Pandemic influenza A(H1N1pdm09) vaccine induced high levels of influenza-specific IgG and IgM antibodies as analyzed by enzyme immunoassay and dual-mode multiplex microarray immunoassay methods
Introduction
Past influenza A virus pandemics have shown that new reassortant viruses have the potential to spread rapidly throughout the world leading to significant morbidity and mortality in humans. The latest influenza pandemic in 2009 was caused by a novel swine-origin reassortant H1N1pdm09 influenza A virus with gene segments originating from avian, human and swine influenza A viruses [1]. The possibility of avian or other animal-origin influenza A viruses to infect and spread among humans has been identified as a potential global threat that could lead to an even more severe pandemic than the previous ones. The genetic determinants responsible for the avian-to-human transmission of influenza A viruses are still partly undetermined and the ability of different strains to infect humans is not fully understood. However, there is serological evidence for bird-to-human transmission of influenza A viruses [2], [3], [4], [5]. This highlights the need to further develop influenza surveillance systems in animals and humans [6] as well as to conduct serological surveys to monitor population immunity to various influenza types and subtypes.
Globally circulating human influenza A and B virus strains are continuously monitored and recommendations for the composition of influenza strains to be included in seasonal vaccines are updated by the World Health Organization (WHO) expert group twice a year. If novel reassortant influenza viruses are found in humans they are often used as basis for the development of pre-pandemic or pandemic (monocomponent) vaccines. The assessment of vaccine immunogenicity and efficacy is essential for successful vaccination campaigns [7]. Vaccines are typically developed and evaluated based on their ability to induce vaccine antigen-specific antibody responses [8]. Demonstrating the presence of antibodies to specific influenza antigens and strains is of great importance since antibodies play a prominent role in the protection against a given influenza virus strain.
The haemagglutination inhibition (HI) assay is the most commonly used method for measuring antibody levels against influenza viruses [9], [10], [11]. Anti-influenza antibodies detected by the HI assay have been shown to correlate well with protective immunity [12], [13]. Another commonly used method to detect antibody responses against microbial pathogens and vaccine antigens is an enzyme immunoassay (EIA). However, there are several limitations in both HI and EIA methods. The limiting factors of these assays are that they are labor- and time-intensive and especially the HI test requires relatively large sample volumes [14], [15]. Conventional EIA allows analysis of different antibody classes, however each immunoglobulin class has to be analyzed separately. HI assay has a strong limitation in analysing currently circulating influenza A (H3N2) viruses. Recent changes in the receptor binding characteristics of seasonal A(H3N2) viruses led to poor agglutination of red blood cells [16]. Therefore, inability of contemporary H3N2 viruses to be analysed by the HI assay requires the development of alternative methods.
In order to better facilitate influenza surveillance and the rapid assessment and development of vaccines, the limiting features of traditional serological assays and enzyme immunoassays have to be overcome. Modern multiplex techniques provide a great opportunity for a more broad-spectrum characterization of humoral immunity induced by vaccines and natural infections. Multiplex technology emerged about 20 years ago, first in the field of genomics and it was later widely used in proteomics, oncology, immunology, and infectious disease research [17], [18], [19], [20], [21], [22]. Emerging multiplex techniques allow researchers to examine vaccine responses with greater throughput and less time [15], [23], [24]. Recently, multiplex protein microarray assays for influenza virus serology have been developed and the assays have shown a great potential in studies of humoral immune responses to influenza infection and influenza vaccines [25], [26], [27]. Influenza hemagglutinin antigen-based microarrays have shown to be a valuable tool in studies of specificity, cross-reactivity and cross-protection of hemagglutinin-specific antibodies [28], [29], [30]. A high density hemagglutinin protein microarray consisting of 127 different hemagglutinin antigens from 60 viruses demonstrated a high-throughput measurement of breadth of antibody diversity induced by vaccination and influenza infection [31]. Of interest is the glycan microarray technology which enables the detection of the specificity of influenza virus strains for different types of glycan structures [32]. The technology allows the analysis the human receptor specificity of avian influenza virus strains.
Here we describe the development, validation, and implementation of an in-house multiplex microarray immunoassay which enables simultaneous quantitative detection of IgM and IgG antibodies against multiple vaccine or viral antigens. In the present study, we analyzed serum specimens collected from 60 individuals before and after vaccination with pandemic influenza vaccine in 2010 in Finland. We analyzed vaccine-induced humoral immune responses and compared antibody responses determined by HI test, EIA and microarray immunoassay. We sought to determine whether the microarray immunoassay could be used instead of other more labor-intensive tests to measure influenza vaccine-induced antibody responses.
Section snippets
Serum samples
A cohort of adults without any immunological disorders was recruited to a clinical and serological follow-up study on a voluntary basis between December 2009 and September 2010 in Tampere, Finland. Vaccinees received one intramuscular dose of Pandemrix™ (GlaxoSmithKlein, Rixenart, Belgium) vaccine in connection with the national pandemic vaccination campaign that was carried out in Finland in 2009 and 2010. The vaccine contained inactivated, split influenza virus propagated in eggs and an
Microarray immunoassay spot signals
The assay principle is shown in Fig. 1. For this study, we designed an array consisting of 12 spots. Two replicate spots of each of 2 antigens and 4 controls were printed on 4 × 3 layout on the bottom of each microtiter well. Upconversion luminescence signals from each sample well were imaged separately at blue 550 nm and green 470 nm emission channels for the detection of the bound Er-UCNP-anti-hIgG and Tm-UCNP-anti-hIgM, respectively. Serum samples collected from the same individual at day 0,
Discussion
Vaccines are typically developed and evaluated based on their ability to induce vaccine antigen-specific antibodies. It is generally assumed that antigen-specific antibody levels correlate with the protection of the host if the vaccine antigen(s) is/are the target(s) for neutralizing antibodies. In the present study, we used the traditional HI test together with EIA and MAIA tests to analyze antibody levels before and after vaccination with Pandemrix (H1N1pdm09) vaccine. The aim of the study
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are grateful to Riitta Santanen for performing the HI assay. We also thank the THL clinical staff who collected the samples and all the volunteers who took part in this study.
Author’s contributions
All authors meet the ICMJE criteria for authorship. AK developed MAIA, performed EIA and MAIA, analyzed serological data and wrote the manuscript together with IJ. TZ, RS and IJ were involved in the Pandemrix vaccination trial and sample collection. AK, LK, HP, TS, MW and IJ were involved in the study design and MAIA development and writing of the manuscript. All authors have read and approved the final version of the manuscript.
Ethics statement
The serum specimens analyzed in the present study were a subcohort of a larger study (Syrjänen et al., 2014) conducted by the National Institute for Health and Welfare (THL) in the city of Tampere, Finland. All participants gave their written informed consent for the study. The study protocols, sample collection, and consents were approved by the Ethics Committee of Pirkanmaa Health District, Finland (Permission ETL R0952M) and the study had received a THL protocol code AH1N1-483-09THL and an
Funding source
The study was supported by THL, University of Turku, Ministry of Social Affairs and Health, Finland and the Medical Research Council of the Academy of Finland. The funding organizations have no role in the design of the study and data analysis and presentation.
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