Search

Abstract The endemic human coronaviruses (HCoVs) circulate worldwide yet remain understudied and unmitigated. The observation of elevated levels of HCoV reactive antibodies in COVID-19 patients highlights the urgent necessity of better understanding of HCoV specific immunity. Here, we characterized in-depth the de novo SARS-CoV-2 specific antibody responses and the boosting of HCoV-reactive antibodies after SARS-CoV-2 vaccination or infection in individuals up to 98 years old. All the vaccinees were home-dwelling with no documented SARS-CoV-2 infection before receiving the COVID-19 mRNA vaccine (BNT162b2). The first two vaccine doses elicited potent SARS-CoV-2 spike binding antibodies in individuals up to 80 years. The third dose largely boosted the previously low S2 domain binding and neutralizing antibodies in elderly 80–90 years old, but less so in those above 90 years. The endemic betacoronavirus (HKU1 and OC43) reactive antibodies were boosted in all vaccinees, although to a lesser extent in those above 80 years old. COVID-19 patients had potent elevation of alpha- and betacoronavirus (229E, NL63, HKU1 and OC43) reactive antibodies. In both patients and vaccinees, S2 domain specific antibody increases correlated with SARS-CoV-2 neutralizing and HCoV-reactive antibody responses in all ages, indicating S2 domain as a candidate for future universal coronavirus vaccine design.

Live attenuated influenza vaccines (LAIV) typically induce a poor hemagglutination inhibition (HI) response, which is the standard correlate of protection for inactivated influenza vaccines. The significance of the HI response is complicated because the LAIV vaccine primarily induces the local mucosal immune system, while the HI assay measures the circulating serum antibody response. However, age and pre-existing immunity have been identified as important factors affecting LAIV immunogenicity. This study aimed to extend our understanding of LAIV-induced immunity, particularly, the impact age and pre-existing immunity have on eliciting functional and neutralising antibody responses in paediatric and adult populations vaccinated with LAIV. Thirty-one children and 26 adults were immunized with the trivalent LAIV during the 2013–2014 influenza season in Norway. Children under 9 years received a second dose of LAIV 28 days after the first dose. Blood samples were collected pre- and post-vaccination. HI, microneutralization (MN) and enzyme-linked lectin assay for neuraminidase (NA) antibodies were measured against the vaccine strains. IgG antibody avidity against hemagglutinin (HA) and NA proteins from the vaccine strains was also assessed. Significant correlations were observed between HI and MN responses to A/California/7/2009 (A/H1N1)pdm09-like strain and B/Massachusetts/2/2012-like strain, suggesting that MN is a potential immunological correlate for LAIV. However, the relationship between recipient age (or priming status) and serological response varied between vaccine strains. There was a notable increase in HI and MN responses in all cohorts except naive children against the H1N1 strain, where most recipients had responses below the protective antibody threshold. NAI responses were generally weak in naive children against all vaccine strains compared with adults or antigen-primed children. Post-vaccination antibody avidity increased only in primed children below nine years of age against the A/H1N1 strain. Overall, our findings indicate that LAIV elicits functional and neutralizing antibody responses in both naive and antigen experienced cohorts, however, the magnitude and kinetics of the response varies between vaccine strains.

Abstract Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 serum samples. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

IntroductionThe haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods.MethodsIn this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes.ResultsWe showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

IntroductionExternal Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods.MethodsWe invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays.ResultsWithin run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization.DiscussionThis study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Abstract Current influenza vaccines have a suboptimal effectiveness. The introduction of a novel A/H1N1 influenza virus in 2009 (H1N1pdm09) provided a unique opportunity to study the humoral response to the AS03-adjuvanted H1N1pdm09 vaccine and repeated annual vaccination with the homologous virus in subsequent influenza seasons. Thirty-two HCWs immunized with the AS03-adjuvanted H1N1pdm09 vaccine in 2009 were divided into four groups based on the longevity of their antibody responses (persistently high or transient), and whether they were repeatedly annually vaccinated in the subsequent four influenza seasons or not. Serological assays were utilized to measure the quantity, quality and functionality of antibodies targeting the major surface glycoprotein hemagglutinin (HA). Persistent high responders (hemagglutination inhibition (HI) titre ≥ 80 at 12 months after H1N1pdm09 vaccination) had protective levels of HI antibodies throughout the study period. In addition, the quality and functionality of these antibodies were greater than the individuals who had a transient antibody response to the pandemic vaccine (HI titre

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

Background Neutralizing antibodies are important for protection against the pandemic SARS-CoV-2 virus, and long-term memory responses determine the risk of re-infection or boosting after vaccination. T-cellular responses are considered important for partial protection against novel variants of concern. Methods A prospective cohort of hospitalized (n = 14) and community (n = 38) patients with rt-PCR confirmed SARS-CoV-2 infection were recruited. Blood samples and clinical data were collected when diagnosed and at 6 months. Serum samples were analyzed for SARS-CoV-2-spike specific antibodies using ELISA (IgG, IgA, IgM), pseudotype neutralization and microneutralization assays. Peripheral blood mononuclear cells were investigated for virus-specific T-cell responses in the interferon-γ and interleukin-2 fluorescent-linked immunosorbent spot (FluroSpot) assay. Results We found durable SARS-CoV-2 spike- and internal protein specific T-cellular responses in patients with persistent antibodies at 6 months. Significantly higher IL-2 and IFN-γ secreting T-cell responses as well as SARS-CoV-2 specific IgG and neutralizing antibodies were detected in hospitalized compared to community patients. The immune response was impacted by age, gender, comorbidity and severity of illness, reflecting clinical observations. Conclusions SARS-CoV-2 specific T-cellular and antibody responses persisted for 6 months post confirmed infection. In previously infected patients, re-exposure or vaccination will boost long-term immunity, possibly providing protection against re-infection with variant viruses.

Background: Households studies reflect the natural spread of SARS-CoV-2 in immunologically naive populations with limited preventive measures to control transmission.We hypothesise that seropositivity provides more accurate household attack rates than RT-PCR. Here, we investigated the importance of age in household transmission dynamics. Methods: We enroled 112 households (291 participants) in a case-ascertained study in Bergen, Norway from 28th February to 4th April 2020, collecting demographic and clinical data from index patients and household members. SARS-CoV-2-specific antibodies were measured in sera collected 6–8 weeks after index patient nasopharyngeal testing to define household attack rates. Findings: The overall attack rate was 45% (95% CI 38–53) assessed by serology, and 47% when also including seronegative RT-PCR positives. Serology identified a higher number of infected household members than RT-PCR. Attack rates were equally high in children (48%) and young adults (42%). The attack rate was 16% in asymptomatic household members and 42% in RT-PCR negative contacts. Older adults had higher antibody titres than younger adults. The risk of household transmission was higher when the index patient had fever (aOR 3.31 [95% CI 1.52–7.24]; p = 0.003) or dyspnoea (aOR 2.25 [95% CI 1.80–4.62]; p = 0.027) during acute illness. Interpretation: Serological assays provide more sensitive and robust estimates of household attack rates than RT-PCR. Children are equally susceptible to infection as young adults. Negative RT-PCR or lack of symptoms are not sufficient to rule out infection in household members. Funding: Helse Vest (F-11628), Trond Mohn Foundation (TMS2020TMT05). Abstrakt: Bakgrunn: Studier av husstander gjenspeiler den naturlige spredningen av SARS-CoV-2 blant ikke-immune populasjoner med begrensede tiltak for å forebygge smittespredning. Vår hypotese er at antistoff-påvisning gir mer nøyaktige angrepsrater i husstander sammenliknet med RT-PCR. Her undersøker vi betydnignenngen betydningen av alder i smittespredningen. Metoder: Vi rekrutterte 112 husstander (291 studiedeltakere) i en indeks kasus-bekreftet studie i Bergen, Norge fra 28.02.2020 til 04.04.2020, og samlet inn demografiske og kliniske data fra indekspasienter og deres husstandsmedlemmer. Angrepsrate i husstander ble beregnet ved å måle SARS-CoV-2-spesifikke antistoffer i sera samlet 6–8 uker etter nasofarynksprøve av indekspasienten. Funn: Den totale angrepsraten var 45% (95% KI 38–53) vurdert ved serologi, og 47% ved å inkludere antistoff negative, RT-PCR positive husstandsmedlemmer. Spesifikke antistoffer identifiserer en høyere andel infiserte husstandsmedlemmer sammenliknet med RT-PCR. Angrepsraten var like høy hos barn (48%) og unge voksne (42%). Angrepsraten var 16% hos personer uten symptomer og 42% hos RT-PCR negative husstandsmedlemmer. Eldre voksne hadde høyere antistoff titre enn yngre voksne. Risiko for smitte i husstander var høyere når indekspasienten hadde feber (aOR 3.31 [95% KI 1.52–7.24]; p = 0.003) eller dyspne (aOR 2.25 [95% KI 1.80–4.62]; p = 0.027) under akuttfasen. Tolkning: Serologiske analyser gir mer sensitive og robuste estimater av angrepsrate i husstander sammenliknet med RT-PCR. Barn er like utsatt for infeksjon som voksne. Negativ RT-PCR eller fravær av symptomer er ikke tilstrekkelige for å utelukke infeksjon blant husstandsmedlemmer. Finansiering: Helse Vest (F-11628), Trond Mohn Stiftelse (TMS2020TMT05).

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

Influenza vaccination often results in a large percentage of low responders, especially in high-risk groups. As a first line of defense, natural killer (NK) cells play a crucial role in the fight against infections. However, their implication with regard to vaccine responsiveness is insufficiently assessed. Therefore, this study aimed at the validation of essential NK cell features potentially associated with differential vaccine responsiveness with a special focus on NKG2C- and/or CD57-expressing NK cells considered to harbor memory-like functions. To this end, 16 healthy volunteers were vaccinated with an adjuvanted pandemic influenza vaccine. Vaccine responders and low responders were classified according to their hemagglutination inhibition antibody titers. A majority of responders displayed enhanced frequencies of NKG2C-expressing NK cells 7- or 14-days post-vaccination as compared to low responders, whereas the expression of CD57 was not differentially modulated. The NK cell cytotoxic potential was found to be confined to CD56dimCD16+ NKG2C-expressing NK cells in the responders but not in the low responders, which was further confirmed by stochastic neighbor embedding analysis. The presented study is the first of its kind that ascribes CD56dimCD16+ NKG2C-expressing NK cells a crucial role in biasing adaptive immune responses upon influenza vaccination and suggests NKG2C as a potential biomarker in predicting pandemic influenza vaccine responsiveness.

Summary: A systematic infection control program is found to be an important tool to reduce hospital-associated infections (HAIs) and surveillance of infection is a significant part of it. The aim of this paper was to present the result from 17 years continuous prevalence studies after implementation of a systematic infection control program, to examine trends of hospital-associated infections and to study possible risk factors for different sites of infection.Data from 61 399 in-patients at Haukeland University Hospital, Norway, from 1994 to 2010 was included in the study. Overall prevalence of HAIs was 7.6%. There was a reduction in HAIs from 8.3% in 1994 to 7.1% in 2010 (relative decrease 14.4%), mostly attributable to a significant reduction in the prevalence of urinary tract infections (UTI). For surgical site infections (SSI) we found a borderline significant increase (p = 0.05). Male gender (except for UTI), urinary tract catheter and surgical operation were all strong predictors for HAIs. Higher age was a risk factor for all infection types, except for BSI.In conclusion, repeated prevalence surveys demonstrated a significant reduction in HAIs but no decrease in hospital-associated BSI, LRTI and SSI. There was, however, a rapid decline of UTI and other less severe HAIs. Keywords: Hospital-associated infection, Prevalence, Risk factors, Infection control program

Influenza is a major respiratory pathogen causing annual outbreaks and occasional pandemics. Influenza vaccination is the major method of prophylaxis. Currently annual influenza vaccination is recommended for groups at high risk of complications from influenza infection such as pregnant women, young children, people with underlying disease and the elderly, along with occupational groups such a healthcare workers and farm workers. There are two main types of vaccines available: the parenteral inactivated influenza vaccine and the intranasal live attenuated influenza vaccine. The inactivated vaccines are licensed from 6 months of age and have been used for more than 50 years with a good safety profile. Inactivated vaccines are standardized according to the presence of the viral major surface glycoprotein hemagglutinin and protection is mediated by the induction of vaccine strain specific antibody responses. In contrast, the live attenuated vaccines are licensed in Europe for children from 2–17 years of age and provide a multifaceted immune response with local and systemic antibody and T cell responses but with no clear correlate of protection. Here we discuss the immunological immune responses elicited by the two vaccines and discuss future work to better define correlates of protection.

ABSTRACT The immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines. IMPORTANCE Influenza viruses are a serious concern for public health and cause a large number of deaths worldwide every year. Current influenza virus vaccines can confer protection from disease, but they often show low efficacy due to the ever-changing nature of the viruses. Novel vaccination approaches target conserved epitopes of the virus, including the hemagglutinin stalk domain, to elicit universally protective antibodies that also bind to mutated viruses or new subtypes of viruses. Importantly, the hemagglutination inhibition assay—the only assay that has been accepted as a correlate of protection by regulatory authorities—cannot measure antibodies against the hemagglutinin stalk domain. Therefore, novel correlates of protection and assays to measure vaccine immunogenicity need to be developed. In this study, we correlated the results from multiple assays with protection in mice after transfer of human serum and a lethal virus challenge to investigate potential novel serological surrogate markers for protection.

The need for more effective influenza vaccines is highlighted by the emergence of novel influenza strains, which can lead to new pandemics. There is a growing population of susceptible subjects at risk for severe complications of influenza, such as the elderly who are only in part protected by current licensed seasonal vaccines. One strategy for improving seasonal and pandemic vaccines takes advantage of adjuvants to boost and modulate evoked immune responses. In this study, we examined the capacity of the recently described adjuvant cyclic di-adenosine monophosphate (c-di-AMP) to serve as an adjuvant for improved mucosal influenza vaccines, and induce effective protection against influenza H5N1. In detail, c-di-AMP promoted (i) effective local and systemic humoral immune responses, including protective hemagglutination inhibition titers, (ii) effective cellular responses, including multifunctional T cell activity, (iii) induction of long-lasting immunity, and (iv) protection against viral challenge. Furthermore, we demonstrated the dose-sparing capacity of the adjuvant as well as the ability to evoke cross-clade protective immune responses. Overall, our results suggest that c-di-AMP contributes to the generation of a protective cell-mediated immune response required for efficacious vaccination against influenza, which supports the further development of c-di-AMP as an adjuvant for seasonal and pandemic influenza mucosal vaccines.

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010–2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region.

ABSTRACT In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. IMPORTANCE Despite the existence of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to cause morbidity and mortality in the human population, emphasizing the continued need for research in the field. While the majority of influenza vaccine strategies target the viral hemagglutinin, the immunodominant antigen on the surface of the influenza virion, antibodies against the viral neuraminidase (NA) have been correlated with less severe disease and decreased viral shedding in humans. Nevertheless, the amount of NA is not standardized in current seasonal vaccines, and the exact breadth of NA-based protection is unknown. Greater insight into the cross-protective potential of influenza virus NA as a vaccine antigen may pave the way for the development of influenza vaccines of greater breadth and efficacy.

Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets.The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus.Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate.ClinicalTrials.gov NCT00868218.