Investigations On Humoral And Cellular Immunity Responses To Highly Pathogenic Avian Influenza A (H5N1) Infection In Chinese Population

[Background and Objective]Outbreaks of highly pathogenic H5N1 avian influenza have been linked to the death of millions of poultry in Asia and has spread to the Middle East, Europe and Africa with more than 50 countries affected. It has recently become apparent that the H5N1 avian influenza virus not only affects the bird population but also poses a significant threat to the human population as this disease is transmitted from birds to humans and other mammals. As of April 2008, a total of 378 human cases of avian influenza had been reported throughout the world with a greater than 60% fatality rate. The ability of the avian influenza to cross species from birds to humans has led to great concern. Even worse, the increasing possibility of human-to-human transmission of this disease has caused worldwide anxiety over the potential of a new influenza pandemic.While some researchers express concern over an avian influenza pandemic, others argue that there is still a lack of evidence to predict if H5N1 will become a pandemic influenza strain. The dispute partially stems from the possibility that some cases of H5N1 infections may be asymptomatic and, therefore, remain unreported. Avian influenza infection in humans generally presents with symptoms similar to the seasonal influenza including a high fever, headache, abdominal pain, vomiting and lower respiratory tract symptoms. Rapid failure of multiple organ systems quickly follows, resulting in death in a high percentage of infected people. Whether or not subclinical cases of avian influenza infection occur in humans is unclear and inherently difficult to determine due to the limitation of assay techniques. Although it has been difficult to ascertain the occurrence of asymptomatic cases of H5N1 infection in humans, there have been case study reports of atypical avian influenza where no respiratory symptoms were present but infection was confirmed through laboratory tests.Investigations on the seroprevalence of H5N1 infection are imperative to understanding the incidence of both symptomatic and asymptomatic H5N1 infection and its full clinical spectrum. The importance of such a study is evident in light of the continuous evolution of the H5N1 virus from the H5N1 strain that was found to be responsible for the 1997 Hong Kong outbreak to several new clades that are distinctively different, based on both gene sequences and on functional antibody analysis.The above illustrates the need for robust methods for detection of exposure of humans to avian influenza viruses. The latter, however, is not straightforward for avian influenza viruses. For human influenza virus infection the hemagglutination inhibition (HI) assay and microneutralization assay (MN) are often used to detect a serological response. However, routine HI has been shown to be insensitive to measure human antibody response against avian influenza. Developing sensitive, practical and rapid serological tests is eminent to this study and possible future pandemic.In addition, antibodies against influenza virus surface proteins, hemagglutinin (HA) and neuraminidase (NA) are the most important protection antibody to influenza infection. However HA and NA often undergo antigenic drift or shift, which may lead to immuno-evade of hosts in virus infection. It may be important for the cell-mediated immunity. IFN-γproducing T cells mediated immunity is vital for influenza infection.In overall, the primary objective of this work is to study whether there is asymptomatic human infection of H5N1 avian influenza.[Methods]A large scale sero-epidemiological survey on population with different exposure to poultry in different regions of China and on high risk population with flu-like illness was conducted. The functional (modified HI, MN) and solid-phase (enzyme-linked immunosorbent assay, ELISA and Western blot, WB) antibody assays were all performed to detect antibody against H5N1 viruses. In addition, avian influenza specific T cell response were screened in population using IFN-γenzyme-linked immunospot assay (ELISPOT) to further confirm H5N1 virus exposure in human.[Results]Using fluorescence activated cell sorting (FACS) technique, horse erythrocytes with more abundant sialic acidsa2, 3 galactose linkages were selected to replace the traditional chicken erythrocytes with less such type of receptors in HI assay. We confirm that using horse erythrocytes can increase the detection of HI activities in rabbit immune sera, and further show that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, using horse erythrocytes increased the sensitivity of detecting positive HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people. Furthermore, as fresh red blood cell (RBC) is easy to be contaminated and hemolytic, aldehyde fixed RBC by two kinds of aldehyde fixing methods, glutaraldehyde and formaldehyde fixing, were used to improve modified HI assay. It showed that aldehyde fixed RBC can be used in HI assay and glutaraldehyde fixing RBC with glycerin reagent showed more stable agglutination pattern and longer preservation.A rapid microneutralization assay combing cell culture and ELISA readout using anti-nucleoprotein were established. Compared with the traditional MN using cytopathogenic effect (CPE) as endpoint, the modified MN showed similar detection ability but more rapid and practicable in testing large size of samples. This technique had applied for the patent for invention.Our sero-epidemiological research provided an overall description of H:5 sero-antibodies in population from different temporal and geographical outbreak areas in China. Four types of subjects were selected: 1). General residents living in H5N1 outbreak areas (Xinjiang and Liaoning); 2). Highly risk population with consistent poultry contacts (Xinjiang and Shandong); 3). Flu-like illness patients during flu season (Anhui and Beijing); 4). General residents living in non-H5N1 outbreak areas (Shanxi). One thousand eight hundred and twenty five volunteers took part in our research. No significant differences on demographic characteristics of subjects were found.Our results indicated that a small portion of Xinjiang general residents, while asymptomatic, may have been infected with H5N1 influenza virus, as shown by the presence of H5N1-specific antibody responses confirmed by both functional and solid-phase antibody assays. As a control, there was no detectable H5-specific antibody in the population of a non-epizootic area of H5N1 infections. On the other hand, there were subjects with positive avian influenza HA peptides (H5, H7, and H9) stimulated IFN-γ~+ T cell immunity using IFN-γELISPOT assay. However, as a significant overlap of HA sequence of positive peptides between H5 and H3, it was difficult to identify H5 specific T cells. As there is less HA overlap between H7, H9 and H3 proteome, positive H7 and H9 specific T cell responses were detected in the subjects.The magnitude and frequency of T cell response against seasonal influenza (H3N2) peptides was significantly declined in the old people and the decline was largely contributed by HA and M peptides responses. We found that IFN-γT cell response was continuously decreased with increasing age to conserve peptides (NP, M, NS and PO), but to the peptides HA and NA, there was the "∧"and "∨"shaped curve respectively No correlation between T cell responses using ELISPOT assay and HI antibody including avian (H5, H7 and H9) and season influenza (H3) were observed.[Conclusions]The modified serological methods should be used as part of a standardized protocol to measure avian influenza antibody responses and should be recommended to local health agencies.Our findings suggest that asymptomatic H5N1 human infection can happen in a small fraction but the overall rate of infection is very low, especially when this result is put in the context of a large, low risk population. These results argue that extensive seroepidemiology investigations need to be conducted in other affected areas to examine whether asymptomatic cases of the avian influenza exist elsewhere and to monitor the risk of bird-to-human transmission of H5N1 virus in order to prevent or control a potential avian pandemic in humans.Our screening on T cell response to influenza in population provides a basic understanding on the virus stimulated T cell pattern. Further work will be focused on identifying the specific avian influenza peptide response and the age-related antibody distribution and cell immunity.