A/H1N1pdm09 LAIV strain with enhanced human cell replication shows reduced antigenic match to wild-type virus

To evaluate the relative contributions of LAIV replication and antigenicity to protection from wt challenge in a ferret model, the previously described approach to optimising the HA protein of A/H1N1pdm09 strains²¹ was applied to A/Norway/31694/2022 (A/NOR22). The optimised H1N1pdm09 LAIV strain (LAIV_H1.opt) carried amino acid substitutions at residues 127 (D127E), 222 (D222G) and 223 (R223Q) in the HA protein, relative to the egg-derived, A/NOR22 parental LAIV (LAIV_H1.par), which maintained the wt HA sequence (Table 1).

To quantify human cell replication, both A/NOR22 variants were used to infect hNEC cultures at a low MOI of 0.01 FFU/well. Viral titres were determined daily over a four-day time course by TCID₅₀ assay. The pre-2009 A/H1N1 LAIV strain, A/NC99, was used as a positive control due to its known high levels of hNEC replication¹⁸.

LAIV_H1.opt replicated to high titres under these conditions in hNEC (Fig. 1a), resembling the control A/NC99 growth curve and peaking at a titre of 6.7 Log₁₀ TCID₅₀/ml on day 4 post-infection, compared to 7.2 Log₁₀ TCID₅₀/ml for A/NC99, also at day 4 post-infection. In contrast, LAIV_H1.par did not replicate efficiently, with viral titres between 2.0 and 2.2 Log₁₀ TCID₅₀/ml across the four-day time course. Geometric mean titre per day was calculated for each virus, to describe virus replication over time as previously reported¹⁸, to aid in statistical comparison (Fig. 1b). By this measure, LAIV_H1.opt (5.6 Log₁₀ TCID₅₀/ml/day) was not statistically different from A/NC99 (6.3 Log₁₀ TCID₅₀/ml/day), while LAIV_H1.par replication was significantly lower (2.0 Log₁₀ TCID₅₀/ml/day, p < 0.01). This indicated that HA optimisation had improved A/NOR22 hNEC titres over 3000-fold in this model. Moreover, LAIV_H1.opt replicated to similar titres in eggs as LAIV_H1.par (Supplementary Fig. 1).

a Four-day time-course for infection in human nasal epithelial cells (hNECs) for LAIV_H1.par and LAIV_H1.opt, at an MOI of 0.01 FFU/cell. Data represent three independent experiments of two hNEC transwells per experiment (total six transwells per virus), measured by TCID₅₀. The pre-2009 H1N1 LAIV strain, A/NC99, was included as a positive control for high hNEC replication. b Average virus titre per day calculated from (a), for A/NC99, LAIV_H1.par and LAIV_H1.opt. Columns show GMT of individual experiments (symbols) and error bars show standard deviation. Groups were compared with one-way ANOVA and all groups compared against each other with Tukey–Kramer HSD post-test **p < 0.01. c Antigenic analyses of LAIV_H1.par and LAIV_H1.opt viruses against the A/NOR22 egg wildtype (e-wt) and A/VIC22 WHO wildtype reference strains (WHO-wt). Numbers indicate HAI titres. Underlined values indicate homologous titres. Results provided by the Worldwide Influenza Centre, The Francis Crick Institute, UK. d Ferret serum HAI titres generated for LAIV_H1.par (n = 2) and LAIV_H1.opt (n = 3) against the relevant homologous LAIV strain, the e-wt and the WHO-wt. Antisera were produced using a high LAIV dose, reflective of the routine seasonal CVV selection process. Dotted line indicates lower limit of detection LoD. e Fold-difference in HAI titres for LAIV_H1.par and LAIV_H1.opt relative to A/NOR22 e-wt and WHO-wt, A/VIC22. Grey line indicates WHO threshold for antigenic match (<4-fold). Columns in all cases show GMT of individual ferrets, distinguished by unique symbols. Lines show geometric standard deviation. A/NC99: A/New Caledonia/20/1999; A/NOR22: A/Norway/31694/2022; A/VIC22: A/Victoria/4897/2022.

For antigenic characterisation of LAIV_H1.par and LAIV_H1.opt, groups of two (LAIV_H1.par) or three (LAIV_H1.opt) ferrets were intranasally vaccinated with a dose of approximately 6–7 Log₁₀ FFU/ferret and antisera were harvested at day 21 post-vaccination. Testing of antisera was performed at the Worldwide Influenza Centre, WHO Collaborating Centre for Influenza Virus Reference and Research, The Francis Crick Institute, by HAI assay (Fig. 1c). HAI titres were produced against the egg-derived parental wt virus, A/NOR22 egg-wt (e–wt); the WHO recommended, egg-based wt reference strain, A/Victoria/4897/2022 (WHO-wt); and the homologous vaccine virus delivered to that animal. Using the raw data generated by the Crick Institute, geometric mean HAI titre (GMT) and standard deviation (SD) was calculated against the vaccine virus and both wt strains (Fig. 1d). Fold-differences between GMT were used to establish antisera cross-reactivity (Fig. 1e).

LAIV_H1.par and LAIV_H1.opt both induced measurable serum immune responses in ferrets, as determined by HAI (Fig. 1d), with LAIV_H1.opt producing a 3-fold higher response than LAIV_H1.par (11.3 vs 8.3 Log₂ HAI). However, against e-wt, LAIV_H1.par and LAIV_H1.opt GMTs were comparable (7.3 Log₂ HAI vs 7.6 Log₂ HAI; SD, 0.6). This was also true against the WHO-wt (7.8 Log₂ HAI; SD, 0.7 vs 6.6 Log₂ HAI; SD, 0.6). As the GMTs were ≤2-fold different between LAIV_H1.par and both e-wt and WHO-wt (Fig. 1e), it was confirmed to be antigenically matched. Conversely, LAIV_H1.opt antisera had an approximately 13-fold difference in cross-reactivity against e-wt virus, and 26-fold against the WHO-wt, relative to LAIV_H1.opt itself.

In summary, due to incorporation of HA protein residue changes D127E, D222G, and R223Q, LAIV_H1.opt possessed significantly improved human replicative fitness, relative to LAIV_H1.par, but reduced antigenic match to both its parent e-wt and the reference WHO-wt.

A/NOR22 monovalent LAIV_H1.opt sheds to higher vaccine virus titres in ferrets than LAIV_H1.par

With confirmation that LAIV_H1.par and LAIV_H1.opt had contrasting human cell replication and antigenicity characteristics, a ferret study was designed to evaluate whether LAIV_H1.opt, due to its improved human cell replication, could provide increased immunogenicity or protection from wt influenza virus infection, despite its reduced antigenic match to wt reference strains.

Based on our recently published clinically translatable A/H1N1pdm09 LAIV ferret efficacy model²⁰, groups of four male/female ferrets (musetla putorius furo) aged over 5 months were intranasally vaccinated with the LAIV or Mock vaccine formulations (Fig. 2). Monovalent LAIV_H1.par (MP-4) and LAIV_H1.opt (MO-4), delivered at a ferret-optimised dose of 4.0 Log₁₀ FFU/ferret, would examine the ability of each strain to induce an immune response and protect from wt challenge in isolation, while quadrivalent (QLAIV) formulations (QP-4, QO-4) would assess immunogenicity and protection in the context of inter-strain competition, as previously described¹⁹.

a Timeline of in vivo study. Animals were intranasally vaccinated at Study Day 0 with monovalent or quadrivalent LAIV formulations. All groups were challenged with wt virus at day 28 post-vaccination, and study termination occurred at day 33. Vertical grey arrows highlight the major interventions during the study time period. Red boxes indicate blood collection. Blue boxes indicate nasal wash sample collection. Horizontal blue arrow indicates ferret core body temperature readings, taken hourly during the study time period. Horizontal yellow arrow indicates ferret bodyweight observations, taken daily during the study time-period. b Table outlining inocula formulations and challenge virus used for assessment of LAIV_H1.par and LAIV_H1.opt efficacy in ferrets. Rows detail vaccine composition. Ferrets were vaccinated with a defined dose (4.0 Log₁₀ FFU/ferret) of either LAIV_H1.par (shaded yellow) or LAIV_H1.opt (shaded purple). Vaccine groups included Monovalent LAIV_H1.par (MP-4) and LAIV_H1.opt (MO-4) LAIV, along with quadrivalent formulations for each LAIV strain (QP-4, QO-4), to assess any impact of inter-strain competition. Both an unvaccinated (Mock) and high-dose (7.0 Log₁₀ FFU/ferret) LAIV_H1.par (MP-7) group were included as controls (shaded white). Groups were challenged with cell-derived A/NOR22 wildtype virus (c-wt) at 5.0 Log₁₀ FFU/ferret (shaded grey)¹⁹.

As well as an unvaccinated control group (Mock), a ferret group was dosed with monovalent LAIV_H1.par at 7.0 Log₁₀ FFU/ferret (MP-7) to mimic conditions used for production of post-infection ferret antisera during CVV development³⁰, as seen in Fig. 1. All ferrets were challenged with A/NOR22 cell-wildtype (c-wt) virus at 5.0 Log₁₀ FFU/ferret at day 28 post-vaccination (Fig. 2). This was used in place of the parental e-wt, as Schewe et al. showed that egg-derived A/H1N1pdm09 wt viruses can be non-pathogenic in ferrets due to a Q223R egg-adaptation in the HA protein²⁰. A/NOR22 e-wt contained this Q223R egg-adaptation and so c-wt A/NOR22 was used as the challenge agent, ensuring induction of a symptomatic influenza infection. Nasal washes were taken daily for 5 days post-vaccination and post-challenge to measure LAIV or wt virus shedding, respectively. Serum bleeds were taken at day 14 and day 21, with data primarily generated from day 21 samples as a representative timepoint, based on previous results¹⁹. Fever and body weight were monitored throughout the study to evaluate the development of influenza-like illness (ILI). Previously, it was shown that measurement of wt shedding and fever in this model could broadly reproduce A/H1N1pdm09 LAIV clinical VE data²⁰. As such, protection from these endpoints was the primary readout of efficacy in this study. Additional clinical signs of infection were also observed for up to 5 days post-challenge.

Following vaccination, to determine whether the enhanced hNEC replication of LAIV_H1.opt would translate to improved replication in the ferret upper-respiratory tract, shedding of the monovalent LAIV viruses post-vaccination was assessed. Monovalent viral titres in nasal washes collected daily for 5 days post-vaccination were measured using TCID₅₀ assay. LAIV_H1.par shedding was not detectable in MP-4 ferrets, while MO-4 ferrets produced consistent, detectable virus shedding across multiple days post-vaccination, with a peak mean viral titre of 2.5 Log₁₀ TCID₅₀/ml at day 3 post-vaccination (Fig. 3a). Interestingly, even LAIV_H1.par delivered at a 1000-fold increased dose (MP-7) did not produce detectable virus shedding. Only LAIV_H1.opt (MO-4 group) demonstrated average geometric mean shedding per day above the assay’s limit of detection (1.8 TCID₅₀/ml/day, Fig. 3b). These data confirmed that LAIV_H1.opt replicated more efficiently than LAIV_H1.par in the ferret nasal-epithelial environment, corroborating observations from the hNEC infection assay in vitro.

Animals were vaccinated at a dose of 4.0 Log₁₀ FFU/ferret with monovalent LAIV_H1.par (MP-4), LAIV_H1.opt (MO-4), or with a high dose (7.0 Log₁₀ FFU/ferret) of monovalent LAIV_H1.par (MP-7) control. Nasal washes were taken at days 1–5 post-vaccination and virus shedding was determined by TCID₅₀ assay in MDCK cells. a Virus titre measurements for each time point generated from four ferrets (symbols) per vaccination group. b LAIV virus geometric mean shedding per ml per day calculated from data in (a). Individual ferrets are indicated by unique symbols. Columns show group geometric mean with error bars representing geometric standard deviation. Horizontal dotted lines indicate the assay’s LoD. Statistical comparisons were not applied due to two of three vaccination groups generating data at the LoD. LoD limit of detection.

LAIV_H1.opt produces higher serum antibody titres than LAIV_H1.par but with reduced cross-reactivity to wt viruses

To confirm whether differences in vaccine replication properties impacted the ferret humoral immune response, the immunogenicity and antigenicity of LAIV_H1.par and LAIV_H1.opt variants were measured. Magnitudes of anti-HA serum antibody responses were assessed from bleeds taken at day 21 post-vaccination using two assays: HAI (Fig. 4a–d) and Microneutralisation (MN, Fig. 4e–h). Vaccine immunogenicity was measured against the homologous A/NOR22 LAIV strains and antisera cross-reactivity was assessed against both the parental e-wt and the c-wt challenge virus.

Serum immune responses were measured post-vaccination at day 21 through HAI, MN and NAI ELLA. For HAI and MN, titres were measured against LAIV_H1.par and LAIV_H1.opt, along with e-wt and c-wt viruses. For NAI ELLA, groups were assessed against a recombinant H2N1_NorNA virus. HAI (a–d). a HAI GMT for monovalent LAIV_H1.par and LAIV_H1.opt antisera and (b) fold-difference in HAI titres in (a) between homologous LAIV strains and wt viruses. Grey line and arrow represent WHO threshold for antigenic match with HAI assay (<4-fold difference vs homologous virus). Mock group HAI titres were not applicable (N/A) in HAI fold-difference calculations. c HAI GMT for quadrivalent LAIV_H1.par (QP-4) and LAIV_H1.opt (QO-4) antisera and (d) fold-difference in quadrivalent HAI titres between homologous LAIV strains and wt viruses. MN (e–h). e Neutralisation GMT for monovalent LAIV_H1.par and LAIV_H1.opt antisera and (f) fold-difference in monovalent MN titres between homologous LAIV strains and wt viruses. g Neutralisation GMT for quadrivalent LAIV_H1.par and LAIV_H1.opt antisera and (h) fold-difference between homologous LAIV strains and wt viruses. Cross-reactivity against virus strains where antisera failed to neutralise classified as ‘Did Not Neutralise’ in MN fold-difference calculations. i NAI GMT for LAIV_H1.par and LAIV_H1.opt in monovalent and quadrivalent formulation. Groups were assessed for equal variance by Levene’s test and intra-group normality using the Shapiro-Wilk test. The geometric mean NAI₅₀ of each group was compared to the geometric mean of the Mock vaccination control using the Welch t-test with the Holm-Šídák correction for multiple comparisons. ****p < 0.0001. Bars show GMT from individual animals (unique symbols) with SD. Dotted horizontal line indicates LoD. Values that failed to elicit an antibody response were plotted as 0.5 x lower LoD. Values that exceeded the upper LoD of assays denoted with #. HAI, MN and NAI GMT for high dose (7.0 Log₁₀ FFU/ferret) monovalent LAIV_H1.par (MP-7) and mock-vaccinated ferrets included as controls. HAI: hemagglutination inhibition; MN: microneutralisation; NAI: Neuraminidase inhibition; ELLA: Enzyme-linked Lectin Assay; LoD: limit of detection; GMT: geometric mean titres; SD: Geometric standard deviation.

LAIV_H1.par vaccinated MP-4 ferrets gave generally low HAI GMT, with those against both homologous LAIV_H1.par (3.6 Log₂ HAI; SD, 1.5) and c-wt challenge virus (<3 Log₂ HAI) approaching or below the limit of detection of the assay. In contrast, LAIV_H1.opt vaccinated MO-4 animals produced the highest HAI antibody GMTs recorded across all groups (Fig. 4a); against homologous LAIV_H1.opt (≥12 Log₂ HAI), e-wt (10.5 Log₂ HAI; SD; 0.6) and c-wt (10.25 Log₂ HAI; SD; 1.0). LAIV_H1.opt homologous GMTs were saturated, meaning that true GMTs may have been underestimated in these data. The MP-7 high dose control group confirmed that a greatly increased dose of LAIV_H1.par in this study produced GMTs against LAIV_H1.par (10.0 Log₂ HAI; SD; 1.4) and both wt viruses (e-wt; 9.5 Log₂ HAI; SD, 1.3, c-wt; 8.8, Log₂ HAI, SD; 1.7) that resembled those seen during initial strain characterisation (Fig. 1). Overall, these HAI results suggested that a low dose of LAIV_H1.opt generated a robust antibody response, whereas LAIV_H1.par at the same dose elicited minimal antibody responses.

While values for the cross-reactivity of MP-4 antisera against e-wt and c-wt viruses were calculated (Fig. 4b), the low and variable titres measured make these fold-difference values unreliable. The high dose LAIV_H1.par control group (MP–7), which induced higher GMTs, remained antigenically matched to both e-wt (1.5-fold) and c-wt (2.75-fold) viruses, despite two ferrets reaching the WHO antigenicity threshold (4-fold difference) against the c-wt. MO-4 antisera were less cross-reactive with e-wt (2–4-fold) and c-wt (2–8-fold) viruses (Fig. 4b). However, due to the saturated homologous HAI titres for LAIV_H1.opt, the fold differences calculated relative to wt strains were likely to have been underestimated.

In quadrivalent formulation (Fig. 4c), LAIV_H1.opt (QO-4) again produced markedly higher HAI GMTs than LAIV_H1.par (QP-4), with both groups comparable to their monovalent equivalents (MP-4 and MO-4, Fig. 4a). Antigenic similarity was also comparable, with LAIV_H1.opt giving fold-differences of 4–8-fold (e-wt) and 4–16-fold (c-wt). Again, these values were likely to be underestimated due to saturated homologous QO-4 GMTs against LAIV_H1.opt.

To give a clearer picture of the magnitude and cross-reactivity of antibodies raised by LAIV_H1.par and LAIV_H1.opt, functional antibodies induced by both LAIV variants were assessed by MN, with half-maximal inhibitory-dilution values (MN₅₀) measured against the relevant homologous LAIV strain and both wt strains (Fig. 4e–h).

In contrast to the HAI data, MP-4 antisera did not detectably neutralise any test virus and gave responses comparable to the Mock vaccinated group (Fig. 4e). LAIV_H1.par antisera from the MP-7 control group produced detectable but variable neutralising antibody GMTs against the homologous LAIV_H1.par virus (10.0 Log₂ MN₅₀; SD, 1.5), the e-wt (9.5 Log₂ MN₅₀; SD, 2.0) and c-wt (8.6 Log₂ MN₅₀; SD, 1.8). MO-4 antisera had markedly higher GMTs than MP-4 against LAIV_H1.opt (14.4 Log₂ MN₅₀; SD, 0.4) as well as e-wt (8.6 Log₂ MN₅₀; SD, 0.7) and c-wt (9.2 Log₂ MN₅₀; SD, 1.0). MN₅₀ neutralisation curves with detectable GMT are shown in Supplementary Fig. 2.

Cross-reactivity could not be assessed for LAIV_H1.par based on MP-4 antisera, due to the absence of measurable neutralisation. When measured by MN, MO-4 antisera exhibited clearly reduced neutralisation capacity against both e-wt (61.4-fold difference) and c-wt (43.3-fold difference), relative to the homologous LAIV_H1.opt virus (Fig. 4f). These cross-reactivity differences were considerably higher than those observed in the HAI assay and were above the 4-fold threshold commonly applied to antigenic comparisons. In contrast, LAIV_H1.par MP-7 antisera were similarly cross-reactive against LAIV_H1.par and both e-wt (1.5-fold) and c-wt (2.8-fold) viruses, with fold-differences of <4-fold in both cases, as seen for HAI data. Finally, QP-4 and QO-4 MN data corroborated monovalent vaccination observations (Fig. 4g). QP-4 antisera did not neutralise LAIV or wt strains, while QO-4 antisera gave elevated MN titres relative to QP-4 but with reduced cross-reactivity against e-wt (58.1-fold different) and c-wt (25.1-fold different) viruses when compared to the homologous LAIV_H1.opt strain.

Taken together, these data indicated that LAIV_H1.opt was considerably more immunogenic than LAIV_H1.par in both monovalent and quadrivalent formulations, with a 4 Log₁₀ dose of LAIV_H1.opt generally inducing higher levels of serum antibodies than a 7 Log₁₀ dose of LAIV_H1.par. In addition, LAIV_H1.opt in this study was confirmed to be antigenically distinct from the parental e-wt virus, as observed during initial CVV characterisation (Fig. 1), as well as the c-wt virus used for ferret challenge. These antigenic differences appeared most pronounced when measuring functional, neutralising antibody responses by MN assay, as compared to HAI.

Higher titres of A/NOR22 NA-specific antibodies were detected in ferrets vaccinated with LAIV_H1.opt compared to LAIV_H1.par

Alongside analysis of anti-HA antibody responses by HAI and MN assays, alternative, non-HA-mediated mechanisms of protection were also investigated. NA-inhibiting (NAI) antibody titres have been demonstrated to correlate with protection against flu infection^31,32. All LAIV and wt viruses in this study shared the same NA sequence, so it was hypothesised that more efficient induction of anti-NA antibodies by LAIV_H1.opt could also contribute to protection from the ensuing challenge.

Assessment of serum anti-NA antibody responses in the same day 21 post-vaccination antisera as for HAI and MN was performed using an NAI ELLA. The source of NA protein used in the assay was a recombinant LAIV virus, A/H2N1_NorNA, carrying the A/NOR22 NA protein sequence common to all LAIV and wt viruses in the study, combined with the antigenically distinct H2 HA protein of A/Ann Arbor/6/60 to isolate anti-NA antibody responses. In brief, the ability of serially diluted ferret antisera to inhibit NA-induced cleavage of fetuin, expressed as a percentage of virus-only positive control wells, was measured. NAI titres were calculated as the reciprocal of the dilution required to produce 50% inhibition (here referred to as NAI₅₀) using a three-parameter logistic curve. Example raw data curves used to calculate the NAI₅₀ values are shown in Supplementary Fig. 3. The magnitude of NAI titres for all LAIV_H1.par and LAIV_H1.opt study groups were then compared to those for Mock vaccinated animals (Fig. 4i).

Antisera from mock-vaccinated control animals showed a low level of NA inhibition, with a mean NAI titre of 5.0 Log₂ NAI₅₀ (SD, 0.7), indicating the presence of background levels of non-specific NA-inhibitors.

The LAIV_H1.par vaccinated MP-4 group gave detectable but variable NAI responses between individual ferrets (GMT 7.3 Log₂ NAI₅₀; SD, 2.9) that were not statistically different from Mock animals. By comparison, the LAIV_H1.opt vaccinated MO-4 group showed significantly increased NA inhibition compared to the Mock group (p < 0.0001). The mean MO-4 NAI titre of 14.5 Log₂ NAI₅₀ (SD, 1.2) was >2-fold higher than that of MP-4 ferrets.

The LAIV_H1.par quadrivalent group QP-4 also failed to display any significant NAI antibody response, with an NAI titre comparable to that of mock-vaccinated ferrets. In contrast, LAIV_H1.opt continued to show induction of NAI antibodies in the QO-4 group, with a significantly increased NAI titre compared to the Mock group of 13.5 Log₂ NAI₅₀ (SD, 0.8; p < 0.0001). This showed that, unlike LAIV_H1.par, the LAIV_H1.opt virus delivered at a low, ferret-optimised dose, induced a substantial NA-specific immune response even in quadrivalent formulation.

Similarly to the results seen for HAI and MN, the LAIV_H1. The MP-7 control group induced a significantly higher NA-specific immune response than Mock animals, with a mean NAI titre of 12.3 Log₂ NAI₅₀ (SD, 0.7; p < 0.0001). This confirmed that when administered at a sufficiently high dose, LAIV_H1.par was also able to induce a robust anti-NA serum immune response. Even so, this was approximately 4-fold lower than the titres seen for MO-4 ferrets.

In summary, the LAIV_H1.opt virus induced higher NAI titres than LAIV_H1.par in both monovalent and quadrivalent formulations as measured by ELLA, with LAIV_H1.par only able to induce robust NAI antibody responses at a greatly increased dose.

LAIV_H1.opt provided improved protection against challenge virus shedding and influenza-like illness

Following confirmation of differences in vaccine shedding and serum antibody responses from ferrets vaccinated with the LAIV_H1.par and LAIV_H1.opt, protection from c-wt challenge was assessed (Fig. 5).

Following vaccination, groups were challenged with A/NOR22 c-wt virus 28 days post-vaccination. Nasal washes were taken at days 1–5 post-challenge and viral titres measured by TCID₅₀ assay. a Virus titre measurements from each time point post-challenge from four ferrets (symbols) per vaccination group. b Geometric mean wt shedding per day of virus titre recorded over 5 days. For statistical analysis the MO-4 vaccination group excluded from group analysis as it did not measure values above LoD. The remaining groups were compared to the Mock control by Welch t test with Holm-Šídák adjustment for multiple comparisons. MO-4 was compared to the Mock control with a one-sample t test, using LoD values. **p < 0.01. c Influenza–like illness in ferrets was monitored through measurement of Fever, as previously described¹⁹. Fever is calculated as the average increase from the pre-challenge baseline body temperature during a defined ‘fever period’, post-challenge. d Changes in animal bodyweight were also assessed. Weight changes are expressed as differences relative to a ferret’s average pre-challenge weight. Columns in all cases represent mean value, with error bars representing standard deviation. Each treatment group was assessed for equal variance using Levene test and intra-group normality using Shapiro–Wilk test. Fever and bodyweight changes for each treatment group were compared to the Mock control using Welch t test with Holm–Sidak’s post-test correcting for multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001. c–wt: Cell-derived wildtype; LoD: Limit of Detection.

Unprotected mock-vaccinated animals shed c-wt virus detectably for 5 days post-challenge, with shedding peaking at approximately day 2 (Fig. 5a). MP-4 and QP-4 groups, vaccinated with LAIV_H1.par, gave c-wt shedding profiles similar to Mock. In contrast, LAIV_H1.opt vaccinated groups MO-4 and QO-4 produced almost no detectable c-wt shedding post-challenge. Statistical comparison of geometric mean shedding per day confirmed these differences (Fig. 5b), with MP-4 (2.8 Log₁₀TCID₅₀/ml/day) and QP-4 (2.1 Log₁₀TCID₅₀/ml/day) groups indistinct from Mock (2.6 Log₁₀ TCID₅₀/ml/day). Ferrets from the MO-4 group with titres at the LoD gave significantly reduced c-wt shedding (p < 0.01) compared to the Mock. Reduced c-wt geometric mean shedding was also observed for QO-4 (1.2 Log₁₀ TCID₅₀/ml/day), but this change was not statistically significant.

To assess fever development, changes in core temperature were monitored hourly up to 5 days post-challenge, using intraperitoneal data loggers. Full temperature traces for all animals are shown in Supplementary Fig. 4. From these data, a value for ‘Fever’ was calculated for each animal. As previously described^19,20, Fever was calculated by first defining a ‘fever period’: a window post-challenge during which Mock-vaccinated animals experienced temperatures elevated by >1.5 standard deviations vs pre-challenge baseline. For all study animals, the average temperature difference vs pre-challenge baseline was then calculated during this fever period. An average Fever of + 1.3 °C was observed in the Mock group post-challenge (Fig. 5c), in keeping with prior A/H1N1pdm09 challenge viruses^21,22. The Fever responses measured for both LAIV_H1.par groups, MP-4 (+1.0 °C) and QP-4 (+1.2 °C), were comparable to the Mock group. Conversely, ferrets vaccinated with LAIV_H1.opt, in groups MO-4 and QO-4, both gave average Fever values of −0.1 °C, which was significantly reduced relative to Mock animals (p < 0.01).

In alignment with temperature data, no loss of bodyweight was measured for LAIV_H1.opt MO-4 (+0.8 g/day) and QO-4 (+2.3 g/day) groups (Fig. 5d). This was significantly different from the average bodyweight loss of −24.1 g/day for the Mock group (MO-4; p < 0.05, QO-4; p < 0.01). In contrast, the loss of bodyweight observed from both LAIV_H1.par groups, MP-4 (−22.2 g/day) and QP-4 (−17.6 g/day), was comparable to Mock-vaccinated animals. Full bodyweight data are shown in Supplementary Fig. 5.

For the MP-7 control group, a limited degree of detectable c-wt virus shedding was observed (1.35 Log₁₀ TCID₅₀/ml/day), along with a minor temperature increase post-challenge (Fever + 0.2 °C, p < 0.05) that was still significantly reduced relative to Mock. Similarly, MP-7 animals were significantly protected from weight loss (−3.2 g/day, p < 0.05), albeit with a greater loss of weight than LAIV_H1.opt vaccinated animals.

In summary, at a 4 Log₁₀ FFU ferret-optimised dose, ferrets vaccinated with both monovalent and quadrivalent LAIV_H1.opt formulations received significant protection from ILI. LAIV_H1.par did not provide significant protection from challenge at an equivalent dose despite being antigenically matched to wt reference strains. However, a 1000-fold increase in dose was able to overcome the limited protection conferred by LAIV_H1.par.