Vaccination against H5 HP avian influenza virus leads to persistent immune response in wild king penguins

Domain: neuroscience
Published: 2026-02-09
Editorial outcome: minor_revision

Abstract
Since 2021, the panzootic nature of high pathogenicity avian influenza (HPAI) represents an increasing threat to wild vertebrate populations. In this context, recent vaccines developed for poultry could provide tools for the conservation of wild endangered birds populations. The king penguin (Aptenodytes patagonicus), a long-lived seabird breeding in dense colonies with an extended chick-rearing period, has been identified as a possible surrogate species for a vaccination trial in a sub-Antarctic natural setting. Here we investigate the immune response of king penguin chicks to a self-amplifying mRNA vaccine against a H5 HPAI clade 2.3.4.4b protein. The cohort entails thirty vaccinated chicks (primo- and boost-injections), and 20 unvaccinated controls. Along 250 days of monitoring, the vaccinated chicks show a high and persistent immune response, granting a strong sero-neutralisation capacity against the virus, up to fledging. No adverse effects are observed. Screening for antibodies against unspecific avian influenza viruses suggest that no natural infection has occurred over the entire trial. The emergence of HPAI in the Southern Indian Ocean in October 2024 highlight the timeliness of such experimental tests. Our results thus show the vaccine could provide a potentially powerful tool for mitigation of avian flu outbreaks in the wild.

Introduction
High pathogenicity avian influenza (HPAI) H5Nx has become a critical threat to biodiversity, affecting hundreds of species and hundreds of thousands of individuals of wild species1,2. Since 2021, H5Nx clade 2.3.4.4b HPAI viruses have been responsible for a panzootic that has spread to all continents except Australia, representing an increasing threat to wild species, domestic animals and humans3. HPAI H5Nx viruses have emerged from farmed poultry, spreading globally. They are associated with high mortality rates, leading to massive die-offs in densely breeding species, and could potentially threaten small populations with extinction4–6. Among newly affected populations, colonial long-lived species such as seabirds4,7–9, condors10, vultures11 and pinnipeds5, are highly sensitive to adult mortality12. This is notably relevant for remote and threatened populations, such as those from Antarctic and sub-Antarctic oceanic regions, where the HPAI H5N1 virus has been first detected in 202313,14. In such areas, the spread of the virus could induce an additional threat to marine vertebrate populations, already under pressure from invasive species, fishing activities or climate change15,16. In particular, penguin populations appear to be of prime concern17.

For threatened populations, vaccination has been identified as a potentially powerful mitigation and protective measure against HPAI in emergency situations18. However, a critical challenge is the limited availability of scientific data on the suitability of vaccines for wild species in natural settings10. Studies have investigated HPAI vaccination in wild species held in captive conditions, notably in zoos following European Commission directives resulting from first HPAI epidemics in the early 2000s. The use of heterologous inactivated vaccines for captive birds appeared to induce a strong immune response, although temporary and heterogeneous among species19–24. Serological studies notably suggested that captive Sphenisciformes (penguins) did not show a strong immune response following vaccination using inactivated vaccines, while flamingos developed an antibody response lasting up to seven years25,26. Most of the vaccines used in this context are nevertheless not homologous with the currently circulating viruses from the H5Nx 2.3.4.4b clade. Two decades later, following the arrival of HPAI in South Africa in 2022, an investigation was conducted in adult captive individuals of the critically endangered African penguins (Spheniscus demersus) using two types of vaccines (an inactivated H5N8 and a H5 avian influenza virus-like particle vaccine)27. This latter study highlighted a potential antibody persistence over several months in this species27, although the sample size was limited. More recently, an emergency vaccination programme was launched for more than 200 captive and free-living California condors (Gymnogyps californianus), after HPAI-related die-offs had been detected in a wild population. In that case, the vaccine was a H5N1 subtype, reverse genetics-derived, inactivated product approved for chickens in the USA, and heterologous to the currently circulating 2.3.4.4b clade viruses28. Immune response, vaccine safety and practicability were first tested on the black vulture (Coragyps atratus) as a surrogate species and on captive individuals10. This latter study described an antibody persistence up to 42 days and suggested the need for a second boost-injection upon recapture10.

The use of vaccination as a conservation strategy for wild populations in natural settings raises ethical, practical and technical challenges. In addition to the availability of safe vaccines leading to an appropriate immunological response, practical issues related to the ecology and biology of the targeted host species have been identified as major challenges29. It may then be especially important to identify key model species that can be studied in natural conditions and considered as surrogates for related endangered species facing conservation issues. Conducting experiments in natural settings provides the opportunity to practically assess the possible implementation of vaccination protocols. In some situations, carefully designed vaccination trials could enable the monitoring of wild individuals over periods of months, while ensuring suitable animal welfare conditions (limited handling, no artificial housing, natural feeding…). It could also allow to quantify the immune response of individuals in their natural environment, including difficult-to-control extrinsic factors, such as natural feeding, harsh winter weather conditions, exposure to predation risks and other infectious agents, that are usually not considered in vaccination trials using captive animals. Monitoring the serological status of individuals, and the local epidemiological situation in the field, would also enable to assess the practical feasibility of post-vaccination surveillance in the event of anti-HPAI vaccination being used for conservation purposes18.

The king penguin (Aptenodytes patagonicus) is a widespread and abundant species in the sub-Antarctic area, with an ecology potentially prone to high circulation of the HPAI virus given the size and density of its colonies, that can reach tens to hundreds of thousands individuals30–32. As a long-lived species, the slow pace of life of king penguins may be associated with long lasting immune processes, as predicted for some of those species33,34. In this context, the long chick-rearing period of the king penguin on land30,35 (c. 300 days) make the species especially suited for investigating the dynamics of the immune response of chicks to vaccination. This least concerned species according to IUCN36 could thus represent a suitable surrogate for endangered penguin species, such as the Northern rockhopper penguin (Eudyptes moseleyi), but also for related species sharing comparable life history traits and potential similar immune system particularities (Diomedeidae, Procellariidae…). We thus decided to investigate the immune response of king penguin chicks to vaccination against a H5Nx HPAI virus in a natural setting.

Planning a HPAI vaccination program on wild birds should be preceded by an evidence-based risk assessment and an integrated cost-benefit analysis adapted to each situation18, including critical considerations of ecological and epidemiological issues, and the identification of a potentially suitable vaccine29,37. The choice of the tested vaccine was made in light of the recent developments in RNA vaccine technologies and their wide applications to humans and domestic animals. In particular, messenger RNA vaccines have been approved and massively used as a fast and efficient response to SARS-CoV-2 pandemic. Next generations of self-amplifying messenger RNA (saRNA, also known as “replicons”) vaccine show a great deal of potential advantages. The principle is based on introducing the encoding of a gene (i.e., RNA), associated with the essential molecular elements required for its self-amplification: the host cell can thus produce the targetted protein itself38. Therefore, a minimal dose of saRNA vaccine could induce an efficient humoral and cellular response, leading to complete immunisation. In the case of fast evolving viruses, like Influenza viruses, saRNA vaccine could be efficiently used to match a recently circulating viral genotype. With regard to Influenza, this approach had been identified before the SARS-CoV-2 pandemic as a possible game changer39. In recent applications, saRNA vaccination was shown to provide an efficient protection against H5Nx virus in ducks40 and domestic geese41, and has subsequently been used to vaccinate millions of ducks in France since 202442. The saRNA vaccine ‘ResponsAI H5’ (Ceva Santé Animale) had notably been shown to reduce virus excretion and transmission, as measured under experimental settings in ducks43. This study had also shown that a boost-injection applied 28 days after the primo-injection led to detectable anti-H5 antibody levels in ducks, however decreasing relatively fast afterwards. Because they specifically target the H5 haemagglutinin protein, such vaccines enable the Differentiation of Infected from Vaccinated Animals (DIVA vaccines): individuals exposed naturally to an unspecific influenza A virus would produce antibodies against the virus nucleoprotein (NP), while only antibodies against the H5 haemagglutinin protein would be expected in vaccinated ones44. This DIVA property is particularly important for vaccination campaigns in wild settings as it would allow the monitoring of the natural field exposure to avian influenza viruses of vaccinated individuals18. Nevertheless, a critical constraint posed by RNA vaccines is that they require ultra-low temperatures (−80 °C) for long-term storage, which can add to other practical constraints, such as the need for a booster injection for instance.

In this study, we report the results of a vaccination trial using a saRNA vaccine against H5 HPAI clade 2.3.4.4b protein conducted on king penguin chicks in a sub-Antarctic colony of the Southern Indian Ocean. A group of chicks receives a primo-injection at about 45 days post-hatching (once emancipated from their parents), and a booster 37 days later, while a group of chicks is used as controls. Along 250 days of fine-scale monitoring, the vaccinated individuals show a high and persistent anti-H5 immune response, granting a strong sero-neutralisation capacity against a H5N1 2.3.4.4b clade virus, up to fledging. We report no adverse effects due to the vaccination treatment on chick health and survival. Overall, our results suggest that the saRNA H5 vaccine could be a valuable and powerful tool for wildlife conservation efforts in the current context of panzootic HPAI.

Results
Safety of the vaccine
No short-term adverse effects on king penguin chicks have been observed following the intra-muscular primo-injection (t0) and the boost-injection (t0+37 days) of the saRNA H5 vaccine (see Fig. 1 for details of the experimental design). Chick body mass and flipper length increased similarly with age in both treatment groups (Fig. 2A and B). The posterior distribution of the interaction between days post-injection and treatment group on both traits fell entirely within the predefined Region of Practical Equivalence (100% inside ROPE). This provides strong evidence for the absence of a biologically meaningful vaccination effect on chick body mass and flipper growth over time.

Chicks survival rates remained very high for both treatment groups until t0+164 days (97% and 95% respectively for vaccinated and control chicks). As expected, some mortality occurred over the harsh Austral winter, but 19/30 vaccinated chicks and 10/20 control chicks could be recaptured for sampling at t0+252 days. Given the high expected recapture rate, this showed a minimum chick survival of at least 50% for both treatment groups (63% and 50% for the vaccinated and control group, respectively), from the primo-injection (chicks of c. 45 days post-hatching) until just before fledging. Accordingly, no effect of the vaccination treatment on chick survival was detected over the period (Hazard Ratio HR = 1.50 [CI = 0.64-3.53, p = 0.35]) (Fig. 2C; and Supplementary Table 1).

Anti-H5 antibody response following vaccination
All chicks from the vaccinated group showed a clear humoral immune response against the H5 protein, measured via a competitive H5 ELISA assay, while no anti-H5 antibodies were detected in control chicks along the whole trial (Fig. 3; and Supplementary Table 2; Supplementary Table 3; Supplementary Table 4). In order to account for a potential strong increase in anti-H5 antibody level at the start of the experiment, followed by a slow decay over time after reaching a peak, the dynamics of antibody levels of vaccinated and control birds were fitted using a Holling IV curve (Fig. 3), with the following equation (see Supplementary Table 5):

f(days) = (a_control + da * treatment) * days^2 / ((b_control + db * treatment) + (c_control + dc * treatment) * days + days^2) (1)

The non-linear modelling analysis conducted to compare the dynamics of antibody responses between the vaccinated and control groups showed that the model that distinguished the two treatments had substantially better support (AICc = −355.65, p < 0.0001), and AICc = 274.72, ΔAICc = 631). For the model distinguishing the two groups, a lower AICc was estimated (ΔAICc = 2.05) when the curve parameters db and dc were fixed to 0, indicating no difference between treatment groups in the offset and linear terms of the curve. A positive da value was highly significant (t = 21.463, p < 0.0001), showing a higher response in antibody level for the vaccinated group (~16x higher scaling response, a_control ~ 0.03; a_vaccinated = a_control + da ~ 0.52), and a higher antibody level over time following vaccination. All parameters contributed meaningfully to the selected model (all p-values < 0.001). After the boost-injection, a higher anti-H5 antibody level was measured in vaccinated chicks at t0+51 days, compared to levels measured at t0+37 days. The Holling IV curve indicated a predicted maximum antibody level reached at t0+83 days (−2 * b_control / c_control). Antibody levels of vaccinated chicks remained relatively high over time following the boost-injection, with 17/19 vaccinated chicks still seropositive at t0+252 days. The antibody levels of only two vaccinated birds decreased post boost-injection: one showed a rapid and permanent decrease, while the other showed a single intermediate value before increasing again at t0+252 days.

The analyses conducted on values resulting from the indirect H5 ELISA assay showed similar results (Supplementary Fig. 1; and Supplementary Table 2; Supplementary Table 6; Supplementary Table 7). The significant positive da values showed a higher response in anti-H5 antibody level (~16x higher scaling response, a_control ~ 0.12; a_vaccinated = a_control + da ~ 1.99) for the vaccinated group, and a higher titre over time following vaccination (Supplementary Table 8). In addition, the fitted Holling IV curve appeared to predict a similar maximum anti-H5 antibody level reached at 83 days post primo-injection.

Seroneutralisation response following vaccination
The linear mixed-effects model on chick plasma seroneutralisation level revealed a significant interaction between treatment and time (χ2 test, p = 0.012). Estimated marginal means showed that vaccinated individuals had substantially higher seroneutralisation titres than controls, at both 51 days (log(titre) = 11.13 ± 0.32 vs. 2.40 ± 0.46; p < 0.0001), and 252 days post primo-injection (log(titre) = 9.43 ± 0.32 vs. 2.60 ± 0.46; p < 0.0001) (Fig. 4; and Supplementary Table 9; Supplementary Table 10). Within-group comparisons revealed no significant change in seroneutralisation activity of control individuals between 51 and 252 days (p = 0.98), whereas vaccinated individuals showed a significant but moderate decline in antibody titres from 51 to 252 days (log(titre) = 11.13 ± 0.32 vs. 9.43 ± 0.32; p = 0.0029). These results show that the saRNA H5 vaccination provided an important seroneutralisation capacity to king penguin chicks, likely conferring an efficient level of protection of vaccinated chicks against HPAI virus along a large part of their rearing-period.

Differentiating vaccinated individuals from potentially naturally exposed ones
The results of the ELISA analysis for anti-NP antibody showed that the levels remained relatively low and non-detectable for both treatment groups across the experiment, showing the lack of immune responses of the chicks to natural exposure to avian influenza A viruses (Fig. 5; and Supplementary Table 2; Supplementary Table 11). It is important to note that the natural circulation of the HPAI H5N1 virus was first detected on the island on October 17th 2024, and more specifically on October 20th in the study colony14, a few weeks before the last sampling point (t0+252 days). Abnormal die-offs of southern elephant seals (Mirounga leonina) associated to the detection of HPAI H5N1 virus in brain samples were observed, but also of adult king penguins14. The serological analyses nevertheless suggested that no chick had mounted a response until that last sampling point, possibly meaning that none had been exposed over the whole experiment trial.

Methods
Ethical issues and permits
Permits to implement the experiment were granted from the French Ministry of Research (APAFIS #45548-2023102712468223) after evaluation by the Comité Régional d’Ethique d’Occitanie and from Préfecture des TAAF (A-2024-95) after evaluation by the Conseil de l’Environnement Polaire and Conseil National de la Protection de la Nature. After the suspicion of HPAI emergence on Crozet, permits were granted by the Préfecture des TAAF to pursue eco-epidemiological fieldwork (A-2024-144).

Model species and study population
The king penguin is a long-lived colonial seabird, breeding in colonies that can reach tens to hundreds of thousands of individuals, with an extended reproductive cycle of ca. 14 months35,60. A single egg is laid, followed by an incubation time of 54 days and an extended rearing period of c. 11 months. Partners share alternatively the incubation of the egg, then the care of the chick, until its full thermal emancipation (around 30–40 days post-hatching). Chicks are then left ashore in the colony by their parents for several months before fledging to sea at around 300 days post-hatching. Once the chicks are emancipated, they often gather in groups (‘crèches’) to withstand the harsh conditions of the Austral winter. Over that time period, they can be captured, resighted and recaptured, allowing good conditions for their monitoring over several months61.

The experimental vaccination trial on king penguin chicks was conducted from February to November 2024 on Possession Island (Crozet archipelago, 46° 24′ 41″ S, 51° 45′ 22″ E, Southern Indian Ocean), within the colony of Baie du Marin.

saRNA vaccine and practical issues
The saRNA RESPONSAI H5 vaccine (Ceva Santé Animale) was used to explore the immune response of the king penguin chick against a H5 HPAI clade 2.3.4.4b protein in natural settings. The vaccine was provided by the producer under a Material Transfer Agreement (MTA) for exclusive use in the trial. Stock vaccine vials were transported and stored at −80 °C during the whole travel to reach the lab facilities on Possession Island, until processing for the field experiment.

Prior to any injection, stock vaccine vials were placed at 4 °C for 12 h before each session of injections, to ensure a complete defrosting. The thawed stock vaccine solution was then diluted freshly with a provided diluent according to the manufacturer's guidelines. The diluted vaccine was administered within the recommended 2 h post-dilution (0.2 ml intra-muscular injection of 3 µg in the thigh). The use of the saRNA vaccine under field conditions in sub-Antarctica was facilitated by the proximity of the study colony to the scientific station, where −80 °C storage facilities were available. However, the possibility to keep the stock vaccine vials at 4 °C during 24 h before conducting a session of injections could allow some flexibility to organise vaccination trials in remote colonies.

Experimental vaccination design
For the experiment, 50 emancipated chicks of approximately 45 days post-hatching were selected and randomly assigned to two treatment groups, and they were monitored along their rearing period, until fledging to sea. Those chicks were temporally captured and tagged in batches using alphanumeric plastic flipper bands and coloured flags allowing efficient and individual tracking during the whole chick rearing-period. Microtransponders (BioTherm 13® 13 mm 134.2 kHz ISO FDX-B, Biomark, Idaho, USA) were subcutaneously inserted into the back of the neck, each one related to a unique and permanent identification number. Birds received either a primo-injection (t0) of the saRNA H5 vaccine in the thigh muscle (n = 30 chicks, vaccinated group), or a placebo injection using the vaccine diluent (n = 20 chicks, control group). A boost-injection was applied approximately 37 days later to all vaccinated chicks, while control chicks received a second placebo injection, under the same handling conditions as at t0. The bird immune responses were monitored along the entire rearing period through blood sampling at 7 capture times. For clarity of presentation and statistical analyses, sampling days were grouped into seven categories: t0, t0+37 days, t0+51 days, t0+66 days, t0+86 days, t0+164 days and t0+252 days (Fig. 1; and Supplementary Table 12). Blood samples (0.2 to 1 ml) were collected from the ulnar vein using a 1 ml heparinized syringe. Plasmas were separated from red blood cells by centrifugation within a few h. They were immediately frozen and stored at −20 °C before conducting serological analyses. All chick external tags (coloured flags and flipper bands) were removed at the end of the experiment, at t0+252 days. Both groups of control and vaccinated penguin chicks likely contained animals of both sexes, but immune responses of the animals were not differentiated by sex. The immune response of the mixed-sex animal group as a whole was analysed.

Evaluation of the vaccine safety
To assess potential adverse effects of the vaccine, we first evaluated local short-term effects after each intra-muscular injection: chicks were kept a few minutes before release to check for any sign of limping, local swelling, or typical distress behaviour (hyperventilation, lack of reactivity…). Chick condition was also monitored at each capture. The chick survival rate in both treatment groups was evaluated regularly along the entire trial through remote checks from the edge of the colony, until all chick external tags were removed at t0+252 days. We assessed potential long-term adverse effects by monitoring the change in chick body mass and body size (flipper length), as morphometric indicators of chick growth, until t0+164 days. In the context of the HPAI outbreak detected locally in late October 2024 on Possession Island, we could not record the chick body masses/sizes at the last sampling point (t0+252 days) due to biosecurity restrictions. To evaluate whether vaccination affected chick body growth trajectories, we used a Bayesian linear mixed-effects model implemented in the brms package (version 1.22.0) of the R software49. The model included body mass or flipper length as the response variables, with days post-primo-injection, treatment group (vaccinated vs. control), and their interaction as fixed effects. Chick identity was included as a random intercept to account for repeated measures of individuals. To formally assess the absence of a biologically meaningful difference between treatment groups over time, we used a Region of Practical Equivalence (ROPE) approach. The ROPE for the interaction term (days: treatment) was set to ±0.2 kg. Posterior distributions and ROPE inclusion percentages were computed using the bayestestR package (version 0.16.0).

To evaluate whether the vaccination treatment had affected the chick survival over time, we fitted a Cox proportional hazards model with treatment group (vaccinated vs. control), as a categorical predictor. Time-to-event was measured from t0 to the last capture at t0+252 days, when external identification of individuals where removed. We used the coxph() function from the survival package of the R software (version 3.8-3). We assessed the proportional hazards assumption using Schoenfeld residuals. Model results are reported as hazard ratio (HR) with 95% confidence interval (CI). A two-sided p-value < 0.05 was considered statistically significant.

Quantification of specific antibody levels
The production and temporal persistence of specific H5 antibody levels were measured in plasma samples using commercially-available enzyme-linked immunosorbent assays (ELISA) kits. First, plasma samples were analysed using a competitive ELISA assay targetting the H5 protein of the clade 2.3.4.4b virus (H5-cELISA) and suitable for the monitoring of vaccination of domestic and wild birds (ID Screen® Influenza H5 Antibody Competition 3.0 Multi-species, reference no. FLUH5V3-5P, Innovative Diagnostics SARL, Grabels, France). Given previous results obtained on other seabird species suggesting that anti-chicken conjugate antibodies could bind with seabird antibodies62, we complemented our results using an anti-H5 indirect ELISA assay (H5-iELISA) (ID Screen® Influenza H5 Indirect, reference no. FLUH5S-2P, Innovative Diagnostics SARL, Grabels, France). In order to evaluate the natural exposure of chicks to unspecific influenza A viruses potentially circulating in the colony (DIVA approach), we also tested plasma samples using a competitive ELISA assay targetting the non-specific nucleoprotein (NP-cELISA) (ID Screen® Influenza A Antibody Competition Multi-species, reference no. FLUACA-5P, Innovative Diagnostics SARL, Grabels, France). ELISA assays were conducted according to instructions provided by the manufacturer. Plasma anti-H5 and anti-NP antibody levels were obtained by reading absorbance values at 450 nm using a microplate reader (Tecan Infinite® 200 Pro; Tecan Group Ltd., Mannendorf, Switzerland), and correcting raw absorbance values using optical densities (OD) of positive (PC) and negative (NC) controls provided for each kit (OD_PC and OD_NC respectively). Anti-H5 and anti-NP antibody levels obtained with competitive ELISA are expressed as inhibition values (inhibition_sample = OD_sample / OD_NC), while anti-H5 antibody levels obtained with indirect ELISA are expressed as S/P ratio (S/P_sample = OD_sample / OD_PC). We referred to each threshold value provided by each ELISA kit instructions to discriminate seropositive from seronegative samples: H5-cELISA = 0.50; H5-iELISA = 0.50; NP-cELISA = 0.45. Analyses conducted on repeated sub-samples allowed us to assess the between- and within-assay repeatability for each ELISA test (see Supplementary Table 13 for detailed coefficients of variation).

HP H5N1 seroneutralisation assay
A seroneutralisation assays was ran on a subset of plasma samples to explore the capacity of specific antibodies to seroneutralise a HPAI H5 virus of clade 2.3.4.4b. The assay was conducted under Biosafety Level 3 conditions using the A/chicken/France/D2107428/2021_2.1b H5N1 HP virus from 2.3.4.4b clade. Plasmas were heat-inactivated (56 °C for 60 min) and twofold serial dilutions were performed in a 50-μl volume in microtiter plates. Sera obtained from experimentally immunized chicken with the homologous inactivated (Sp_H5N1_A/chicken/France/D2107428/2021_1) (positive control serum) and obtained from SPF chicken (negative control serum) were also used. Diluted plasmas were mixed with an equal volume containing 100 TCID50 of the H5N1 virus. Four control wells of virus alone (positive for the cytopathogenic effect (CPE) control) and four control wells of dilution medium alone (negative CPE control) were included. After 1 h incubation at 37 °C and 5% CO2 for neutralisation, 100 μl of MDCK cells at 2 × 10^5/ml were added to each well. Plates were incubated at 37 °C and 5% CO2 for 72 h, after which the CPE was read under the microscope. For each plasma, the seroneutralisation titer was expressed as the mean of the last dilution factor for which no CPE was observed of two independent experiments. The positive control serum has a titer greater than 2048, the negative control serum has a titer less than 8.

Antibody levels comparison
Statistical analyses were conducted using R63 (R Core Team, 2023, version 4.3.1). We compared the antibody level at each time point between vaccinated and control individuals using analyses of variance. The same model was used for both competition and indirect ELISA results. To assess the effect of vaccination on antibody dynamics, we used linear mixed-effects models (LMMs) fitted with the lme4 package in R. Antibody levels were modeled as the response variable, with treatment group (vaccinated vs. control), sampling day (as a factor), and their interaction as fixed effects. Individual identity was included as a random effect to account for repeated measures of individuals.

We tested the significance of main effects and interactions using Type III ANOVA with Satterthwaite’s approximation for denominator degrees of freedom, implemented in the lmerTest package. When significant interactions were detected, we conducted post-hoc pairwise comparisons between treatment groups at each sampling day using estimated marginal means (EMMs, emmeans package), with Tukey adjustment for multiple testing.

Fitted models for antibody levels
To model the dynamics of anti-H5 antibody levels, we fitted the sequential values from each individual with a Holling IV rational function64 in order to account for the expected sigmoid early build-up of antibody level following the primo- and boost-injections, followed by a decay of antibody level towards a potential asymptotic value for large numbers of days post vaccination. A fixed treatment effect was set to compare the vaccinated and control groups (stable low level was expected for control chicks).

We fitted a non-linear regression model to the time series data (exact time series) using the nlsLM() function from the minpack.lm R package (version 1.2-4). The Holling IV function adapted to our modelling situation is:

f(days) = (a * days^2) / (b + c * days + days^2) (2)

with f(days), the antibody level (calculated from ELISA titre values); days, the number of days since the primo injection; a, the value of the function at the asymptotic reached at high values of days; (−2b/c), the value of day at the peak antibody level. We compared a pooled model, in which vaccinated and control groups shared the same temporal response function, to a group model in which curve parameters were allowed to vary between the two groups (vaccinated vs placebo).

We compared three competing models:
• Pooled model: vaccinated and control chicks shared the same response curve (no group effect);
• Full group model: curve parameters were allowed to differ between groups, with the following parameterization:

f(days) = ((a_control + da * treatment) * days^2) / ((b_control + db * treatment) + (c_control + dc * treatment) * days + days^2) (3)

where days is the number of days since the primo-injection and treatment is a binary indicator of group membership (0 = control, 1 = vaccinated). We parameterized the Holling type IV function such that the control group served as the reference, with parameters a_control, b_control and c_control. For the vaccinated group, parameters were expressed as additive deviations from the control: a_control + da, b_control + db, and c_control + dc.
• Reduced group model: a nested version of the group model where only the asymptote differed between groups (da ≠ 0, db = dc = 0).

Model comparisons were conducted using the corrected Akaike Information Criterion (AICc) to account for differences in model complexity and sample size, using the AICcmodavg R package (version 2.3-4). The model with the lowest AICc value was retained as the most parsimonious representation of the data. A difference in AICc greater than 2 was considered evidence of meaningful support for the better-fitting model.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Mathilde and co-authors investigates the immune response of king penguin chicks vaccinated with an\nH5Nx highly pathogenic avian influenza (HPAI) virus vaccine under natural environmental conditions. This is a study of\nconsiderable significance. The authors demonstrated both the safety of vaccination in king penguin chicks and the\npersistence of IgG and neutralizing antibody responses for up to 252 days. These findings are consistent with previous\nstudies validating the efficacy of saRNA vaccines in other target species, underscoring the broad application potential\nof AIV saRNA vaccines. Furthermore, differential diagnostic analyses confirmed that the king penguin\nchicks were not naturally infected with AIV during the experimental period, highlighting the value of DIVA vaccines in distinguishing natural infection\nfrom vaccine-induced immunity.\nThe manuscript is very well written, particularly in its detailed description of the materials and methods. Overall, the findings\nare robust and provide valuable insights that may benefit future H5 vaccine research in other endangered penguin species.\nSome of concerns are outlined below.\n1. As the authors noted, a key scientific limitation of this study is the absence of a challenge experiment. This omission\nposes a challenge in establishing the correlation between antibody titers and protective efficacy, which should be further\nevaluated under controlled laboratory conditions in future studies.\n2. Considering that neither the authors nor previous studies have detected AIV infection in king penguins (DOI:\n10.1007/s00300-009-0587-4; DOI: 10.1111/j.1751-0813.1981.tb05839.x), it remains uncertain whether this species is\nsusceptible to HP H5N1. This question warrants further investigation and ongoing surveillance, as local outbreaks may pose\npotential threats to the species and could directly affect the future applicability and value of the vaccine.Some discussions\nneed to be strengthened, such as the fact that certain wild animals can be infected with the H5 subtype of avian influenza,\nand the broad host range of the H5 subtype should be addressed and discussed with references from top-tier journals. Such\nas : doi: 10.1016/j.cell.2025.08.016 doi: 10.1016/j.cell.2024.07.024; doi: 10.1016/j.cell.2023.04.002\n3. The initial sample size was relatively small and further decreased by the end of the experiment, which may have affected\nthe assessment of antibody persistence. This concern is supported by Figure 3, in which one vaccinated individual exhibited\nantibody levels comparable to those of the control group.\n4. Although the authors discussed the challenges of assessing long-term antibody persistence (e.g., over one year), it is\nimportant to consider that penguins are long-lived species. Therefore, the duration of antibody responses should ideally be\nevaluated through to adulthood or the breeding period. Alternatively, the title could be refined to specify the actual time frame\ncovered in this study, for example: “…persistent immune response in wild king penguin fledglings prior to sea departure.”\n5. A minor issue in the figures is that the use of light red and light green makes it difficult to distinguish overlapping data\npoints. It is recommended that the color of the vaccinated group be changed to a darker shade to improve visual clarity and\ndifferentiation.", "reviewer_2": "This manuscript describes the field evaluation of an H5N1 influenza saRNA vaccine in penguins. The impact of the work\nlies in the fact that the H5N1 virus is decimating wild bird populations, it is encroaching upon likely naïve bird populations in\nthe Antarctica region, and interventions are being sought. Data supporting immunization of wildlife against H5N1 influenza\nis scant and this manuscript is therefore highly significant, important, and will be of broad interest.\nOverall the data presented is simple and clear and the conclusions drawn are sound.\nThe following are minor comments for the authors’ consideration.\n1. Pg7, ln 186. It is a little unclear if survival rates presented are a result of bird deaths or bird deaths and/or inability to\nrecapture specific animals. I realize this may be a difficult question for the authors to definitively answer, but some general\ncomment on expected recapture rates in the absence of likely death would be useful. I guess the question is “how hard is it to\nrecapture all birds in a study group this size”.\n2. Ln 244. 1 out of 19 birds did not appear to respond to boosting. At face value, 5% of non-responders is large. Was there\nanything in particular noted about the non-responding bird (low body weight, issues noted with vaccination etc) that might\nhelp explain this result?\n3. What is missing from the manuscript is a way to link the observed antibody titers with expected protection. Is there any\nway to infer this link? Possibilities could be the use of a hemagglutination inhibition assay where there is an accepted\nprotective correlate (in humans and chickens at least). Or correlation of the titers detected in this paper with protections\nobserved in the captive duck and geese studies that were conducted with the same vaccine.\n4. More detail is needed on the seronuetralization assay. For example, what infectious dose of virus was used, how were\ntiters read etc."}, "author_response": "POINT-BY-POINT RESPONSES TO THE REFEREES’ COMMENTS: AUTHORS RESPONSES IN BLUE BELOW,\nwith line numbers for the file with track changes.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Mathilde and co-authors investigates the immune response of king penguin chicks\nvaccinated with an H5Nx highly pathogenic avian influenza (HPAI) virus vaccine under natural\nenvironmental conditions. This is a study of considerable significance. The authors demonstrated\nboth the safety of vaccination in king penguin chicks and the persistence of IgG and neutralizing\nantibody responses for up to 252 days. These findings are consistent with previous studies validating\nthe efficacy of saRNA vaccines in other target species, underscoring the broad application potential\nof AIV saRNA vaccines. Furthermore, differential diagnostic analyses confirmed that the king penguin\nchicks were not naturally infected with AIV during the experimental period, highlighting the value of\nDIVA vaccines in distinguishing natural infection from vaccine-induced immunity.\nThe manuscript is very well written, particularly in its detailed description of the materials and\nmethods. Overall, the findings are robust and provide valuable insights that may benefit future H5\nvaccine research in other endangered penguin species.\n# AUTHOR RESPONSE: We thank the referee for these very positive comments.\nSome of concerns are outlined below.\n1. As the authors noted, a key scientific limitation of this study is the absence of a challenge\nexperiment. This omission poses a challenge in establishing the correlation between antibody titers\nand protective efficacy, which should be further evaluated under controlled laboratory conditions in\nfuture studies.\n# AUTHOR RESPONSE: We agree that this is a scientific limitation of the study, but it is difficult to\nimagine to conduct a virus challenge in controlled conditions for ethical and practical reasons given\nthe considered penguin species. We now outlined in the Discussion that the correlation between\nantibody titers and protective efficacy could need to be further evaluated under controlled\nlaboratory conditions, most conveniently in another species, by inserting ‘Alternatively, experimental\nviral challenge might be considered in confined settings in other surrogate species, notably to\nascertain the correlation between measures of immune responses and protection’ (lines 347-349).\n2. Considering that neither the authors nor previous studies have detected AIV infection in king\npenguins (DOI: 10.1007/s00300-009-0587-4; DOI: 10.1111/j.1751-0813.1981.tb05839.x), it remains\nuncertain whether this species is susceptible to HP H5N1. This question warrants further\ninvestigation and ongoing surveillance, as local outbreaks may pose potential threats to the species\nand could directly affect the future applicability and value of the vaccine.Some discussions need to\nbe strengthened, such as the fact that certain wild animals can be infected with the H5 subtype of\navian influenza, and the broad host range of the H5 subtype should be addressed and discussed with\nreferences from top-tier journals. Such as : doi: 10.1016/j.cell.2025.08.016 doi:\n10.1016/j.cell.2024.07.024; doi: 10.1016/j.cell.2023.04.002\n# AUTHOR RESPONSE: There is indeed no evidence that king penguins on Crozet had been exposed to\navian influenza virus before 2024 (see the two reference cited by the referee), but as we outlined in\nthe manuscript the virus emerged locally on Crozet in October 2024 and was associated with die-offs\nof elephant seals and king penguins, as we reported in Clessin et al. 2025 (Circumpolar spread of\navian influenza H5N1 to southern Indian Ocean islands. Nature Communications 16: 8463.\ndoi.org/10.1038/s41467-025-64297-y), which was available only as a pre-print at the time we had\nsubmitted the current Lejeune et al. manuscript. The detection of H5N1 HP avian influenza of clade\n2.3.4.4b virus in the brain of several individuals, together with individuals showing symptoms\nsuggestive of HP avian influenza infection, makes us confident that this species is susceptible to HP\nH5N1. We do agree with the referee that it is of outmost importance to continue investigation on\nthis issue, notably to evaluate what proportion of individuals of this species have been exposed and\nmounted an immune response, and also how other wild species, notably in sub-Antarctic marine bird\nand mammal communities, may be exposed, and to infer between-species transmission pathways\n(we have much on-going work on those issue, with sampling efforts at broad spatial scales). We are\nnow specifying in the last part of the Results that ‘Abnormal die-offs of southern elephant seals\n(Mirounga leonina) associated to the detection of HPAI H5N1 virus in brain samples were observed,\nbut also of adult king penguins[14]’ (lines 310-312). Further, we now stressed in the Discussion that\nmany wild species appear susceptible to HP H5Nx viruses, which warrant further extensive\nmonitoring of the eco-epidemiological situation, with implications for the potential use of a vaccine\nas a mitigation tool (with a citation of Moratorio et al. 2024 Cell, as suggested by the referee; lines\n381-387).\n3. The initial sample size was relatively small and further decreased by the end of the experiment,\nwhich may have affected the assessment of antibody persistence. This concern is supported by\nFigure 3, in which one vaccinated individual exhibited antibody levels comparable to those of the\ncontrol group.\n# AUTHOR RESPONSE: The sample size was contrained by animal experimentation considerations,\nbut it was clearly sufficient to show antibody persistence in a high proportion of individuals. The\nrepeated sampling of individuals over the course of the experiment allowed us making a robust\ninvestigation of the antibody dynamics. The occurrence of one of the vaccinated individuals showing\nan early increase in anti-H5 antibody level, followed by a fast decline to an antibody level comparable\nto those of the control group, raises the question of the underlying variability of humoral immune\nresponse in this species, suggesting the interest of more extrensive immunology studies. Following\nthis remark, we now outline in the Discussion that larger sample sizes could be useful for this reason\nin future studies (lines 416-430).\n4. Although the authors discussed the challenges of assessing long-term antibody persistence (e.g.,\nover one year), it is important to consider that penguins are long-lived species. Therefore, the\nduration of antibody responses should ideally be evaluated through to adulthood or the breeding\nperiod. Alternatively, the title could be refined to specify the actual time frame covered in this study,\nfor example: “…persistent immune response in wild king penguin fledglings prior to\nsea departure.”\n# AUTHOR RESPONSE: We fully agree with the referee that the fact that the studied species is long-\nlived is of outmost importance. Given that such long lived species have defereed maturity (breeding\nfor the first time at age 2 or 3 for penguins, or when more than ten years old in some albatross\nspecies), studying the duration of antibody responses through to adulthood in such wild species\nwould be especially challenging (even when individuals would come back to breed, it would be\ndifficult to find them among the very large number of conspecifics in the breeding colonies,\nespecially in penguins that cannot be marked with outside marks). Studying antibody persistence\namong years in adult is indeed the next logical step. This is a perspective specifically discussed in a\nwhole paragraph of the Discussion (lines 388-412) and now we added ‘Given the particularly\nthreatened status of some populations of albatrosses [33], their longevity, site fidelity and likely\nsusceptibility to the virus [13], conducting such an experiment in a large albatross species could be a\nnext logical step’. (lines 412-415). Regarding a potential change to the title, we think that the current\ntitle conveys the main content of the manuscript while respecting the requested number of words\n(15), but we would be ready to change it to something like ‘Vaccination against H5 HP influenza virus\nleads to persistent immune response in wild king penguin chicks’ or ‘Vaccination against H5 HP\ninfluenza virus leads to persistent immune response in wild king penguin fledglings prior to sea\ndeparture’ if found more approriate.\n5. A minor issue in the figures is that the use of light red and light green makes it difficult to\ndistinguish overlapping data points. It is recommended that the color of the vaccinated group be\nchanged to a darker shade to improve visual clarity and differentiation.\n# AUTHOR RESPONSE: This can be changed accordingly.\nReviewer #2 (Remarks to the Author):\nThis manuscript describes the field evaluation of an H5N1 influenza saRNA vaccine in penguins. The\nimpact of the work lies in the fact that the H5N1 virus is decimating wild bird populations, it is\nencroaching upon likely naïve bird populations in the Antarctica region, and interventions\nare being sought. Data supporting immunization of wildlife against H5N1 influenza is scant and this\nmanuscript is therefore highly significant, important, and will be of broad interest.\nOverall the data presented is simple and clear and the conclusions drawn are sound.\n# AUTHOR RESPONSE: We thank the referee for these very positive comments.\nThe following are minor comments for the authors’ consideration.\n1. Pg7, ln 186. It is a little unclear if survival rates presented are a result of bird deaths or bird\ndeaths and/or inability to recapture specific animals. I realize this may be a difficult question for the\nauthors to definitively answer, but some general comment on expected recapture rates in the\nabsence of likely death would be useful. I guess the question is “how hard is it to recapture\nall birds in a study group this size”.\n# AUTHOR RESPONSE: The expected recapture rate in the absence of likely death is close to 1 given\nthe field situation of the colony (close to the base), the type of marking used on the chicks while they\nare on land, the resighting efforts and the skills of the field workers. Formal capture-mark-recapture\nanalyses on an independent but comparable data set gathered in a previous year confirmed that the\ndetectability was close to 1 if the individuals were still alive. If an individual was not recaptured, it\nhad most likely died given the observation pressure, but we cannot be sure of this so we keep the\nstatement that it is a minimum observed survival rate, but we now specify ‘Given the high expected\nrecapture rate,….’ (lines 186-187). Of note, the situation at the end of the experiment was particular\nbecause the starting outbreak of HPAI (that had started in October 2024) had constrained accesses to\nthe colony. We now outline this in the Discussion (lines 421-430).\n2. Ln 244. 1 out of 19 birds did not appear to respond to boosting. At face value, 5% of non-\nresponders is large. Was there anything in particular noted about the non-responding bird (low body\nweight, issues noted with vaccination etc) that might help explain this result?\n# AUTHOR RESPONSE: Nothing particular was noted about the non-responding bird that might help\nexplain this result. We agree with the referee that exploring potential causes of non-responses in\nsuch a wild species would be of value. We now mention this in the Discussion (lines 416-421) as this\nwould be important to consider for further work on the topic.\n3. What is missing from the manuscript is a way to link the observed antibody titers with\nexpected protection. Is there any way to infer this link? Possibilities could be the use of a\nhemagglutination inhibition assay where there is an accepted protective correlate (in humans and\nchickens at least). Or correlation of the titers detected in this paper with protections observed in the\ncaptive duck and geese studies that were conducted with the same vaccine.\n# AUTHOR RESPONSE: It is always difficult to infer the expected level of protection with observed\nantibody titers, especially in a (wild) species for which little is known. The observed antibody titers\nare nevertheless high compared to what has been reported for captive ducks for which infectious\nchallenges were conducted after vaccination with the vaccine we used (Grasland et al. 2023a and\n2023b). They are also high compared to the captive geese vaccination study we cite that considered\nseveral vaccines among which the one we used (Piesche et al. 2025). Also, we present both anti-H5\nELISA results as well as seroneutralisation results against a 2.3.4.4b clade HP AI virus. We prefer not\nto go behond the statements we are already making in the main text (lines 280-286, 298-300, 337-\n341, .\n4. More detail is needed on the seronuetralization assay. For example, what infectious dose of\nvirus was used, how were titers read etc.\n# AUTHOR RESPONSE: More detail is now provided on the seroneutralization assay. The following\nparagraph was added in the Methods section (590-606):\n“A virus seroneutralisation assay was conducted under Biosafety Level 3 conditions using the\nA/chicken/France/D2107428/2021_2.1b H5N1 HP virus from 2.3.4.4b clade. Plasmas were heat-\ninactivated (56°C for 60 min) and twofold serial dilutions were performed in a 50-μL volume in\nmicrotiter plates. Sera obtained from experimentally immunized chicken with the homologous\ninactivated virus (Sp_H5N1_A/chicken/France/D2107428/2021_1) (positive control serum) and\nobtained from SPF chicken (negative control serum) were also used. Diluted plasmas were mixed\nwith an equal volume containing 100 TCID of the H5N1 virus. Four control wells of virus alone\n50\n(positive for the cytopathogenic effect (CPE) control) and four control wells of dilution medium alone\n(negative CPE control) were included. After 1h incubation at 37°C and 5% CO for neutralization, 100\n2\nμL of MDCK cells at 2 × 105/mL were added to each well. Plates were incubated at 37°C and 5% CO\n2\nfor 72h, after which the CPE was read under the microscope. For each plasma, the seroneutralisation\ntiter was expressed as the mean of the last dilution factor for which no CPE was observed of two\nindependent experiments. The positive control serum has a titer greater than 2048, the negative\ncontrol serum has a titer less than 8.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have no other comments.", "reviewer_2": "the authors have responded well to my suggestions."}, "author_response": "AUTHOR RESPONSE: We thank reviewer #1 for the re-review of our manuscript.\nAUTHOR RESPONSE: We thank reviewer #2 for the re-review of our manuscript."}]

Decision letter