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postgraduate thesis: Neuraminidase inhibiting antibodies to influenza A virus
Title | Neuraminidase inhibiting antibodies to influenza A virus |
---|---|
Authors | |
Issue Date | 2017 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Karunarathna, H. M. T. K.. (2017). Neuraminidase inhibiting antibodies to influenza A virus. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Annual vaccination is the cornerstone global strategy utilized worldwide to prevent influenza virus infection in human. Although, antibodies raised against both hemagglutinin and neuraminidase are important in protection from disease, there is a paucity of studies done on anti-neuraminidase antibodies, especially in relation to protection from pandemic H1N1 2009 influenza A virus infection in natural infection. Therefore in this study, our aims were i. optimization of enzyme linked lectin assay (ELLA) using different types of NA antigens, including VLP as a source of antigen ii. development of a new method to calculate input dose of NA antigen for neuraminidase inhibition enzyme linked lectin (NI ELLA) assay and iii. detection of anti-neuraminidase antibody response (anti-NA N1) against pandemic H1N1 2009 influenza A virus infection in human subjects.
Two reverse genetic live viruses; H6N1 (pandemic), H6N1 (seasonal) and two VLPs expressing NA protein from A/California/04/2009 and A/Cambodia/JP52a/2005 were generated and showed a sigmoidal shape NA activity titration curve in ELLA. All four antigens have shown a specificity to anti-NA N1 antibody when standard antisera are used. A panel of human serum samples having different levels of anti-NA N1 antibodies were tested with different levels of each antigen input doses. We devised a method for estimating input dose of both antigens types and demonstrated that the antigen dose has a marked effect on the NA antibody titers. We have suggested normalizing the NA antigen dose at 32 antigen units. The anti-NA N1 titers observed with NA-VLP pdm as an antigen source were markedly lower than the titers observed with compatible H6N1 virus. Western blotting confirmed that NA protein levels are higher in NA-VLP pdm than H6N1pdm virus in a solution of comparable NA activity in ELLA. This led us to conclude that reverse genetic live viruses H6N1 are a better NA antigen source compared with VLPs expressing NA protein only as NA sources for ELLA test.
Anti-NA N1 titers of eighteen paired serum (acute and convalescent) samples from patients PCR confirmed for pandemic H1N1 2009 influenza A virus infection confirmed the increase of level of anti-NA N1 titers with the infection. The change of fold titer was higher in those serum samples with lower baseline levels of anti-NA N1 titers in acute serum samples compared to those serum samples with higher level of anti-NA N1 titers in acute serum samples. There was a correlation between anti-NA N1 titers in acute serum samples and disease severity of each.
It was found that binding between hemagglutinin and sialic acid (SA) in live viruses on fetuin increases the NA activity of live viruses compare to the NA only expressing VLPs. In summary we have optimized the ELLA assay, explored, but rejected the use of VLP as an antigen source for the ELLA assay, and demonstrated the anti N1 antibody responses and cross reactive responses in patients with pandemic H1N1 2009 influenza A virus infection in humans and correlated anti NA antibody responses with disease severity. |
Degree | Master of Philosophy |
Subject | Neuraminidase Influenza A virus |
Dept/Program | Public Health |
Persistent Identifier | http://hdl.handle.net/10722/251298 |
HKU Library Item ID | b5864137 |
DC Field | Value | Language |
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dc.contributor.author | Karunarathna, Herath Mudiyanselage Thusitha Kumara | - |
dc.date.accessioned | 2018-02-24T08:55:44Z | - |
dc.date.available | 2018-02-24T08:55:44Z | - |
dc.date.issued | 2017 | - |
dc.identifier.citation | Karunarathna, H. M. T. K.. (2017). Neuraminidase inhibiting antibodies to influenza A virus. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/251298 | - |
dc.description.abstract | Annual vaccination is the cornerstone global strategy utilized worldwide to prevent influenza virus infection in human. Although, antibodies raised against both hemagglutinin and neuraminidase are important in protection from disease, there is a paucity of studies done on anti-neuraminidase antibodies, especially in relation to protection from pandemic H1N1 2009 influenza A virus infection in natural infection. Therefore in this study, our aims were i. optimization of enzyme linked lectin assay (ELLA) using different types of NA antigens, including VLP as a source of antigen ii. development of a new method to calculate input dose of NA antigen for neuraminidase inhibition enzyme linked lectin (NI ELLA) assay and iii. detection of anti-neuraminidase antibody response (anti-NA N1) against pandemic H1N1 2009 influenza A virus infection in human subjects. Two reverse genetic live viruses; H6N1 (pandemic), H6N1 (seasonal) and two VLPs expressing NA protein from A/California/04/2009 and A/Cambodia/JP52a/2005 were generated and showed a sigmoidal shape NA activity titration curve in ELLA. All four antigens have shown a specificity to anti-NA N1 antibody when standard antisera are used. A panel of human serum samples having different levels of anti-NA N1 antibodies were tested with different levels of each antigen input doses. We devised a method for estimating input dose of both antigens types and demonstrated that the antigen dose has a marked effect on the NA antibody titers. We have suggested normalizing the NA antigen dose at 32 antigen units. The anti-NA N1 titers observed with NA-VLP pdm as an antigen source were markedly lower than the titers observed with compatible H6N1 virus. Western blotting confirmed that NA protein levels are higher in NA-VLP pdm than H6N1pdm virus in a solution of comparable NA activity in ELLA. This led us to conclude that reverse genetic live viruses H6N1 are a better NA antigen source compared with VLPs expressing NA protein only as NA sources for ELLA test. Anti-NA N1 titers of eighteen paired serum (acute and convalescent) samples from patients PCR confirmed for pandemic H1N1 2009 influenza A virus infection confirmed the increase of level of anti-NA N1 titers with the infection. The change of fold titer was higher in those serum samples with lower baseline levels of anti-NA N1 titers in acute serum samples compared to those serum samples with higher level of anti-NA N1 titers in acute serum samples. There was a correlation between anti-NA N1 titers in acute serum samples and disease severity of each. It was found that binding between hemagglutinin and sialic acid (SA) in live viruses on fetuin increases the NA activity of live viruses compare to the NA only expressing VLPs. In summary we have optimized the ELLA assay, explored, but rejected the use of VLP as an antigen source for the ELLA assay, and demonstrated the anti N1 antibody responses and cross reactive responses in patients with pandemic H1N1 2009 influenza A virus infection in humans and correlated anti NA antibody responses with disease severity. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.subject.lcsh | Neuraminidase | - |
dc.subject.lcsh | Influenza A virus | - |
dc.title | Neuraminidase inhibiting antibodies to influenza A virus | - |
dc.type | PG_Thesis | - |
dc.identifier.hkul | b5864137 | - |
dc.description.thesisname | Master of Philosophy | - |
dc.description.thesislevel | Master | - |
dc.description.thesisdiscipline | Public Health | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.mmsid | 991026387279703414 | - |