Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5μg, 7.5μg, or 30μg), or a non-adjuvanted vaccine (30μg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5μg and 30μg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30μg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.


INTRODUCTION
Enveloped RNA viruses, such as influenza viruses and coronaviruses, constantly evolve, thus causing zoonotic outbreaks and occasional pandemics in humans. Mutations accumulate over time and enable the virus to escape existing immunity established from previous infection and/or vaccination. This mechanism leads to the emergence of geographic and temporal novel variants, which hamper the effectiveness and efficacy of the vaccines designed based on ancestral viruses. As a result, vaccines targeting enveloped RNA viruses need to be updated at regular intervals. Vaccines capable of inducing broadly cross-protective immune responses are urgently needed.
Since its first isolation in 1996, the highly pathogenic avian influenza (HPAI) H5N1 virus have caused outbreaks in domestic and wild birds worldwide, as well as sporadic animal-human transmissions. To date, 862 human infections have been laboratory confirmed which resulting in 455 deaths (1). Tens of thousands of HPAI H5N1 virus strains have emerged in the last two decades. These variant strains are grouped into 10 clades and dozens of subclades according to the main surface glycoprotein hemagglutinin (HA) gene sequences. All the variants isolated from human infections are from clades 0, 1, 2 and 7 (2)(3)(4). To combat the HPAI H5N1 viruses in situations of potential human-to-human transmission, a panel of prepandemic H5N1 vaccine candidates from each of the most common (sub)clades have been prepared (3). Different vaccines formats, including subunit, live attenuated, and adenoviral vectores have been tested in clinical trials alone, or in combination with adjuvants such as AS03 and MF59 (5)(6)(7)(8)(9). These vaccines elicited protective homologous antibody responses and low to moderate levels of neutralizing antibodies to closely related strains. However, the breadth of crossneutralizing antibody responses after vaccination has not been fully elucidated.
Compared to the highly variable HA head domain, HA stalk and neuraminidase (NA) are more conserved among circulating strains across different continents and seasons (10)(11)(12). Recent studies have revealed functions of non-neutralizing antibodies targeting these more conserved domains. For example, HA stalk specific antibodies can block viral genome release into the cytoplasm; whilst NA specific antibodies reduce progeny virion release from infected cells (13). In addition, non-neutralizing antibodies can trigger cytotoxicity and phagocytosis to clear infected cells (14,15). However, whether these nonneutralizing antibodies correlate with in vivo protection against the highly pathogenic H5N1 virus remains unclear.
We have conducted a clinical trial with a virosomal H5N1 vaccine with Matrix M adjuvant in 60 adults. We have previously demonstrated that the adjuvanted H5N1 vaccines elicited potent vaccine specific neutralizing antibodies, and to a lesser extent cross-reactive hemagglutination inhibition (HI) antibodies and Th1 and Th2 CD4+ T cell responses against closely related strains (16)(17)(18). Here, we established an expanded panel of H5N1 pseudotypes covering all (sub)clades isolated from human infections; and characterized the kinetics and breadth of antibody responses after vaccination, including dissection of the multifaceted non-neutralizing antibody responses. We also assessed the in vivo protection from vaccine induced antibodies in a passive transfer murine model and investigated immunological candidates for correlates of protection.

Study Design
Sixty healthy adult volunteers (20- (16). The study was approved by the Regional Committee for Medical Research Ethics, Northern Norway and the Norwegian Medicines Agency. All participants provided written informed consent before inclusion into the study.
Participants were randomized into 4 groups, and intramuscularly vaccinated twice with the H5N1 virosomal vaccine (Crucell Berna Biotech) containing 30mg HA alone, or 1.5, 7.5 or 30mg HA with 50mg Matrix M adjuvant (Novavax) at 3-week interval (16). None of the participants had previously received an H5N1 vaccine.

Vaccine and Sampling
A monovalent inactivated virosomal H5N1 vaccine, containing vaccine strain NIBRG-14, a virus derived from A/Vietnam/1194/ 2004 (H5N1) and A/Puerto Rico/8/1934 (H1N1) using reverse genetics, was used in the clinical trial. The influenza surface antigens HA and NA were purified from beta-propiolactone (BPL) inactivated egg grown viruses, mixed with lecithin and incorporated into the phospholipid bilayer by spontaneous formation of the virosomes. The HA content of the vaccine was quantified by single radial diffusion, and the presence of NA was confirmed.
The adjuvant Matrix M used in the trial was the 3 rd generation immune stimulating complex, which contains Matrix-A and Matrix-C fractions produced from purified Quillaja saponin fractions A and C, at the proportion of 91:9. The vaccine was formulated as 30mg HA alone, or 1.5, 7.5 or 30mg HA with 50mg Matrix M adjuvant, filled into single use syringes, and stored at 4°C until use. All participants (n=60) received 2 doses of the vaccine at a 3-week interval, except one withdrawal from the 7.5µg HA adjuvanted vaccine group.
Blood samples were collected before, and up to 42 days after vaccination. Sera were separated, aliquoted and stored at -80°C until use.

Hemagglutination Inhibition Assay
All sera were treated with receptor-destroying enzyme (RDE, Seiken) at a ratio of 1 in 4 at 37°C for 18h, and heat treated at 56°C for 1h. The treated sera were analysed in duplicate (2-fold serial dilution, starting from 1:10) with 4 hemagglutinating units of viruses and 0.8% horse red blood cells, as previously described (16,17 Pseudotype-Based Neutralization Assay H5N1 pseudotypes were generated by co-transfecting lentiviral vectors pHR'CMV-Luc, pCMVRD8.2, pCMVR-H5HA, and pCMVR-N1NA into HEK293T cells as previously described (19,20 All sera were heat inactivated and analysed in duplicate (4fold serial dilution, starting from 1:10) with H5N1 pseudotypes corresponding to 20,000 to 200,000 relative luciferase activity (RLA), as previously described (20). The pseudotype-based neutralization (PN) titer (IC 80 ) was determined as the reciprocal of the sera dilution giving 80% reduction of RLA. PN titers were normalized based on HIV-1 gag p24 quantities for different pseudotypes. Non-detected samples were assigned a value of 2 for calculation purpose.

Enzyme-Linked Immunosorbent Assay
H5HA stalk and N1NA specific immunoglobulin G (IgG) were quantified in sera using the enzyme-linked immunosorbent assay (ELISA) developed in-house, as previously described (21). Serially diluted sera were analysed in Maxi Sorp 96-well plates coated with 1 mg/ml recombinant chimeric HA (cH9/5HA) that combines the H5HA stalk domain from NIBRG-14 strain with an HA globular head domain from A/guinea fowl/Hong Kong/ WF10/1999 H9 influenza A virus, or 1 mg/ml recombinant N1 neuraminidase. Immunoglobulin concentrations were interpolated from standard human IgG curves. For calculation purposes, non-detected samples were assigned as 0.04 mg/ml against cH9/5HA and 0.005 mg/ml against N1NA.

Enzyme-Linked Lectin Assay
All sera were treated at 56°C for 45min and analysed in duplicate (3-fold serial dilution, starting from 1:5) with a reassortant NIBRG-73 virus, as previously described (22). The NIBRG-73 virus has N1NA from the NIBRG-14 strain and H7HA from A/ equine/Prague/1956 virus. The neuraminidase enzymatic activity was measured with fetuin, horseradish peroxidase-conjugated peanut agglutinin and o-phenylenediamine dihydrochloride (Sigma-Aldrich), and read as optical density (OD) value at 490nm. The neuraminidase inhibition (NI) titer (IC 50 ) was calculated as the reciprocal dilution of sera giving 50% reduction in enzymatic activity. Non-detected samples were assigned a value of 2 for calculation purpose.

Antibody-Dependent Cellular Cytotoxicity Reporter Assay
All sera were heat inactivated, serially diluted and analysed using ADCC Reporter Bioassay kit (Promega), as previously described (22). MDCK cells infected with NIBRG-14 virus at multiplicities of infection (MOI) 0.34 were used as target cells, and Jurkat/ NFAT-luc cells were used as effect cells. The antibody-dependent cellular cytotoxicity (ADCC) reporter activity was measured with Bio-Glo Luciferase Assay Reagent (Promega) as relative luciferase activity (RLA). ADCC titer (EC 50 ) was calculated as the reciprocal dilution of sera giving 50% of maximum RLA. Non-detected samples were assigned a value of 2 for calculation purpose.

Passive Transfer and Viral Challenge in Mice
Six to eight weeks old female BALB/c mice (Taconic, Denmark) were housed under specific-pathogen free conditions at Rikshospitalet, Oslo University Hospital. All animal experiments were approved by the Norwegian Food Safety Authority.
Pooled human sera or saline (400µl/mouse) were injected intraperitoneally (i.p.) into mice (n=12/group) 1 day prior to viral challenge. Mice were anaesthetized by subcutaneous (s.c.) injection of Hypnorm/Dormicum (0.05ml working solution/10g) and infected intranasally (i.n.) with 5 MLD 50 of NIBRG-14 virus in 20 µl/mouse (10µl/nostril). Mice were monitored for survival and weight loss for 2 weeks after challenge, with an endpoint of 20% weight reduction, as required by the Norwegian Food Safety Authority. Mice that lost more than 20% body weight were euthanized by cervical dislocation. For mice that did not lose weight during the 2-week monitoring, the maximum body weight loss was assigned as 0.01% for calculation purpose. At days 3 and 5 post challenge, both lobes of lungs were harvested (n=3 mice/group), snap frozen and stored at -80°C until use.

Virus Quantification
Frozen lung tissues were weighted and homogenized in DMEM with 1% antibiotics. NIBRG-14 virus was quantified in TCID 50 on MDCK cells (23). Hemagglutination assay with 0.7% human red blood cells was used to measure the viral load. The limit of detection was 22.49 TCID 50 /ml of homogenate.

Phylogenetic Tree
Full-length HA protein amino acid sequences from influenza type A viruses were downloaded from NCBI Influenza Virus Database. Phylogenetic analyses were performed at ngPhylogeny.fr using MAFFT (Multiple Alignment using Fast Fourier Transform, default settings), BMGE (Block Mapping and Gathering with Entropy, default settings), and PhyML (Phylogeny software based on the Maximum-likelihood, default settings) (24,25). See Supplementary Table 1 for the accession no. of all HA amino acid sequences used.

Statistical Analyses
Biological replicates were used in all experiments, unless otherwise stated. Antibody quantification results including HI, PN, VN, NI, ADCC titers, and IgG concentrations, and lung viral load results were Ln transformed prior to statistical tests. Turkey's multiple comparisons and Fisher's LSD test were performed in two-way analysis of variance (ANOVA). False Discovery Rate controlled multiple comparisons were performed in Nonparametric Kruskal-Wallis test. Nonparametric Spearman correlations and Pearson correlations were tested, linear fitting curves were plotted when Spearman or Pearson P <0. 10. In multiple linear regression analyses, all independent variables were centered prior to test. * P<0.05, ** P<0.01, *** P<0.001, all P values are two-tailed. All statistical analyses were performed with GraphPad Prism 7 and SPSS 25.

Study Design
Sixty adults were divided into 4 groups and vaccinated with the H5N1 virosomal vaccine alone or escalating doses of adjuvanted vaccine: Group 30mg -(30mg HA alone, 15 subjects), Group 1.5mg + (1.5mg HA with adjuvant Matrix M, 15 subjects), Group 7.5mg + (7.5mg HA with adjuvant Matrix M, 15 subjects), and Group 30mg + (30mg HA with adjuvant Matrix M, 15 subjects). All subjects received a boosting dose 3 weeks after the first priming dose, except for one withdrawal after the first dose in 7.5mg + group ( Figure 1A). Twenty-five subjects had earlier received seasonal influenza vaccine(s) or reported influenza virus infection ( Table 1). Sequential pre-and post-vaccination serum samples up to 42 days were collected from all vaccinees.

Adjuvanted H5N1 Vaccines Rapidly Elicits Strain Specific and Broadly Cross-Clades Neutralizing Antibodies
We assessed HA specific antibodies using the HI assay. None of the vaccinees had detectable antibodies before vaccination. After priming, only the 30mg + group showed potent increases of vaccine specific antibodies. After boosting, all 3 adjuvanted groups had antibodies above the protective level (HI titer ≥32), while the non-adjuvanted 30mg -group remained below the protective level ( Figure 1B). The 1.5mg + group had significantly lower antibody fold-induction after priming compared to the 30mg + group, while after boosting the difference diminished.
On the contrary, the 30mg -group showed significant antibody induction after priming but limited further increase after boosting ( Figure 1C).
We further analysed antibody responses using pseudotypebased neutralization (PN) assay. In 24 subjects, low levels of preexisting vaccine specific neutralizing antibodies were detected. Of note, the vaccine elicited potent homologous antibody responses 7 days after priming in all 4 groups, which were further significantly elevated to higher level in the 1.5mg + and 7.5mg + groups after boosting. The 1.5mg + group had lower antibody fold-induction after priming compared to the 30mg + group but reached similar neutralizing antibody titer after boosting; whilst the 30mg -group had rapid antibody increase 7 days after priming but no significant boost after the second dose ( Figures 1E, F).
To explore the breadth of the vaccine elicited crossneutralizing antibodies, we developed a panel of H5N1 pseudotypes expressing divergent H5 hemagglutinins covering multiple (sub)clades of H5N1 viruses isolated from humans ( Figure 1D and Supplementary Table 1). Remarkably, crossneutralizing antibodies were elicited to equivalent titers as compared to vaccine specific antibodies in all 4 groups when tested against the 3 closely related pseudotypes, namely HK5052 (subclade 2.3.2.1), Turkey (subclade 2.2.1) and Shenzhen (subclade 2.3.4). Meanwhile, although significantly boosted by vaccination, lower levels of cross-neutralizing antibodies were detected to the 2 more distant pseudotypes: Indonesia (subclade 2.1.3.2) and Shanxi (clade 7). Nevertheless, broadly neutralizing antibodies were detected in all the subjects in the 30mg + group across all the (sub)clades ( Figure 1G).

Multifaceted Antibody Responses After H5N1 Vaccines
Total HA stalk specific antibodies were measured in ELISA against the recombinant chimeric protein cH9/5HA, which consists of the HA stalk domain from the vaccine strain and an irrelevant H9HA head. All subjects were found to have preexisting HA stalk specific antibodies. As early as 7 days after priming, we observed significant antibody increases in all 4 groups. The 30mg + group had significantly higher antibody fold-induction as compared to the 1.5mg + and 30mg -groups (Figures 2A, B).
We next assessed neuraminidase (NA) specific antibodies by ELISA ( Figures 2C, D). In 51 subjects, NA specific antibodies were detected prior to vaccination. The H5N1 vaccines elicited potent antibody increases in all 4 groups at 7 days after priming. Further significant antibody increases were found after boosting in 1.5mg + and 7.5mg + H5N1 groups ( Figure 2C). Overall, the 7.5mg + and 30mg + groups showed equivalently potent responses, while the 1.5mg + and 30mg -groups had lower fold change of NA specific antibodies ( Figure 2D).
To quantify the antibodies that inhibit NA enzymatic activity, we performed enzyme-linked lectin assay (ELLA). Before vaccination, detectable NA inhibition (NI) titers were observed in 36 of 60 subjects. Vaccinees in the groups 30mg -, 7.5mg + and 30mg + had significant antibody increases 7 days after priming. While vaccinees in the 1.5mg+ group showed antibody induction 21 days after priming, which increased significantly after boosting ( Figure 2E). The kinetics of NI antibody fold change among 4 vaccine groups was similar to the kinetics of NA specific binding antibodies by ELISA ( Figures 2D, F). Lastly, we measured ADCC inducing antibodies. Thirty-seven out of 60 subjects had pre-existing ADCC inducing antibodies. The H5N1 vaccines further elevated the antibody levels, which remained high after boosting in all groups, especially the 30mg+ group ( Figures 2G, H).
In summary, the virosomal H5N1 vaccines elicited potent and multifaceted antibody responses. The adjuvanted intermediate (7.5mg +) and high (30mg +) dose vaccines potently induced antibody increases 7 days after priming, which were further elevated or maintained after boosting. By comparison, the adjuvanted low (1.5mg +) dose vaccine showed delayed antibody kinetics; whilst the non-adjuvanted (30mg -) vaccine elicited antibodies of an overall lower magnitude.

H5N1 Vaccines Induced Robust Antibody Responses
As the majority of our subjects had some detectable antibodies against the H5N1 vaccine components prior to vaccination, we investigated whether pre-existing immunity hampers the H5N1 vaccine elicited antibody responses. Firstly, the age of the subjects enrolled in this study ranged from 20 to 49 years old ( Table 1). We saw significant correlations between the age and pre-existing level of neutralizing antibodies, HA stalk and NA specific binding antibodies ( Figure 3A). In contrast, the antibody levels after H5N1 vaccination had inverse or no correlation with age ( Figure 3B). Next, subjects who had had seasonal influenza vaccination or infection within 5 years prior to the current study showed significantly higher levels of pre-existing neutralizing, NA specific and ADCC inducing antibodies. H5N1 vaccination boosted antibodies to comparably high levels in all subjects ( Figure 3C). Collectively, the H5N1 vaccines induced robust and multifaceted antibody responses regardless of age, vaccination/infection history or pre-existing antibody levels.

Human Immune Post H5N1 Vaccination Sera Confer In Vivo Protection
To study whether the H5N1 vaccine induced antibodies are protective against infection, we performed passive serum transfer and virus challenge in a murine model. Briefly, we pooled prevaccination sera from groups 1.5mg + and 30mg + as D0 sera, and post-vaccination sera were pooled within each group from all vaccinees in the 1.5mg + and 30mg + groups for days 7, 21 and 42. Pooled immune sera or saline were then transferred into mice one day prior to a viral challenge (5×MLD 50 ) with the NIBRG-14 virus. Body weight was monitored for 14 days after challenge ( Figure 4A). Mice that received saline or D0 sera became ill and rapidly lost weight. All 5 mice receiving saline and 2 out of 6 mice receiving D0 sera died between days 7 and 8 after challenge. By contrast, all mice that received post-vaccination sera survived. Mice that received sera 7 and 21 days post-vaccination from group 1.5mg + showed mild symptoms and lost a maximum of 16% and 9.3% of body weight, respectively, although they quickly recovered. Mice receiving day 42 sera from the group 1.5mg + and sera at all 3 time points from the group 30mg + displayed no symptoms of disease and had < 3% body weight loss at maximum (Figures 4B-D).
We measured the lung weight and viral load at 3 and 5 days after challenge. Mice that received days 21 and 42 sera from group 30 mg + showed lower lung weight at 5 days post challenge, and lower viral loads at days 3 and 5, compared to the mice receiving saline ( Figures 4E, F). In addition, we observed good correlations between the lung viral load and body weight loss, as well as between the lung viral load and the lung weight ( Figures 4G, H). To summarise, immune sera elicited by H5N1 vaccination, especially the adjuvanted high dose (30mg +), conferred full protection against a lethal in vivo challenge.

Neutralizing Antibody and Neuraminidase Inhibiting Antibody Titers Act as Correlates of Protection
Our final aim was to study which of the H5N1 vaccine elicited antibody responses correlated with in vivo protection. We included the widely used hemagglutination inhibition (HI) titer, neutralizing antibody titer measured in pseudotype-based assay (PN titer), hemagglutinin stalk specific antibody level (HA stalk IgG) and neuraminidase inhibition (NI) titer as candidates for correlates of protection. As the Fc receptors involved in ADCC are different between humans and mice, the ADCC inducing antibody titer was not included in analyses.
Importantly, we observed inverse correlations between the antibody levels in the pooled human sera that mice received by The vaccine was supplied as prefilled syringes of pre-formulated virosomal vaccine with Matrix M. 2 Not applicable (N/A). 3 One withdrawal after first dose vaccine in 7.5mg + group. passive transfer and murine body weight loss, as well as lung viral load after challenge ( Figures 5A, B). Of note, the NI titer had the closest relationship with both murine body weight loss and lung viral load, followed by PN titer. Whilst the HI titer and HA stalk IgG level showed lower degrees of correlation with in vivo protection, determined by the Peason's r values in correlation analyses. We further dissected the contribution of each antibody parameter to the predictions of in vivo protection. The adjusted R square and the standard error of the estimate in the multiple linear regression analyses were used to determine the level of fit in different prediction models. When predicting the maximum body weight loss, PN titer and NI titer alone or combined were among the best-fit models ( Figure 5C and Supplementary Table 2). In the lung viral load predictions, NI titer alone, NI and PN titer combined, and all 4 antibody parameters together were the top 3 models ( Figure 5D and Supplementary Table 2). Notably, NI titer contributed significantly to the top models predicting both body weight loss and lung viral load (Figures 5C, D and  Supplementary Table 3). Together, our correlation and regression analyses on the in vivo protection provided by pooled human sera in murine challenge model cohesively demonstrated that PN and NI titers act as correlates of protection. Further study is needed to confirm the roles of PN and NI titers as correlates of protection in human infection scenario.

DISCUSSION
Since 1996, highly pathogenic avian influenza (HPAI) H5N1 have caused outbreaks in domestic and wild birds worldwide, as well as sporadic zoonotic-human transmissions (3,26). The high mortality rate in confirmed human infections raised alarms about the global pandemic preparedness (1). Dozens of pre-pandemic H5N1 candidates have been developed, and clinical trials have been conducted with vaccines developed in various platforms (3,5). Here, we investigated in-depth the multifaceted antibody responses after a virosomal H5N1 vaccine alone or with the Matrix M adjuvant. Our data showed that the adjuvanted H5N1 vaccines elicited potent vaccine-specific and broadly cross-neutralizing antibodies, as well as HA stalk and NA specific functional antibodies. The multifaceted antibody responses were found as early as 7 days after the first dose and were further boosted and  (PN titer, left), total HA stalk specific antibodies (IgG, center) and total NA specific antibodies (IgG, right) tested in pre-vaccination sera (pre-existing, D0, A) correlate with age. Inverse or no significant correlation with age is found in sera after vaccination (post-vac, D42, B). (C) Subjects with previous vaccination and/or infection had significantly higher pre-existing antibodies, compared to subjects without previous vaccination or infection. After vaccination, the difference was abolished. HA specific neutralizing antibodies (PN titer, far left), total NA specific antibodies (IgG, central left), neuraminidase enzymatic activity inhibiting antibodies (NI titer, central right), and antibody-dependent cell-mediated cytotoxicity (ADCC) inducing antibodies (ADCC titer, far right) were tested in sera before (pre-existing) and 42 days after vaccination (post-vac). The geometric mean titers are shown as bars (C), and each symbol represents one subject (A-C). Linear fitting curve was plotted as dotted line when Pearson correlation P < 0,05. Pearson r and P values are noted for each correlation (A, B). *P < 0.05, **P < 0.01, ***P < 0.001 (Antibody titers and concentrations were Ln transformed in statistical analyses. Fisher's LSD test between subjects with previous vaccination/infection and subjects without was performed in two-way ANOVA in (C).
maintained at high levels after the second dose. These antibodies provided full protection against H5N1 virus challenge in mice. Of note, the levels of neutralizing antibodies and NA inhibiting antibodies could predict in vivo protection against infection and disease progression.
Like other enveloped RNA virus, influenza viruses evolve continuously due to its error-prone replication and large host reservoirs. Newly emerged strains may thus escape existing immunity established from previous infection and vaccination. The HPAI H5N1 viruses have evolved into multiple clades and Pre-vaccination sera from subjects in 1.5mg + and 30mg + groups were pooled together as D0 sera. Sera from days 7, 21 and 42 after vaccination from subjects in 1.5mg + or 30mg + groups were pooled separately. Group-and time-point-wise pooled sera were administered intraperitoneally to female BALB/c mice (n=12 per group). NaCl was given as control. One day later, the mice were infected intranasally with 5× 50% mice lethal dose ( subclades. Therefore, vaccines capable of eliciting crossprotective immunity are highly desired. Previous studies on cross-neutralizing antibodies after H5N1 vaccines were mostly limited to clades 1 and 2 (8,(27)(28)(29)(30) 2). In contrast, our early studies using HI, microneutralization and single radial hemolysis assays showed no measurable preexisting antibodies; and 2 doses of vaccine were required for induction of low to moderate levels of antibodies that could cross neutralize clades 1 and 2 [ Supplementary Figure 1 and references (16)(17)(18)]. The detection of pre-existing antibodies in this study could be attributed to the higher sensitivity of pseudotype-based neutralization compared to the traditional assays used earlier. The protective potential of these preexisting antibodies has not yet been fully elucidated, but our results from the sera passive transfer and viral challenge in mice indicate that efficient vaccines will still be needed for protection. The different magnitude and kinetics of the antibody response emphasize the necessity of implementing and harmonizing highly sensitive serological assays to fully understand the immune responses. For example, a more balanced distribution of surface glycoproteins and sensitive luminescence readout  Tables 2-3. Antibody parameters from each individual subject in clinical trial were put together in silico the same way as sera were combined to make group-and-time-point-wise pooled human sera for mice passive transfer. Geometric mean of antibody parameter was calculated, Ln transformed and centered before being used as independent variables. Maximum body weight loss and Ln-transformed lung viral load were used as dependent variables in regression analyses in SPSS 25.
make pseudotype-based neutralization assay more sensitive, quantitative and better suited in detecting zoonotic virus specific antibodies (19,31,32 (33). In our study, we observed potent increases of HA stalk specific IgGs and ADCC inducing antibodies, which peaked after priming with no further boost after the second vaccine dose. By contrast, cross-neutralizing and NA inhibiting antibodies were significantly elicited after both the priming and boosting dose, especially in the low and intermedium adjuvanted (1.5mg + and 7.5mg +) groups (Figures 1, 2).
Adjuvants enhance vaccine immunogenicity, especially in the absence of pre-existing immune memory. The Matrix M has been tested in vaccines against malaria and is currently used in the NVX-CoV2373 vaccine against COVID-19 (34)(35)(36)(37)(38). Here we demonstrated that the Matrix M adjuvanted H5N1 influenza vaccine induced potent increases of broadly cross-neutralizing antibodies, as well as HA stalk and NA specific non-neutralizing antibodies. The adjuvanted 7.5mg + and 30mg + vaccines induced significantly higher titers and better breadth of antibodies as compared to the non-adjuvanted 30mg -group. More importantly, days 21 and 42 post-vaccination sera from 30mg + group provided full protection in mice against both disease progress and viral infection ( Figure 4).
The controlled human influenza virus infection model and vaccine field studies are useful in assessing the vaccine effectiveness and establishing correlates of protection (39,40). Unfortunately, it is difficult to conduct such studies with HPAI H5N1 viruses due to the high morbidity and mortality rate associated. We therefore transferred human sera into naïve mice before an in vivo challenge to study relevant correlates of protection. Other studies applying human sera transfer and mice challenge have shown that HA stalk specific antibodies predict in vivo protection against heterologous virus challenge (41,42). Here, our results demonstrated that PN and NI titers had closer correlations with in vivo protection against a homologous virus challenge, as compared to HI titer or HA stalk specific IgG ( Figure 5). These results are in agreement with other human challenge (43) and cohort studies (44)(45)(46). A caveat here is that our multiple linear regression analyses suffer from inherited collinearity issues due to the similar kinetics of vaccine-elicited antibody responses. Therefore, the coefficients of each predictor may be subject to variance inflation (Supplementary Table 3). Nevertheless, the overall level of predictions should hold accurate ( Figures 5C, D and Supplementary Table 2).
In this study, we assessed the kinetics, magnitude and in vivo protection efficacy of the multifaceted antibody responses after the adjuvanted virosomal H5N1 vaccines. This vaccine was developed in preparation for a potential H5N1 pandemic. Nevertheless, lessons learnt from H5N1 vaccine development could help preparation for the ongoing and future pandemics: 1) Highly sensitive assays help better understand the breadth of cross-reactive immune responses after infection and vaccination. 2) Adjuvants allow vaccine dose sparing and enhance both the magnitude and breadth of responses against vaccine strain and heterologous variants.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The Regional Committee for Medical Research Ethics, Northern Norway and the Norwegian Medicines Agency. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Norwegian Food Safety Authority.

AUTHOR CONTRIBUTIONS
FZ and RC conceived the project. FZ, GG, and RC designed the experiments. RC and GP conducted the clinical study and collected samples. FZ, LH, and GG conducted the experiments. FZ analyzed the data. FZ, LH, GP, GG, and RC prepared and edited the manuscript. All authors contributed to the article and approved the submitted version.
FUNDING antigens used in HI assay. We thank Prof. Florian Krammer for providing the baculoviruses used in purifying cH9/5HA and N1NA proteins. We also thank the staff at the department of comparative medicine at Oslo University Hospital for technical help in association with the mouse experiments.