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 strains was applied to A/Norway/31694/2022 (A/NOR22). The optimised H1N1pdm09 LAIV strain (LAIV) 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), 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 TCID assay. The pre-2009 A/H1N1 LAIV strain, A/NC99, was used as a positive control due to its known high levels of hNEC replication.
LAIV 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 Log TCID/ml on day 4 post-infection, compared to 7.2 Log TCID/ml for A/NC99, also at day 4 post-infection. In contrast, LAIV did not replicate efficiently, with viral titres between 2.0 and 2.2 Log TCID/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 reported, to aid in statistical comparison (Fig. 1b). By this measure, LAIV (5.6 Log TCID/ml/day) was not statistically different from A/NC99 (6.3 Log TCID/ml/day), while LAIV replication was significantly lower (2.0 Log TCID/ml/day, p < 0.01). This indicated that HA optimisation had improved A/NOR22 hNEC titres over 3000-fold in this model. Moreover, LAIV replicated to similar titres in eggs as LAIV (Supplementary Fig. 1).
For antigenic characterisation of LAIV and LAIV, groups of two (LAIV) or three (LAIV) ferrets were intranasally vaccinated with a dose of approximately 6-7 Log 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 and LAIV both induced measurable serum immune responses in ferrets, as determined by HAI (Fig. 1d), with LAIV producing a 3-fold higher response than LAIV (11.3 vs 8.3 Log HAI). However, against e-wt, LAIV and LAIV GMTs were comparable (7.3 Log HAI vs 7.6 Log HAI; SD, 0.6). This was also true against the WHO-wt (7.8 Log HAI; SD, 0.7 vs 6.6 Log HAI; SD, 0.6). As the GMTs were ≤2-fold different between LAIV and both e-wt and WHO-wt (Fig. 1e), it was confirmed to be antigenically matched. Conversely, LAIV antisera had an approximately 13-fold difference in cross-reactivity against e-wt virus, and 26-fold against the WHO-wt, relative to LAIV itself.
In summary, due to incorporation of HA protein residue changes D127E, D222G, and R223Q, LAIV possessed significantly improved human replicative fitness, relative to LAIV, but reduced antigenic match to both its parent e-wt and the reference WHO-wt.
With confirmation that LAIV and LAIV had contrasting human cell replication and antigenicity characteristics, a ferret study was designed to evaluate whether LAIV, 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 model, 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 (MP-4) and LAIV (MO-4), delivered at a ferret-optimised dose of 4.0 Log 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 described.
As well as an unvaccinated control group (Mock), a ferret group was dosed with monovalent LAIV at 7.0 Log FFU/ferret (MP-7) to mimic conditions used for production of post-infection ferret antisera during CVV development, as seen in Fig. 1. All ferrets were challenged with A/NOR22 cell-wildtype (c-wt) virus at 5.0 Log 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 protein. 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 results. 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 data. 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 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 TCID assay. LAIV 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 Log TCID/ml at day 3 post-vaccination (Fig. 3a). Interestingly, even LAIV delivered at a 1000-fold increased dose (MP-7) did not produce detectable virus shedding. Only LAIV (MO-4 group) demonstrated average geometric mean shedding per day above the assay's limit of detection (1.8 TCID/ml/day, Fig. 3b). These data confirmed that LAIV replicated more efficiently than LAIV in the ferret nasal-epithelial environment, corroborating observations from the hNEC infection assay in vitro.
To confirm whether differences in vaccine replication properties impacted the ferret humoral immune response, the immunogenicity and antigenicity of LAIV and LAIV 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.
LAIV vaccinated MP-4 ferrets gave generally low HAI GMT, with those against both homologous LAIV (3.6 Log HAI; SD, 1.5) and c-wt challenge virus (<3 Log HAI) approaching or below the limit of detection of the assay. In contrast, LAIV vaccinated MO-4 animals produced the highest HAI antibody GMTs recorded across all groups (Fig. 4a); against homologous LAIV (≥12 Log HAI), e-wt (10.5 Log HAI; SD; 0.6) and c-wt (10.25 Log HAI; SD; 1.0). LAIV 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 in this study produced GMTs against LAIV (10.0 Log HAI; SD; 1.4) and both wt viruses (e-wt; 9.5 Log HAI; SD, 1.3, c-wt; 8.8, Log 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 generated a robust antibody response, whereas LAIV 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 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, the fold differences calculated relative to wt strains were likely to have been underestimated.
In quadrivalent formulation (Fig. 4c), LAIV (QO-4) again produced markedly higher HAI GMTs than LAIV (QP-4), with both groups comparable to their monovalent equivalents (MP-4 and MO-4, Fig. 4a). Antigenic similarity was also comparable, with LAIV 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.
To give a clearer picture of the magnitude and cross-reactivity of antibodies raised by LAIV and LAIV, functional antibodies induced by both LAIV variants were assessed by MN, with half-maximal inhibitory-dilution values (MN) 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 antisera from the MP-7 control group produced detectable but variable neutralising antibody GMTs against the homologous LAIV virus (10.0 Log MN; SD, 1.5), the e-wt (9.5 Log MN; SD, 2.0) and c-wt (8.6 Log MN; SD, 1.8). MO-4 antisera had markedly higher GMTs than MP-4 against LAIV (14.4 Log MN; SD, 0.4) as well as e-wt (8.6 Log MN; SD, 0.7) and c-wt (9.2 Log MN; SD, 1.0). MN neutralisation curves with detectable GMT are shown in Supplementary Fig. 2.
Cross-reactivity could not be assessed for LAIV 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 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 MP-7 antisera were similarly cross-reactive against LAIV 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 strain.
Taken together, these data indicated that LAIV was considerably more immunogenic than LAIV in both monovalent and quadrivalent formulations, with a 4 Log dose of LAIV generally inducing higher levels of serum antibodies than a 7 Log dose of LAIV. In addition, LAIV 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.
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. 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 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, 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 NAI) using a three-parameter logistic curve. Example raw data curves used to calculate the NAI values are shown in Supplementary Fig. 3. The magnitude of NAI titres for all LAIV and LAIV 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 Log NAI (SD, 0.7), indicating the presence of background levels of non-specific NA-inhibitors.
The LAIV vaccinated MP-4 group gave detectable but variable NAI responses between individual ferrets (GMT 7.3 Log NAI; SD, 2.9) that were not statistically different from Mock animals. By comparison, the LAIV 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 Log NAI (SD, 1.2) was >2-fold higher than that of MP-4 ferrets.
The LAIV 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 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 Log NAI (SD, 0.8; p < 0.0001). This showed that, unlike LAIV, the LAIV 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. The MP-7 control group induced a significantly higher NA-specific immune response than Mock animals, with a mean NAI titre of 12.3 Log NAI (SD, 0.7; p < 0.0001). This confirmed that when administered at a sufficiently high dose, LAIV 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 virus induced higher NAI titres than LAIV in both monovalent and quadrivalent formulations as measured by ELLA, with LAIV only able to induce robust NAI antibody responses at a greatly increased dose.
Following confirmation of differences in vaccine shedding and serum antibody responses from ferrets vaccinated with the LAIV and LAIV, protection from c-wt challenge was assessed (Fig. 5).
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, gave c-wt shedding profiles similar to Mock. In contrast, LAIV 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 LogTCID/ml/day) and QP-4 (2.1 LogTCID/ml/day) groups indistinct from Mock (2.6 Log TCID/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 Log TCID/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, 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. The Fever responses measured for both LAIV groups, MP-4 (+1.0 °C) and QP-4 (+1.2 °C), were comparable to the Mock group. Conversely, ferrets vaccinated with LAIV, 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 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 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 Log TCID/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 vaccinated animals.
In summary, at a 4 Log FFU ferret-optimised dose, ferrets vaccinated with both monovalent and quadrivalent LAIV formulations received significant protection from ILI. LAIV 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