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

Abstract

Influenza A viruses continue to circulate throughout the world as yearly epidemics or occasional pandemics. Influenza infections can be prevented by seasonal multivalent or monovalent pandemic vaccines. In the present study, we describe a novel multiplex microarray immunoassay (MAIA) for simultaneous measurement of virus-specific IgG and IgM antibodies using Pandemrix-vaccinated adult sera collected at day 0 and 28 and 180 days after vaccination as the study material. MAIA showed excellent correlation with a conventional enzyme immunoassay (EIA) in both IgG and IgM anti-influenza A antibodies and good correlation with hemagglutination inhibition (HI) test. Pandemrix vaccine induced 5–30 fold increases in anti-H1N1pdm09 influenza antibodies as measured by HI, EIA or MAIA. A clear increase in virus-specific IgG antibodies was found in 93–97% of vaccinees by MAIA and EIA. Virus-specific IgM antibodies were found in 90–92% of vaccinees by MAIA and EIA, respectively and IgM antibodies persisted for up to 6 months after vaccination in 55–62% of the vaccinees. Pandemic influenza vaccine induced strong anti-influenza A IgG and IgM responses that persisted several months after vaccination. MAIA was demonstrated to be an excellent method for simultaneous measurement of antiviral IgG and IgM antibodies against multiple virus antigens. Thus the method is well suitable for large scale epidemiological and vaccine immunity studies.

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.