresponses,knownas
immunosenescence, impairs antibody avidity and B- and T-cell
responses to vaccination as we age (32,33). This phenomenon could
be one of the reasons for the reduction in baseline levels with age in
the case of group 1 HA anti-stalk antibodies. However, we cannot
explain why group 2 HA responses seem not to be affected by
immunosenescence in a similar way. Nevertheless, responses in the
younger populations are more uniform, while responses in the
elderly seem to have higher variability. This could be explained by
the variability in the degree of immunosenescence, which has
been recently proposed not to be a strict decline but a dynamic
balance that might be necessary for an adequate response to known
antigens but detrimental to responses to new antigens in most
circumstances (34).
Our analysis by birth year did not show a pattern according to
the likely first exposure to each HA group of viruses in our age
groups. However, the group of 50–65 year olds who could have first
encountered A(H2N2) had higher pre-existing immunity, while the
elderly (≥65 years old) showed unexpected results with lower
baseline anti-stalk antibody levels against this group and like
those against HA group 2 levels. These findings could be
explained by immunosenescence in the elderly population.
However, further studies should be performed to understand the
effect of imprinting on age.
To conclude, our results show that, in general, modest responses
are elicited against both HA groups 1 and 2 and that consecutive
exposures to substantially different strains drive responses against the
HA stalk domain. This concept is already being used for universal
vaccine approaches that aim at eliciting broad, long-lasting, cross-
reactive protection with chimeric HA designs (35). However, our
findings suggest that immunosenescence, especially in older patients,
could drive lower responses to seasonal vaccination. Therefore,
strategies that aim to enhance immune responses in the elderly
should be considered for future vaccine designs (36).
Our study has several limitations. First, it was designed as a
sero-epidemiological study of vaccine responses, and only serum
samples were available. Second, the cohorts analyzed differed in the
type of vaccine recommended by the Spanish health agencies and
sometimes were not strictly followed. Third, the lack of information
on previous exposures to influenza virus makes it difficult to
interpret results, although the likelihood of priming could be
inferred from the year of birth.
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
Ethics statement
This research was performed according to the Declaration of
Helsinki and was approved by the Ethics Committee of East-
Valladolid health area under the code PI 21-2314. The patients/
participants provided their written informed consent to participate
in this study.
Author contributions
TA and AG-S conceived, designed, and supervised the study.
TA provided training to LS-dP. WS and PP generated the viruses.
LS-dP performed the experiments, including the growth of viral
stocks and ELISAS. Samples were provided by IS-M, JE, and RO.
LS-dP and TA analyzed data, wrote the manuscript, and prepared
the figures. TA, AG-S, and PP provided reagents, methods, and
expertise. TA, AG-S, JE, and IS-M supervised the study. All authors
contributed to the article and approved the submitted version.
Funding
This work was supported by the “Sociedad Española de
Enfermedades Infecciosas y Microbiologı
aClı
nica,”SEIMC
mobility grant awarded to LS-dP. LS-dP received a Rı
o Hortega
grant (CM20/00138) from Instituto de Salud Carlos III (co-funded
by the European Regional Development Fund/European Social
Fund, “A way to make Europe”/”Investing in your future”). This
work was also partly supported by the CRIPT (Center for Research
on Influenza Pathogenesis and Transmission), a National Institute
of Allergy and Infectious Diseases (NIAID)-funded Center of
Excellence for Influenza Research and Response (CEIRR, contract
75N93021C00014), the Collaborative Influenza Vaccine Innovation
Sa
´nchez-de Prada et al. 10.3389/fimmu.2023.1194073
Frontiers in Immunology frontiersin.org06
Centers (CIVIC, NIAID contract 75N93019C000510), and NIAID
grants P01AI097092, R01AI142086, U01AI165452, and
U19AI168631 to AG-S. This study was also partially supported by
Collaborative Influenza Vaccine Innovation Centers (CIVIC)
contract 75N93019C00051 (PP), the Center for Excellence on
Influenza Research and Response (CEIRR) contract
75N93021C00014 (PP), and NIH grants P01AI097092 (PP),
R01AI145870 (PP), and R01AI141226 (PP). The funders played
no role in the study design, data collection, analysis, and the
interpretation of data, or the writing of this manuscript.
Acknowledgments
We thank Richard Cadagan for technical assistance.
Conflict of interest
The AG-S laboratory has received research support from GSK,
Pfizer, Senhwa Biosciences, Blade Therapeutics, Kenall
Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills
Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix,
Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied
Biological Laboratories and Merck, outside of the reported work.
AG-S has consulting agreements for the following companies
involving cash and/or stock: Castlevax, Amovir, Vivaldi
Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda,
Accurius, Esperovax, Farmak, Applied Biological Laboratories,
Pharmamar, Paratus, CureLab Oncology, CureLab Veterinary,
Synairgen, and Pfizer, outside of the reported work.
AG-S has been an invited speaker in meeting events organized
by Seqirus, Janssen, Abbott, and Astrazeneca.
AG-S is an inventor on patents and patent applications on the
use of antivirals and vaccines for the treatment and prevention of
virus infections and cancer, owned by the Icahn School of Medicine
at Mount Sinai, New York, outside of the reported work.
The remaining authors declare that the research was conducted
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