# Non-conjugated TLR agonists increase the quantity of antibodies but not their quality in a murine immunization model

Nathan Aymerich, Olivia T M Bucheli, Kevin Portmann, Luca J

Schlotheuber, Ingibjörg Sigvaldadóttir, Jean Baudry, Klaus Eyer

► To cite this version:

Nathan Aymerich, Olivia T M Bucheli, Kevin Portmann, Luca J Schlotheuber, Ingibjörg Sigvaldadót-tir, et al.. Non-conjugated TLR agonists increase the quantity of antibodies but not their quality in a murine immunization model. Vaccine, 2025, 62, pp.127518. 10.1016/j.vaccine.2025.127518. hal-05410719

HAL Id: hal-05410719

https://cnrs.hal.science/hal-05410719v1

Submitted on 11 Dec 2025

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# Non-conjugated TLR agonists increase the quantity of antibodies but not their quality in a murine immunization model

Nathan Aymerich<sup>a</sup>, Olivia T.M. Bucheli<sup>b</sup>, Kevin Portmann<sup>b</sup>, Luca J. Schlotheuber<sup>b</sup>, Ingibjörg Sigvaldadóttir<sup>b</sup>, Jean Baudry<sup>a</sup>, Klaus Eyer<sup>b,c,*</sup>

<figure><img src="image_2.png" /></figure>

## ABSTRACT

Bacterial vaccines have largely shifted from whole bacteria to selected proteins or glycans, thereby removing immune-stimulant components such as toll-like receptor (TLR) agonists from the formulation. Therefore, TLR agonists are often added to these vaccines to increase immunogenicity, and their addition has been frequently associated with increased antibody titers. To investigate the effects of TLR agonists at the B cell level, a small exploratory cohort study was performed with a murine immunization model using alum and R-phycoerythrin, with and without different TLR agonists. We observed that, while none of the TLR agonists increased affinity, the addition of TLR2, TLR9, and especially a combination of TLR2/4/9 agonists was able to increase the antibody quantity by increasing the number of secreting cells and/or cellular secretion rates. Our results indicate that the increased antibody titers for various TLR agonists or combinations might stem from an increased antibody magnitude rather than higher affinity antibodies.

## 1. Introduction

Bacterial vaccines for human use have mostly transitioned from utilizing whole bacteria to incorporating specific proteins or glycans. While offering several advantages in terms of safety and production, this removes various immune-stimulant components from the vaccine [1,2]. One important class of immune stimulants that are being removed by switching antigen formats from whole bacteria to proteins are toll-like receptor (TLR) agonists. A variety of agonistic structures present in bacteria are being recognized by TLRs, including triacylated lipopeptides (recognized by TLR1/2 [3] and TLR2/6 [4,5]), lipopolysaccharides (LPS, recognized by TLR4), cytosine-phosphate-guanine sequences (CpG, recognized by TLR9), and flagellin (recognized by TLR5) [6]. Since the omission of TLR agonists diminishes immunogenicity [1,2], vaccine adjuvants and additives that enhance the immune response can be added, and a variety of TLR agonists are currently used or being investigated as additives to vaccines, including natural or synthetic agonistic analogs of the compounds mentioned above, such as Pam3CSK4 (TLR1/2 agonist), Pam2CSK4 (TLR2/6 agonist), monophosphoryl lipid A (MPLA, TLR4 agonist), CpG (TLR9 agonist), among others [7–12].

In most studies where TLR agonists were added, researchers described that their addition led to an increased antibody level in serum

[13–17]. Several mechanisms or combinations could lead to an increase in titer, including an increased affinity or concentration. Indeed, TLR agonists have been associated with increased germinal center responses that might be linked with the generation of higher affinity antibodies [18–21], although there are also reports differing from these observations [22]. Although it seems established that TLR agonists lead to a higher titer in many cases, the reason behind the increased titer is not always understood.

Therefore, to investigate the effects of TLR agonists on the quality, i. e., affinity and functional isotype, and quantity, i.e., secreting cells and their cellular output, of the induced antibody repertoire, we performed a small exploratory cohort study with a murine immunization model using alum and a fluorescent antigen, R-phycoerythrin (PE), commonly used in murine immunizations [23–25]. Specifically, we tested the addition of different TLR agonists to TLR2, 4 and 9 and TLR3, individual or in combination, and compared the results to using PE/alum in female BALB/c mice. The responding humoral immunity was assessed on the level of individual antibody-secreting cells (ASCs) using the high throughput analysis platform DropMap [26,27] to separate measurements of binding strength, antibody isotype, cellular frequency, and secretion rates. To measure antibody secretion, we employed a fluorescence relocation assay within the platform, as described earlier [26–29], using paramagnetic nanoparticles coated with an anti-

<footnote><sup>a</sup> Laboratoire Colloïdes et Matériaux Divisés (LCMD), ESPCI Paris, PSL Research University, CNRS UMR8231, Chimie Biologie Innovation, 75005 Paris, France</footnote>

<footnote><sup>b</sup> Laboratory for Functional Immune Repertoire Analysis, Institute of Pharmaceutical Sciences, D-CHAB, ETH Zurich, 8093 Zurich, Switzerland</footnote>

<footnote><sup>c</sup> Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark</footnote>

<footnote>* Corresponding author at: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark</footnote>

<footnote>E-mail address: eyerk@biomed.au.dk (K. Eyer).</footnote>
antibody immobilization and specific detection reagents. This enabled us to quantify the frequency of antibody-secreting cells, antibody iso-types, and their individual secretion rates. Here, the fluorescence signal in the antibody channel can be calibrated to determine the antibody concentration. Additionally, to measure affinity, we introduced fluo-rescently labeled antigen, and when secreted antibodies bind to the target antigen, here PE, we observed fluorescence relocation in both the antibody detection and antigen channels. Having measured concentra-tion, this approach also allows for the estimation of the antibody's interaction strength with the antigen [26–29], reported here as an af-finity slope due to the absence of calibration antibodies with low affinity towards PE. Higher slopes correlate with higher binding strength [26–29].

## 2. Methods

### 2.1. Immunization of mice and cell preparation

R-phycoerythrin (PE, Sigma-Aldrich) was used as antigen for the immunizations (70 µg per immunization, or 10 µg and 150 µg), added either alone or with Pam3CSK4 (TLR2 agonist, 10 µg), poly(I:C) HMW (TLR3 agonist, 50 µg), monophosphoryl lipid A from S. minnesota R595 (MPLA, TLR4 agonist, 5 µg), CpG ODN 1826 (TLR9 agonist, 15 µg; all from Invivogen) or a combination of Pam3CSK4, MPLA and CpG ODN 1826 (combination of TLR2/4/9 agonists: 10, 5 and 15 µg, respectively) to PBS and mixed 1:1 (ν/ν) with 2 % Alhydrogel adjuvant (alum, Invivogen). 8–10 weeks old female BALB/cJRj mice (Janvier) were immunized i.p. twice, four weeks apart, with 100 µl of the prepared solutions (Fig. 1A). Six days after the second immunization, the animals were euthanized, and spleens and blood were collected. Splenic single-cell suspensions were obtained and then enriched in B cells as described elsewhere [28]. All performed experiments were approved by the Cantonal Ethics Committee of Zurich under the license number ZH215/19. B cells were enriched as described elsewhere [28], resus-pended in 3 ml MACS buffer and the cell number was measured using a Countess 3 automated cell counter (Thermo Fisher). The B cells were used for the droplet-based measurements. The blood was collected by cardiac bleeding, clotted at room temperature (RT), spun, and the serum used for the titer measurements.

5 out of the 50 immunized mice did respond differently to immuni-zation by producing only small frequencies of Immunoglobulin G-

secreting cells (IgG-SCs) compared to the others (≤ 0.05 % IgG-SCs). These mice were excluded from further analysis, following a Grubb's test (α < 0.05) that identified them as outliers in the IgG frequency data.

### 2.2. Droplet generation, aqueous phases and data analysis

The spleen B cells were then analyzed using the DropMap platform and assays to quantify IgM and IgG secretion rates, further detecting the IgG subclass and the binding of the secreted antibodies against PE ac-cording to the literature [29,30].

### 2.3. Titer measurements

We used Maxisorp Nunc-Immuno 96-well plates (Thermo Scientific) for the coating with antigen, diluted to 20 µg/ml in PBS (100 µl per well, incubated overnight at 4 °C). Then, the wells were blocked with 1 % recombinant human serum albumin (rHSA) in PBS for 2 h at RT. After blocking, the plates are washed twice with 0.5 % rHSA in PBS. Serial dilutions of serum or antibodies (in 0.1 % rHSA, 100 µl per well) were added to the wells, including positive and negative controls, and incu-bated for 2 h at RT. Plates are washed again twice with PBS containing rHSA, followed by the addition of goat anti-mouse IgG horseradish peroxidase (HRP, either anti-IgG (H + L), HRP, Invitrogen #62-6520, anti-IgG1-HRP, #1071-05, anti-IgG2a-HRP, #1081-05, or anti-IgG2b-HRP, all three from SouthernBiotech, #1091-05), each diluted to 50 ng/ml in PBS with HSA (100 µl per well). The specificity of the probes was assessed in a anti-IgG ELISA with the use of mouse IgG isotype controls (IgG1, 2a and 2b) and an anti-GFP (MA1-952, IgG2b), anti-CD3 (14-0037-82, IgG2a) anti-TNFα antibody (14-7348-81, IgG1, all ThermoFisher), respectively. As positive control, we used murine anti-PE monoclonal antibody (14-4120-81, IgG1, ThermoFisher). Afterward, the plates are washed three times with PBS. For fluorescence measurement, fresh detection reagent was prepared by mixing PBS with HSA, 30 % H₂O₂ (final concentration 100 µM), and Amplex Red (final concentration 5 µM, Invitrogen A22177), and 100 µl was added per well. Directly afterward, the fluorescence signal (excitation at 535 nm, emission at 590 nm) was recorded in kinetic mode at 10-min intervals for up to an hour using a plate reader (Tecan infinite m200). Data analysis involved the calculation of the slope of the fluorescence increase, which we calibrated with IgG levels.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Percentages of Ig-SCs. (A) Employed immunization schedule in this study. Frequencies of IgM- (B) and IgG-secreting cells (C) within all purified cells from the splenic B cell lineage. Combination in (B) and (C) refers to the combination of TLR2/4/9 agonists. We used Welch's unequal variances t-test to compute the respective *p*-values: \* < 0.05, \*\* < 0.01, \*\*\* < 0.001, \*\*\*\* < 0.0001. N = 5 (PE), 5 (PE + TLR2), 6 (PE + TLR4), 5 (PE + TLR9), 6 (PE + combination), and 5 mice (PE + TLR3). Filled colored circles indicate outliers that were removed from further analysis (see also methods). All displayed standard errors are SD.</figcaption></figure>
## 2.4. Data representation and statistical analysis

In the different bar charts, each dot represents one mouse, and the mean and standard deviation are plotted for each condition. We used Welch's unequal variances t-test to compute the respective p-values when comparing the means of two conditions in predefined comparisons: the p-values were computed to compare each condition with TLR agonists added to the PE/alum condition but not for any other pair of conditions, except when otherwise specified. To compare single-cell distributions in Fig. 2B and 3D, we used the Kruskal Wallis test for multiple comparisons. p-values were denoted as follows: \* < 0.05, \*\* < 0.01, \*\*\* < 0.001, \*\*\*\* < 0.0001.

# 3. Results

## 3.1. PE dose optimization

First, we aimed at optimizing the dose of antigen dose [30], as different ones were found in literature [23,31–33]. We tested 10, 70 and 150 µg as these were doses used in literature. Looking at IgG-secreting cells (IgG-SCs) only, we found 0.18 ± 0.07 %, 0.19 ± 0.15 % and 0.09 ± 0.02 %, and 0.12 ± 0.07 %, 0.13 ± 0.12 % and 0.03 ± 0.01 % of PE-specific cells. Also, within the specific cells, the intermediate cells displayed the highest affinities (SI Fig. 1), and we decided to use this concentration as we were interested in whether the TLR agonists could increase affinities (SI Figs. 2 and 3).

## 3.2. A combination of TLR2/4/9 agonists increased the percentage of IgM- and IgG-SCs, and TLR2 and TLR9 agonists of IgG-SCs only

Immunization with PE and alum only resulted in a frequency of IgM-SCs of 0.38 ± 0.31 % (Fig. 1B), and only the addition of the combination of TLR2/4/9 agonists was able to increase this frequency significantly (about 2×, 0.94 ± 0.30 %, *p* = 0.015). In terms of IgG-SCs (Fig. 1C), we observed 0.19 ± 0.15 % in PE/alum and a 3× increased frequency in immunizations with added TLR2 agonist (*p* = 0.004), TLR9 agonist (*p* = 0.049), and a 4× increased frequency for the combination of TLR2/4/9 agonists (*p* = 0.003), compared to 0.02 ± 0.01 % IgG-SCs in naïve control mice (data not shown).

## 3.3. The addition of TLR2 agonist alone increased the secretion rates of IgG-SCs, but a combination of TLR2/4/9 agonists reduced the secretion rates for both IgM- and IgG-SCs

We next quantified the secretion rates (SR) of the identified IgM- or IgG-SCs to determine whether the addition of the different TLR agonists influenced the cellular output (Fig. 2). The median SR for mice immunized with PE/alum were 86 ± 18 and 94 ± 18 Ig/s for IgM and IgG, respectively. In comparison to PE/alum, we observed no significant effect on the IgM SR when the different TLR agonists were added (Fig. 2A), and the only differences in IgG SR were found in the TLR2 condition (146 ± 28 IgG/s, *p* = 0.01). Given the influences of TLR2/4/9 agonists as individual additions, we would have expected an increased IgG SR for the combination, but this was not the case (78 ± 29 IgG/s), and the median IgM and IgG SR for the combination were significantly different from the average of the individual additions (Fig. 2A).

Lastly, we compared the SR distributions of the TLR agonist conditions with the PE/alum conditions, composed of the individual IgG-SCs, and the increase in IgG SR was also visible at the level of the single-cell distribution (see Fig. 2B). There was a clear difference in the distribution for the TLR2 agonist compared to PE/alum, with a general shift to higher SR but also a new population appearing at around 300 IgG/s. Additionally, the inability of the TLR combination to increase SR was also apparent on the single-cell level where the cells secreting above 100 IgG/s seemed generally reduced and the SR distribution was different from the individual distributions resulting from the TLR2, TLR4, and TLR9 agonists (Fig. 2B).

## 3.4. The addition of TLR agonist had very little effect on the frequency of PE-specific cells, their secreted Ig isotype and their displayed affinities

We next looked at the PE-specific antibody response by measuring the PE-specific IgG-SCs in the spleen as previously described [28,29]. For this analysis, we did not focus on the IgM-SCs as their displayed affinities were not in a detectable range for our technique, and only a few cells were found to bind to PE in our setup (roughly 1:200 IgM-SCs). Within the IgG-SCs, PE-specific cells were detected in all conditions (Fig. 3A), with TLR2 and the combination showing significant increases compared to PE/alum (around 3×). Furthermore, the fraction of specific cells within IgG-secreting cells was 64 ± 12 % in the PE/alum condition and did not change significantly with the addition of the different TLR agonists (Fig. 3B). Overall, slightly more than half of the IgG-secreting cells were PE-specific in the different conditions, and a small, but

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** IgG secretion rates. (A) Median IgM (blue) and IgG (red) SR for each mouse, means and SDs are displayed here. The values for the TLR2, 4 and 9 agonist groups are also averaged for comparison with the combination (bars with dotted outline). (B) IgG SR distributions for each condition, pooled from all the mice in the group. The number of cells that were found to secrete ≥500 IgG/s out of the total number of IgG-secreting cells for each condition is specified at the top, along with the corresponding percentage. Welch's unequal variances t-test (A) and Kruskal Wallis test (B) were used for comparison, *p*-values: \* < 0.05, \*\* < 0.01, \*\*\* < 0.001, \*\*\*\* < 0.0001. *N* = 5 (PE), 5 (PE + TLR2), 6 (PE + TLR4), 5 (PE + TLR9), 6 (PE + combination), and 5 mice (PE + TLR3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** PE affinity-related measurements. The frequency of cells secreting a PE-specific IgG (A) within all assayed cells and (B) as a percentage of all IgG-SCs. (C) Average affinity slope of PE-specific IgG antibodies (a higher slope correlated with higher affinity). (D) Distributions of single-cell affinity slopes, pooled for each condition from all the mice of the group. The number of cells with values >5 is shown at the top, as well as the total number of specific IgG-SCs. Welch's unequal variances t-test (A-C) and Kruskal Wallis test (D) were used for comparison, *p*-values: \* < 0.05, \*\* < 0.01, \*\*\* < 0.001, \*\*\*\* < 0.0001. N = 5 (PE), 5 (PE + TLR2), 6 (PE + TLR4), 5 (PE + TLR9), 6 (PE + combination), and 5 mice (PE + TLR3). Filled colored circles indicate outliers that were removed from further analysis. All displayed standard errors are SD.</figcaption></figure>

significant decrease was observed for the TLR3 agonist (48 ± 8 %, *p* = 0.046).

The relative proportion of secreted isotypes (IgG1 and IgG2a/2b/3) was not significantly affected by the addition of TLR agonists, except for

TLR9 (*p* = 0.002). Within the IgG-SCs, 90 % were of the IgG1 isotype (SI Fig. 4). Whether an IgG-SC was PE-specific was not linked with its IgG isotype as the respective fractions of IgG1 and IgG2a/2b/3 were comparable between the subset of PE-specific and non-specific IgG-SCs (data

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Total splenic immunoglobulin secretion. (A) Total B cell counts after enrichment for the different conditions, and (B) the calculated and normalized output of secreted immunoglobulins in the spleen. The graph displays the total IgM, IgG, and PE-specific IgG molecules secreted in the spleen, considering B cell count after enrichment, normalized by the average IgM and IgG values of the PE group. Welch's unequal variances t-test was used for comparison, *p*-values: \* < 0.05, \*\* < 0.01, \*\*\* < 0.001, \*\*\*\* < 0.0001. N = 5 (PE), 5 (PE + TLR2), 6 (PE + TLR4), 5 (PE + TLR9), 6 (PE + combination), and 5 mice (PE + TLR3).</figcaption></figure>
not shown). Similarly, the IgG SR (median and single-cell level distri-
bution) were essentially similar when considering all the IgG-SCs or only
the PE-specific subset (data not shown).

We next extracted the antigen-binding slope, related to affinity [26,27], for each PE-specific cell, a measure we previously correlated with affinity where a higher slope correlated with higher affinities. First, looking at the means, we did not find any significant increase or decrease in the extracted affinity indicators (Fig. 3C). Indeed, the pooled single-cell distributions did not reveal any increase in mean affinity but displayed a decrease in mean affinities and subtle differences in their shapes (Fig. 3D).

3.5. TLR agonists increased the cellular antibody output

Finally, we aimed to calculate the antibody output standardized by
the total number of B cells present in the spleen to estimate the impact of
the observed changes on the animal. To do so, we first counted the total
number of cells from the B cell lineage after enrichment (Fig. 4A). We
generally observed an increase in the splenic B cell count compared to
PE/alum, and significant ones for the TLR2 agonist and the combination
by a factor of three and five, respectively. Using this information, we
multiplied the average antibody secretion rates per B cell (mean SR
multiplied by frequency of SCs) by the spleen B cell count for each
mouse, and we obtained a correlate of the total amount of the antibodies
secreted by the spleen, distinguishing IgM, total IgG, and PE-specific
IgG, as shown on Fig. 4B. To illustrate the differences better, we
normalized the observed numbers by the values observed in PE alum
mice. Here, the TLR2 agonist and the combination of TLR2/4/9 agonists,
and to a lesser extent the TLR9 agonists, induced a significant increase in
total IgG (by a factor of 16, 24 and 7, respectively) and PE-specific IgG
(by a factor of 13, 11 and 4, respectively), on the day of the measure-
ment. These differences, however, were not visible yet in increased titers
(SI Fig. 5).

4. Discussion

In general, the different TLR agonists and the combination of TLR2/
4/9 agonists altered several aspects of the induced immune response
that could be linked to the common observations of increased titer,
namely cellular secretion rates and cellular frequencies. These changes
were mostly concerning cellular Ig secretion rates, but neither the af-
finity nor the isotype of the antibodies. Indeed, the frequency of IgG-SCs
(TLR2, TLR9, TLR2/4/9 agonists), PE-specific IgG-SCs (TLR2, TLR9 and
TLR2/4/9 agonists) as well as the IgG SR (TLR2 agonist) were affected
by the inclusion of non-covalently linked TLR agonists. Interestingly, the
effect seemed additive when comparing the combination of TLR2/4/9
agonists to the single agonists regarding the frequency of the IgG-SCs,
but not in respect to their SR for which the increase observed for each
of the single agonists was absent in the combination. Within the com-
bination, there was a roughly 3× increase in B cells, and among these
cells, 3× as many secreted PE-specific antibodies, leading to a 10× in-
crease in specific IgG production. Interestingly, this combination is
supposed to be present in bacteria, specifically *N. meningitides* species,
which are described to contain TLR2 agonists (bacterial cell-wall com-
ponents), TLR4 agonists (lipopolysaccharides), and TLR9 agonists
(bacterial DNA, more precisely unmethylated CpG sequences) [34–37].

However, there are also some important differences to note. The IgG
isotype distribution was drastically different from mice immunized with
fixed bacteria (N. meningitidis serotypes B and C and alum, as published
in Aymerich et al.) [30], where the antibody response in the spleen was
largely dominated by IgG-secreting cells (93 ± 7 % and 90 ± 5 %,
respectively) and by functional murine isotypes (IgG2a/2b/3, around
80 %). Using the TLR agonists, we observed here a roughly 50:50 split
between IgM- and IgG-SCs, and, within IgG-SCs, we found that 90 %
were of the IgG1 isotype in all conditions. While TLR agonists have been
described to directly interact with B cells, leading to class-switching and

maturation to antibody-secreting cells [38–44], this might be because,
with full bacteria, the bacterial antigens and TLR agonists are both
present on the bacteria and can co-activate the BCR and TLR on various
B cell subpopulations in parallel [45]. Indeed, studies that have shown
efficient switching *in vivo* usually used immunogens that included an-
tigens and TLR agonists in physical proximity, such as virus-like parti-
cles [43,46,47], which might resemble the bacterial surface.
Additionally, it has been shown that the number of interactions with
receptors on B cells has an important role in immunoglobulin class
switching *in vitro* [48]. These factors would be missing in our experi-
mental conditions, but also in all formulations where a TLR agonist is
simply added.

However, there are some limitations to our study. First, this mea-
surement only quantified the antibody secretion in the spleen and not in
other compartments, where antibody-secreting cells are present, such as
the bone marrow, and we analyzed the cells after extraction ex vivo.
Furthermore, the purification and measurements can introduce noise
due to variations in the processes. In our case, the measurement was also
limited to a single time point so that it cannot consider differences in the
kinetic of the response. We chose day 6 after the second immunization
due to experience from previous experiments, as the maximum fre-
quency of secreting cells was often observed on that day. Furthermore,
differences could be also antigen-, dose- and adjuvant-specific.
Furthermore, all immunization solutions were prepared with alum,
which preferentially induces a Th2 response [49,50] with IgG1 as an
associated IgG subclass in mice, and we used the BALB/c strain for these
experiments, which is generally considered a Th2-dominant strain
[51,52]. To conclude, our results indicate that the increased antibody
titers that have been measured for various TLR agonists or combinations
[13–15] might stem from an increased magnitude of the antibody
response rather than a better quality with higher affinity antibodies, at
least in settings where there is no physical link between the TLR agonists
and the antigen.

Author contributions

N.A. planned, performed and analyzed the experiments, and drafted
the manuscript. O.T.M.B, K.P., L.J.S., and I.S. contributed to the mouse
measurements. K.E. and J.B. supervised the project. All authors com-
mented on and revised the manuscript.

CRediT authorship contribution statement

**Nathan Aymerich:** Writing – original draft, Methodology, Investi-
gation, Formal analysis, Data curation, Conceptualization. **Olivia T.M.**
**Bucheli:** Writing – review & editing, Resources, Methodology. **Kevin**
**Portmann:** Writing – review & editing, Resources, Methodology. **Luca**
**J. Schlotheuber:** Writing – review & editing, Resources, Methodology.
**Ingibjörg Sigvaldadóttir:** Writing – review & editing, Resources,
Methodology. **Jean Baudry:** Writing – review & editing, Supervision,
Conceptualization. **Klaus Eyer:** Writing – review & editing, Supervision,
Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgments

The European Research Council starting grant (grant #803'363) and
the Swiss National Science Foundation (grant #310030_197619) sup-
ported this work. This work has benefited from the technical contribu-
tion of the joint service unit CNRS UAR 3750. We would also like to
thank Guilhem Chenon for the development of the DropMap application
used for image analysis.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2025.127518.

## Data availability

Data will be made available on request.

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