SARS-CoV-2 and influenza virus coinfections in the Tuscan population during the 2021/2022 influenza season.

Introduction: The 2021/2022 influenza season was not characterised by a well-defined incidence peak. As reported by the Italian National Institute of Health, a high value of incidence of influenza cases was recorded in week 13, but it was still lower than in other influenza seasons. This abnormal circulation was probably due to relaxation of the COVID-19 pandemic restriction measures, such as social distancing, smart-working, home leaning and the use of masks, which greatly reduced the circulation of respiratory-transmitted viruses, including human respiratory syncytial virus (HRSV). The symptoms of SARS-CoV-2 and influenza are quite similar, sharing the human-to-human transmission route via respiratory droplets. Methods: The aim of this study was to estimate the rate of coinfection with influenza viruses and/or HRSV in SARS-CoV-2-positive subjects (N = 940) in a population of central Italy during the 2021/2022 season. Results: A total of 54 cases of coinfection were detected during the study period, 51 cases (5.4%) of SARS-CoV-2 and influenza virus and three cases (0.3%) of SARS-CoV-2 and HRSV coinfection. Conclusions: These results highlight the importance of continuous monitoring of the circulation of influenza virus and other respiratory viruses in the context of the COVID-19 pandemic.

Detection of Influenza D Antibodies in Dogs, Apulia Region, Italy, 2016 and 2023.

Dogs are known to be susceptible to influenza A viruses, although information on influenza D virus (IDV) is limited. We investigated the seroprevalence of IDV in 426 dogs in the Apulia region of Italy during 2016 and 2023. A total of 14 samples were positive for IDV antibodies, suggesting exposure to IDV in dogs.

Immunogenicity and safety of COVID-19 booster vaccination: A population-based clinical trial to identify the best vaccination strategy.

BACKGROUND: Various SARS-CoV-2 variants of concerns (VOCs) characterized by higher transmissibility and immune evasion have emerged. Despite reduced vaccine efficacy against VOCs, currently available vaccines provide protection. Population-based evidence on the humoral immune response after booster vaccination is crucial to guide future vaccination strategies and in preparation for imminent COVID-19 waves. METHODS: This multicenter, population-based cohort study included 4697 individuals ≥18 years of age who received a booster vaccination. Antibody levels against SARS-CoV-2 receptor binding domain (RBD) and neutralizing antibodies against wild-type (WT) virus and Omicron variants were assessed at baseline (day of booster vaccination) and after four weeks. Safety was evaluated daily within the first week using a participant-completed electronic diary. Antibody levels were compared across different vaccination strategies, taking into account individual host factors. RESULTS: Our main model including 3838 participants revealed that individuals who received a booster with mRNA-1273 compared to BNT162b2 vaccine had a significantly higher increase (95 %CI) in anti-RBD-antibody levels (37,707 BAU/mL [34,575-40,839] vs. 27,176 BAU/mL [26,265-28,087]), and of neutralization levels against WT (1,681 [1490-1872] vs. 1141 [1004-1278] and Omicron variant (422 [369-474] vs. 329 [284-374]). Neutralizing antibody titres highly correlated with anti-RBD antibodies, with neutralizing capacity 4.4 fold higher against WT compared to Omicron. No differences in safety were found between the two booster vaccines. CONCLUSION: Our study underlines the superiority of a booster vaccination with mRNA-1273, independent of the primary vaccination and therefore provides guidance on the vaccination strategy.

Serological Evidence for Circulation of Influenza D Virus in the Ovine Population in Italy.

Influenza D virus (IDV) is a novel orthomyxovirus initially isolated from pigs exhibiting influenza-like disease in the USA. Since then, IDV has been detected worldwide in several host species, including livestock animals, whilst specific antibodies have been identified in humans, raising concerns about interspecies transmission and zoonotic risks. Few data regarding the seroprevalence of IDV in small ruminants have been available to date. In this study, we assessed the prevalence of antibodies against IDV in ovine serum samples in Sicily, Southern Italy. Six hundred serum samples, collected from dairy sheep herds located in Sicily in 2022, were tested by haemagglutination inhibition (HI) and virus neutralization (VN) assays using reference strains, D/660 and D/OK, representative of two distinct IDV lineages circulating in Italy. Out of 600 tested samples, 168 (28.0%) tested positive to either IDV strain D/660 or D/OK or to both by HI whilst 378 (63.0%) tested positive to either IDV strain D/660 or D/OK or to both by VN. Overall, our findings demonstrate that IDV circulates in ovine dairy herds in Sicily. Since IDV seems to have a broad host range and it has zoonotic potential, it is important to collect epidemiological information on susceptible species.

Flu vaccine administration in the period before SARS-CoV-2 infection and its outcomes: An umbrella review.

Objective: The potential association between influenza vaccination and SARS-CoV-2 infection and related outcomes is still controversial. The aim of this umbrella review is to represent the impact of previous influenza vaccination and COVID-19 outcomes using evidence currently available in literature. Methods: A literature search of MEDLINE, EMBASE, Scopus, Web of Science and Cochrane Library was conducted. The paper selection was conducted using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) method by two-blinded authors. The quality of meta-analyses was assessed using the AMSTAR 2 scale (A MeaSurement Tool to Assess systematic Reviews). The outcomes investigated were SARS-CoV-2 infection after influenza vaccination, hospitalization, intensive care unit admission, mechanical ventilation and mortality. Results: The literature research identified 7 ecological studies and 6 meta-analyses. All the ecological studies show a negative relationship between influenza vaccination and COVID-19. The meta-analyses suggest a protective action of influenza vaccination against SARS-CoV-2 infection. Regarding the outcomes evaluated, only two studies reported a statistically significant reduction of 12% and of 17% in hospitalization and intensive care unit admission, respectively. Regarding mechanical ventilation, three studies showed a risk reduction of 31%, 27% and 28%. A substantial reduction of mortality risk was also observed in one study. Conclusions: These results suggest that influenza vaccination could be associated with reduced susceptibility to SARS-CoV-2 infection, mechanical ventilation and mortality. Our findings highlighted how the administration of flu vaccine in subjects at risk could lead to a reduction in mortality, particularly in the over 65y.

The burden of influenza and the role of influenza vaccination in adults aged 50-64 years: A summary of available evidence.

Influenza is a vaccine-preventable disease and a global public health problem. Although most national influenza vaccination recommendations focus on subjects aged ≥65 years, an extensive burden of influenza has also been reported in those aged ≥50 years and is exacerbated by immune system aging. The main purpose of this review is to provide an overview of the burden of influenza and its potential prevention within the 50-64 age-group. These subjects account for a large proportion of the workforce, and play a central economic and social role. Individuals aged 50-64 years had a 3-times higher rate of hospitalization and a 9-fold higher mortality rate attributable to influenza than those aged 18-49-years, generating higher influenza-related hospitalization costs. Available data suggest that including healthy subjects aged 50-64 years in influenza vaccination recommendations would allow a broader population to be reached, reducing the economic and social burden of influenza.

Evaluation of immune response to SARS-CoV-2 Omicron sublineages six months after different vaccination regimens in Italy.

The Omicron variant is the most divergent, displaying more mutations than previous SARS-CoV-2 variants, particularly in the gene that encodes the spike protein. This study aimed to assess the persistence of neutralizing antibodies towards the SARS-CoV-2 Omicron sublineages (BA.2, BA.5, BQ.1, XBB and XBB1.5) six months after the third dose in different vaccination regimens. Subjects who received 3 doses of mRNA vaccine retained their neutralization activity against BA.2 and BA.5, even though 56.3% and 66.7% showed a ≥ 2-fold reduction in the neutralizing antibody titre, respectively. Subjects who had received the adenovirus-based vaccine plus a booster dose of mRNA vaccine retained their neutralization activity especially against BA.2. With regard to BQ.1, XBB and XBB.1.5, the majority of the subjects showed a ≥ 2-fold reduction in neutralizing antibody titre, with the greatest evasion being observed in the case of XBB. Overall, our results provide further evidence that triple homologous/heterologous vaccination and hybrid immunity result in detectable neutralizing antibodies against the ancestral virus; however, emerging Omicron sublineages, such as XBB and XBB.1.5, show a great evasive capacity, which compromises the effectiveness of current COVID-19 vaccines.

Establishment and validation of a high-throughput micro-neutralization assay for respiratory syncytial virus (subtypes A and B).

The validation of a bioanalytical method allows us to determine its validity for a designated purpose and to guarantee the reliability of its analytical results. The virus neutralization assay has proved to be suitable for the detection and quantification of specific serum-neutralizing antibodies against respiratory syncytial virus subtypes A and B. Respiratory syncytial virus is a negative-sense RNA virus and is responsible for the majority of acute lower respiratory tract infections in infants and older adults worldwide. Owing to its widespread infection, the WHO considers it a target for the development of preventive vaccines. Despite the high impact of its infections, however, only one vaccine has been recently approved. The aim of this paper is to provide a detailed validation process for the microneutralization assay and to demonstrate that this method can effectively support the efficacy assessment of candidate vaccines and the definition of correlates of protection.

Evaluation of Monkeypox- and Vaccinia virus-neutralizing antibodies in human serum samples after vaccination and natural infection.

Introduction: In early to mid-2022, an unexpected outbreak of Monkeypox virus infections occurred outside the African endemic regions. Vaccines originally developed in the past to protect against smallpox are one of the available countermeasures to prevent and protect against Orthopoxvirus infections. To date, there are few studies on the cross-reactivity of neutralizing antibodies elicited by previous vaccinia virus-based vaccination and/or Monkeypox virus infection. The aim of this study was to evaluate a possible approach to performing Monkeypox and vaccinia live-virus microneutralization assays in which the read-out is based on the production of cytopathic effect in the cell monolayer. Methods: Given the complexity of Orthopoxviruses, the microneutralization assay was performed in such a way as to uncover a potential role of complement, with and without the addition of an external source of Baby Rabbit Complement. A set of human serum samples from individuals who had been naturally infected with Monkeypox virus and individuals who may have and not have undergone vaccinia virus vaccinations, was used to evaluate the performance, sensitivity, and specificity of the assay. Results and conclusions: The results of the present study confirm the presence and cross-reactivity of antibodies elicited by vaccinia-based vaccines, which proved able to neutralize the Monkeypox virus in the presence of an external source of complement.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The 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. Methods: In 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. Results: We 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.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External 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. Methods: We 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. Results: Within 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. Discussion: This 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.

SARS-CoV-2 epidemiological trend before vaccination era: a seroprevalence study in Apulia, Southern Italy, in 2020.

Aim: The present study aimed at assessing the prevalence of antibodies against SARS-CoV-2 in the general population in the province of Bari (Apulia region, Southern Italy) during the year 2020. Subject and methods: In this study, 1325 serum samples collected from January to December 2020 were tested for the presence of IgM and IgG antibodies against whole-virus SARS-CoV-2 antigen by commercial ELISA. Positive samples were further tested by in-house ELISA for the detection of anti-receptor binding domain (RBD) IgM and IgG antibodies and by micro-neutralization (MN) assay for the detection of neutralizing antibody. Results: One hundred (7.55%) samples had the presence of at least one antibody class against SARS-CoV-2 by commercial ELISA, of which 88 (6.6%) showed IgG and 19 (1.4%) showed IgM antibodies. The proportion of samples with IgG antibodies increased from 1.9% in January-February to 9.6% in November-December, while no significant increase was observed for IgM. When tested by in-house ELISA and MN assay, 17.0% and 31.6% were found positive to RBD IgG and RBD IgM, respectively, while 12.0% showed neutralizing antibody. Conclusion: The proportion of samples with SARS-CoV-2 IgG antibodies increased during 2020, especially in the second half of the year, consistent with data reported by the routine epidemiological surveillance of SARS-CoV-2 cases. Despite the high number of reported cases, the seroprevalence values are relatively low, and only a small proportion of samples had neutralizing antibodies. Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-023-01834-3.

Neutralizing activity of African lineage Zika virus immune sera towards Asian lineage.

Genetic and phylogenetic studies indicated that Zika virus (ZIKV) has evolved into 2 major lineages, the African and Asian. However, ZIKV has been described as a single serotype. This study aimed at assessing the cross-neutralization between ZIKV African and Asian lineages strains. Sixthy-five samples collected in 2007 and 30 samples collected from the same subjects in 2011/2012 in West Africa and positive to neutralizing antibody against ZIKV MR-766 strain (African lineage) were tested against ZIKV H/PF/2013 strain (Asian lineage) by microneutralization assay. All samples showing neutralizing antibodies against MR-766 strain showed also neutralizing activity against H/PF/2013 strain, although with lower titers. This is consistent with about 120 amino acid differences between the two strains. Despite differences in the magnitude of neutralizing activity against different ZIKV strains, all samples showed neutralizing antibody titers considered to be protective.

Evaluation of antibody response to SARS-CoV-2 variants after 2 doses of mRNA COVID-19 vaccine in a correctional facility.

The SARS-CoV-2 pandemic has posed a challenge for correctional facilities worldwide. People in such settings are more vulnerable to severe forms of infection and it is impossible to completely isolate inmates from the outside world. This study aimed to assess the antibody-mediated immune response in terms of neutralizing antibodies against Alpha, Beta, Gamma and Omicron (sub-lineage BA.1) variants of concern after two doses of mRNA vaccine in correctional officers and inmates from an Italian correctional facility. Most of the correctional officers (56.5%) and inmates (52.3% and 63.6%) retained their neutralizing activity toward the Alpha and Gamma variants, respectively. By contrast, the most striking reduction in comparison with the ancestral virus was found in the antibody response toward the Beta and Omicron variants, in both correctional officers (91.2% and 93.9%) and inmates (85.1% and 92.8%). In addition, subjects who had undergone primary vaccination and had previously been naturally infected had higher neutralizing antibody titers toward the 4 variants than negative subjects. Overall, our findings indicate that primary mRNA vaccination is able to induce neutralizing antibodies toward the ancestral virus, while titers toward variants may vary, depending on the mutations harboring by the variants. Although the correctional setting is often considered distinct or isolated from the wider society and sanitary system, the health of correctional workers and prisoners is inexorably linked to the public health of the country as a whole and it is of paramount importance to monitor the antibody response in these settings.

The neutralizing response to SARS-CoV-2 Omicron variants BA.1 and BA.2 in COVID-19 patients and homologous and heterologous vaccinees.

The rapid replacement of Omicron BA.1 by BA.2 sublineage is very alarming, raising the question of whether BA.2 can escape the immunity acquired after BA.1 infection. We compared the neutralizing activity toward the Omicron BA.1 and BA.2 sub-lineages in five groups: COVID-19 patients; subjects who had received two doses of mRNA vaccine; subjects naturally infected with SARS-CoV-2 who had received two doses of mRNA; and subjects who had received three doses of homologous or heterologous vaccine. The results obtained highlight the importance of vaccine boosters in eliciting neutralizing antibody responses against Omicron sub-lineages, and suggest that the adenovirus vectored vaccine elicits a lower response against BA.1 than against BA.2 sub-lineage.

Immune response to SARS-CoV-2 Omicron variant in patients and vaccinees following homologous and heterologous vaccinations.

The SARS-CoV-2 Omicron variant has rapidly replaced the Delta variant of concern. This new variant harbors worrisome mutations on the spike protein, which are able to escape the immunity elicited by vaccination and/or natural infection. To evaluate the impact and susceptibility of different serum samples to the Omicron variant BA.1, samples from COVID-19 patients and vaccinated individuals were tested for their ability to bind and neutralize the original SARS-CoV-2 virus and the Omicron variant BA.1. COVID-19 patients show the most drastic reduction in Omicron-specific antibody response in comparison with the response to the wild-type virus. Antibodies elicited by a triple homologous/heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1. Overall, these findings confirm that vaccination of COVID-19 survivors and booster dose to vaccinees with mRNA vaccines is the correct strategy to enhance the antibody cross-protection against Omicron variant BA.1.

Antibody Avidity and Neutralizing Response against SARS-CoV-2 Omicron Variant after Infection or Vaccination.

Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods: Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.

COVID-19 Outbreak and BNT162b2 mRNA Vaccination Coverage in a Correctional Facility during Circulation of the SARS-CoV-2 Omicron BA.1 Variant in Italy.

Background. The recent spread of the highly mutated SARS-CoV-2 Omicron variant (B.1.1.529) has raised concerns about protection against COVID-19 in congregate settings such as prisons, characterized by a high risk of transmission and possible difficulties in obtaining adequate vaccination coverage. The present study aims to investigate the spread of an outbreak of COVID-19 in an Italian correctional facility during the dominant circulation of the Omicron BA.1 variant, and also considers BNT162b2 mRNA vaccination coverage among inmates. A COVID-19 screening campaign by RT-PCR was performed on 515 detainees from 4−30 January 2022, in response to an outbreak that began in the correctional facility. Furthermore, 101 serum samples collected from healthy inmates 21 days after having received the second dose of the BNT162b2 vaccine were tested for neutralizing antibodies against both the wild-type SARS-CoV-2 strain and the Omicron BA.1 variant. The global attack rate during the study period was 43.6% (RR 0.8), progressively reducing from unvaccinated inmates (62.7%, RR 1.8) to those who had one dose (52.3%, RR 1.5), two doses (full cycle) (45.0%, RR 1.3), and the third dose (booster) vaccinated group (31.4%, RR 0.7). The percentage of SARS-CoV-2 positive subjects among unvaccinated inmates was significantly higher than in the other groups (p < 0.001), while no significant difference was observed between inmates with one or two vaccine doses. Only two of the positive inmates were hospitalized for COVID-19. The geometric mean titer of neutralizing antibodies in the tested sub-group after two doses of vaccine was lower than in previous studies against the wild-type virus, and showed a complete lack of neutralization against the Omicron variant in 92.1% of individuals. The findings support the need to prioritize vaccination in correctional facilities, as a public health measure to increase the protection of inmates and consequently of prison workers and the community against COVID-19, in coordination with the other prevention strategies.

SARS-CoV-2 Circulation during the First Year of the Pandemic: A Seroprevalence Study from January to December 2020 in Tuscany, Italy.

Italy was the second country affected by the SARS-CoV-2 pandemic; the virus spread mainly in Northern Italy with a subsequent diffusion to the center and southern part of the country. In this study, we aimed to assess the prevalence of antibodies against SARS-CoV-2 in the general population of the Siena province in the Tuscany region (Central Italy) during 2020. A total of 2480 serum samples collected from January to December 2020 were tested for IgM and IgG antibodies against SARS-CoV-2 by a commercial ELISA. Positive and borderline samples were further tested for the presence of anti-receptor-binding domain (RBD) IgM and IgG antibodies by an in-house ELISA and by a micro-neutralization assay. Out of the 2480 samples tested by the commercial ELISA, 81 (3.3%) were found to be positive or borderline for IgG and 58 (2.3%) for IgM in a total of 133 samples (5.4%) found to be positive or borderline for at least one antibody class. When the commercial ELISA and in-house ELISA/micro-neutralization assay results were combined, 26 samples (1.0%) were positive for RBD IgG, 11 (0.4%) for RBD IgM, and 23 (0.9%) for a neutralizing antibody. An increase in seroprevalence was observed during the year 2020, especially from the end of summer, consistent with the routine epidemiological surveillance of COVID-19 cases.

Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

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.