Инфекция и иммунитет. 2019; 9: 763-772
Реакция микронейтрализации в сравнении с реакцией торможения гемагглютинации при оценке иммуногенности гриппозных вакцин и диагностике гриппа
https://doi.org/10.15789/2220-7619-2019-5-6-763-772Аннотация
Целью исследования являлось сравнение результатов реакции торможения гемагглютинации (РТГА) и реакции микронейтрализации при детекции антител к вирусам гриппа А(H1N1), A(H1N1)pdm09 и А(H3N2) в сыворотках людей, а также оптимизация условий постановки реакции микронейтрализации. Предложенный вариант реакции микронейтрализации основан на выявлении снижения репродукции вируса гриппа в инфицированных клетках MDCK в присутствии вирусспецифичных сывороточных антител. Репродукцию вирусов в клетках оценивали иммуноферментным методом в 96-луночных культуральных планшетах с помощью типоспецифичных моноклональных антител, направленных к NP-белку вирусов гриппа А. Параллельно в реакции микронейтрализации и РТГА были проанализированы парные сыворотки 205 волонтеров, привитых инактивированными сезонными гриппозными вакцинами, а также 117 взрослых пациентов, переболевших лабораторно подтвержденным гриппом. Доказана целесообразность обработки сывороток человека рецептор-разрушающим энзимом (RDE) при постановке не только РТГА, но и реакции микронейтрализации. Показана бόльшая чувствительность реакции микронейтрализации по сравнению с РТГА. По данным микронейтрализации показатели частоты сероконверсий и кратности прироста антител к вирусам гриппа А у вакцинированных и переболевших людей превышали в 1,4–2,5 раза результаты, полученные в РТГА. Более высокая чувствительность реакции микронейтрализации имела большое значение при расшифровке заболевания, вызванного новым патогеном. Эффективность серодиагностики гриппа A(H1N1)pdm09 у ПЦРположительных пациентов была в 1,5 раза выше по результатам реакция микронейтрализации по сравнению с РТГА. Согласно традиционным представлениям, основанным на результатах ранних работ, титр гриппспецифичных антител 1/40, выявленный в РТГА, считается защитным. При этом общепринятого протективного уровня вирусспецифичных антител для реакции нейтрализации до сих пор не установлено. Уровни антител в сыворотках, выявленные при использовании предложенного варианта реакции микронейтрализации, были значительно выше, чем по результатам РТГА. В поствакцинальных сыворотках привитых волонтеров среднегрупповые титры вируснейтрализующих антител, соответствующие 1/40 в РТГА, составили 1/195, 1/203 и 1/426–1/430 для вирусов гриппа A(Н1N1), A(Н1N1)pdm09 и A(Н3N2) соответственно. В сыворотках переболевших гриппом пациентов эти же показатели составили 1/285, 1/215 и 1/488. В соответствии с этим для реакции микронейтрализации порогами для «условно протективного» уровня антител у взрослых вакцинированных волонтеров или заболевших пациентов предложено считать в титры 1/160 для вирусов A(Н1N1) и A(Н1N1)pdm09, а также 1/320 для вируса A(Н3N2), что согласуется с данными других исследователей.
Список литературы
1. Изучение популяционного иммунитета к гриппу у населения Российской Федерации: Методические указания МУ 3.1.3490-17 (утв. Главным государственным санитарным врачом РФ 27 октября 2017 г.)
2. Методы определения показателей качества иммунобиологических препаратов для профилактики и диагностики гриппа: Методические указания МУ 3.3.2.1758-03 (утв. Главным государственным санитарным врачом РФ 28 сентября 2003 г.)
3. Alvarez M.M., López-Pacheco F., Aguilar-Yañez J.M., Portillo-Lara R., Mendoza-Ochoa G.I., García-Echauri S., Freiden P., Schultz-Cherry S., Zertuche-Guerra M.I.,Bulnes-Abundis D., Salgado-Gallegos J., Elizondo-Montemayor L., Hern á ndezTorre M. Specific recognition of influenza A/H1N1/2009 antibodies in human serum: a simple virus-free ELISA method. PLoS One, 2010, vol. 5, no. 4: e10176. doi: 10.1371/journal.pone.0010176
4. Benoit A., Beran J., Devaster J.M., Esen M., Launay O., Leroux-Roels G., McElhaney J.E., Oostvogels L., van Essen G.A., Gaglani M., Jackson L.A., Vesikari T., Legrand C., Tibaldi F., Innis B.L., Dewé W. Hemagglutination inhibition antibody titers as a correlate of protection against seasonal A/H3N2 influenza disease. Open Forum Infect. Dis., 2015, vol. 2, no. 2: ofv067. doi: 10.1093/ofid/ofv067
5. Black S., Nicolay U., Vesikari T., Knuf M., Del Giudice G., Della Cioppa G., Tsai T., Clemens R., Rappuoli R. Hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children. Pediatr. Infect. Dis. J., 2011, vol. 30, pp. 1081–1085. doi: 10.1097/INF.0b013e3182367662
6. Centers for Disease Control and Prevention (CDC). Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccination with seasonal influenza vaccine. Morb. Mortal. Wkly. Rep., 2009, vol. 58, no. 19, pp. 521–524.
7. Ehrlich H.J., Müller M., Oh H.M., Tambyah P.A., Joukhadar C., Montomoli E., Fisher D., Berezuk G., Fritsch S., Löw-Baselli A., Vartian N., Bobrovsky R., Pavlova B.G., Pöllabauer E.M., Kistner O., Barrett P.N. Baxter H5N1 Pandemic Influenza Vaccine Clinical Study Team. A clinical trial of a whole-virus H5N1 vaccine derived from cell culture. N. Engl. J. Med., 2008, vol. 358, no. 24, pp. 2573–2584. doi: 10.1056/NEJMoa073121
8. EMA, Guideline on influenza vaccines. Non-clinical and clinical module. Draft. 2016.
9. Grund S., Adams O., Wählisch S., Schweiger B. Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus. J. Virol. Methods, 2011, vol. 171, no. 2, pp. 369–373. doi: 10.1016/j.jviromet.2010.11.024
10. Hobson D., Curry R.L., Beare A.S., Ward-Gardner A. The role of serum haemagglutinatination binhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J. Hyg., 1972, vol. 70, pp. 767–777.
11. Lin Y., Gu Y., McCauley J.W. Optimization of a quantitative micro-neutralization assay. J. Vis. Exp., 2016, vol. 118: e54897. doi: 10.3791/54897
12. Matrosovich M., Gao P., Kawaoka Y. Molecular mechanisms of serum resistance of human influenza H3N2 virus and their involvement in virus adaptation in a new host. J. Virol., 1998, vol. 72, no. 8, pp. 6373–6380.
13. Memoli M.J., Shaw P.A., Han A., Czajkowski L., Reed S., Athota R., Bristol T., Fargis S., Risos K., Powers J.H., Davey R.T., Taubenberger J.K. Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. mBio, 2016, vol. 7, no. 2: e00417-16. doi: 10.1128/mBio.00417-16
14. Nakane P.K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem., 1974, vol. 22, no. 12, pp. 1084–1091. doi: 10.1177/22.12.1084
15. Potter C.W., Oxford J.S. Determinants of immunity to influenza infection in man. Br. Med. Bull., 1979, vol. 35, pp. 69–75.
16. Rowe T., Abernathy R.A., Hu-Primmer J., Thompson W.W., Lu X., Lim W., Fukuda K., Cox N.J., Katz J.M. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol., 1999, vol. 37, pp. 937–943.
17. Rudenko L., Kiseleva I., Naykhin A.N., Erofeeva M., Stukova M., Donina S., Petukhova G., Pisareva M., Krivitskaya V., Grudinin M., Buzitskaya Zh., Isakova-Sivak I., Kuznetsova S., Larionova N., Desheva J., Dubrovina I., Nikiforova A., Victor J., Neuzil K., Flores J., Tsvetnitsky V., Kiselev O. Assessment of human immune responses to H7 avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase I study of live attenuated H7N3 influenza vaccine. PLoS One, 2014, vol. 9, no. 2: е87962. doi: 10.1371/journal.pone.0087962
18. Serological diagnosis of influenza by microneutralization assay. WHO Manual. 2010.
19. Stephenson I., Das R.G., Wood J.M., Katz J.M. Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine, 2007, vol. 25, no. 20, pp. 4056–4063. PMID: 17412461. doi: 10.1016/j.vaccine.2007.02.039
20. Stephenson I., Wood J.M., Nicholson K.G., Zambon M.C. Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin. J. Med. Virol., 2003, vol. 70, pp. 391–398. doi: 10.1002/jmv.10408
21. Trombetta C.M., Montomoli E. Influenza immunology evaluation and correlates of protection: a focus on vaccines. Expert. Rev. Vaccines, 2016, vol. 15, no. 8, pp. 967–976. doi: 10.1586/14760584.2016.1164046
22. Tsang T.K., Cauchemez S., Perera R.A., Freeman G., Fang V.J., Ip D.K., Leung G.M., Malik Peiris J.S, Cowling B.J. Association between antibody titers and protection against influenza virus infection within households. J. Infect. Dis., 2014, vol. 210, no. 5, pp. 684–692. doi: 10.1093/infdis/jiu186
23. Verschoor C.P., Singh P., Russell M.L., Bowdish D.M., Brewer A., Cyr L., Ward B.J., Loeb M. Correction: Microneutralization assay titres correlate with protection against seasonal influenza H1N1 and H3N2 in children. PLoS One, 2015, vol. 10, no. 6: e0131531. doi: 10.1371/journal.pone.0131531
24. Zhu H., Ding X., Chen X., Yao P., Xu F., Xie R., Yang Z., Liang W., Zhang Y., Li Y., Shen J., He P., Guo Z., Su B., Sun S., Zhu Z. Neutralizing antibody but not hemagglutination antibody provides accurate evaluation for protective immune response to H5N1 avian influenza virus in vaccinated rabbits. Vaccine, 2011, vol. 29, no. 33, pp. 5421–5423. doi: 10.1016/j.vaccine.2011.05.067
Russian Journal of Infection and Immunity. 2019; 9: 763-772
Microneutralization reaction compared to hemagglutination inhibition assay to evaluate immunogenicity of influenza vaccines and influenza diagnostics
https://doi.org/10.15789/2220-7619-2019-5-6-763-772Abstract
The aim of this study was to compare the results of the hemagglutination inhibition test (HI-test) and microneutralization reaction in detection of antibodies to influenza A(H1N1), A(H1N1)pdm09, and A(H3N2) viruses in human sera, as well optimize microneutralization reaction conditions. The proposed variant of microneutralization reaction is based on detecting decreased influenza virus reproduction in infected MDCK cells in the presence of virus-specific serum antibodies. Virus reproduction was evaluated by enzyme-linked immunosorbent assay in 96-well cell culture plates using type-specific anti-influenza A viruse NP-protein monoclonal antibodies. In parallel, in microneutralization reaction and HI-test paired sera collected from 205 volunteers inoculated with inactivated seasonal influenza vaccines were analyzed, as well as from 117 adult patients with laboratory-confirmed influenza. The rationale for treatment of human serum with receptor-destroying enzyme (RDE) was proved not only for HI-test, but also for microneutralization reaction. Compared to HI-assay, the microneutralization reaction displayed higher sensitivity. According to microneutralization data, seroconversion rates and increase in antibody titer against influenza A viruses in both vaccinated and infected persons were superior to HI-test data by 1.4–2.5-fold. Moreover, higher sensitivity of this method was of great importance for the diagnostics of disease caused by new pathogens. The efficacy of influenza A(H1N1)pdm09 serodiagnostics in PCRpositive patients was 1.5 times higher based on microneutralization reaction vs. HI-assay data. According to the data from early studies, it is commonly believed that 1/40 titer of flu-specific antibodies detected by HI-test is set as protective. However, a consensus on protective level for virus-specific antibodies in neutralization reaction has not been established yet. It was found that serum antibody levels detected by the proposed version of microneutralization reaction were significantly higher than those in HI-assay. In the post vaccination sera collected from vaccinated volunteers, average titers of virus neutralizing antibodies corresponding to 1/40 in HI-test were 1/195, 1/203, and 1/ 426–1/430 for influenza A(H1N1), A(H1N1)pdm09 and A(H3N2), respectively, whereas in influenza patients they were 1/285, 1/215 and 1/488, respectively. Thus, it was suggested to consider a threshold value for “conditionally protective” level of neutralizing antibodies in adult vaccinated volunteers or infected patients, an average titer 1/160 for A(H1N1) and A(H1N1)pdm09 viruses, as well as 1/320 — for A(H3N2) virus, which agree with data published elsewhere.
References
1. Izuchenie populyatsionnogo immuniteta k grippu u naseleniya Rossiiskoi Federatsii: Metodicheskie ukazaniya MU 3.1.3490-17 (utv. Glavnym gosudarstvennym sanitarnym vrachom RF 27 oktyabrya 2017 g.)
2. Metody opredeleniya pokazatelei kachestva immunobiologicheskikh preparatov dlya profilaktiki i diagnostiki grippa: Metodicheskie ukazaniya MU 3.3.2.1758-03 (utv. Glavnym gosudarstvennym sanitarnym vrachom RF 28 sentyabrya 2003 g.)
3. Alvarez M.M., López-Pacheco F., Aguilar-Yañez J.M., Portillo-Lara R., Mendoza-Ochoa G.I., García-Echauri S., Freiden P., Schultz-Cherry S., Zertuche-Guerra M.I.,Bulnes-Abundis D., Salgado-Gallegos J., Elizondo-Montemayor L., Hern á ndezTorre M. Specific recognition of influenza A/H1N1/2009 antibodies in human serum: a simple virus-free ELISA method. PLoS One, 2010, vol. 5, no. 4: e10176. doi: 10.1371/journal.pone.0010176
4. Benoit A., Beran J., Devaster J.M., Esen M., Launay O., Leroux-Roels G., McElhaney J.E., Oostvogels L., van Essen G.A., Gaglani M., Jackson L.A., Vesikari T., Legrand C., Tibaldi F., Innis B.L., Dewé W. Hemagglutination inhibition antibody titers as a correlate of protection against seasonal A/H3N2 influenza disease. Open Forum Infect. Dis., 2015, vol. 2, no. 2: ofv067. doi: 10.1093/ofid/ofv067
5. Black S., Nicolay U., Vesikari T., Knuf M., Del Giudice G., Della Cioppa G., Tsai T., Clemens R., Rappuoli R. Hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children. Pediatr. Infect. Dis. J., 2011, vol. 30, pp. 1081–1085. doi: 10.1097/INF.0b013e3182367662
6. Centers for Disease Control and Prevention (CDC). Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccination with seasonal influenza vaccine. Morb. Mortal. Wkly. Rep., 2009, vol. 58, no. 19, pp. 521–524.
7. Ehrlich H.J., Müller M., Oh H.M., Tambyah P.A., Joukhadar C., Montomoli E., Fisher D., Berezuk G., Fritsch S., Löw-Baselli A., Vartian N., Bobrovsky R., Pavlova B.G., Pöllabauer E.M., Kistner O., Barrett P.N. Baxter H5N1 Pandemic Influenza Vaccine Clinical Study Team. A clinical trial of a whole-virus H5N1 vaccine derived from cell culture. N. Engl. J. Med., 2008, vol. 358, no. 24, pp. 2573–2584. doi: 10.1056/NEJMoa073121
8. EMA, Guideline on influenza vaccines. Non-clinical and clinical module. Draft. 2016.
9. Grund S., Adams O., Wählisch S., Schweiger B. Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus. J. Virol. Methods, 2011, vol. 171, no. 2, pp. 369–373. doi: 10.1016/j.jviromet.2010.11.024
10. Hobson D., Curry R.L., Beare A.S., Ward-Gardner A. The role of serum haemagglutinatination binhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J. Hyg., 1972, vol. 70, pp. 767–777.
11. Lin Y., Gu Y., McCauley J.W. Optimization of a quantitative micro-neutralization assay. J. Vis. Exp., 2016, vol. 118: e54897. doi: 10.3791/54897
12. Matrosovich M., Gao P., Kawaoka Y. Molecular mechanisms of serum resistance of human influenza H3N2 virus and their involvement in virus adaptation in a new host. J. Virol., 1998, vol. 72, no. 8, pp. 6373–6380.
13. Memoli M.J., Shaw P.A., Han A., Czajkowski L., Reed S., Athota R., Bristol T., Fargis S., Risos K., Powers J.H., Davey R.T., Taubenberger J.K. Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. mBio, 2016, vol. 7, no. 2: e00417-16. doi: 10.1128/mBio.00417-16
14. Nakane P.K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem., 1974, vol. 22, no. 12, pp. 1084–1091. doi: 10.1177/22.12.1084
15. Potter C.W., Oxford J.S. Determinants of immunity to influenza infection in man. Br. Med. Bull., 1979, vol. 35, pp. 69–75.
16. Rowe T., Abernathy R.A., Hu-Primmer J., Thompson W.W., Lu X., Lim W., Fukuda K., Cox N.J., Katz J.M. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol., 1999, vol. 37, pp. 937–943.
17. Rudenko L., Kiseleva I., Naykhin A.N., Erofeeva M., Stukova M., Donina S., Petukhova G., Pisareva M., Krivitskaya V., Grudinin M., Buzitskaya Zh., Isakova-Sivak I., Kuznetsova S., Larionova N., Desheva J., Dubrovina I., Nikiforova A., Victor J., Neuzil K., Flores J., Tsvetnitsky V., Kiselev O. Assessment of human immune responses to H7 avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase I study of live attenuated H7N3 influenza vaccine. PLoS One, 2014, vol. 9, no. 2: e87962. doi: 10.1371/journal.pone.0087962
18. Serological diagnosis of influenza by microneutralization assay. WHO Manual. 2010.
19. Stephenson I., Das R.G., Wood J.M., Katz J.M. Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine, 2007, vol. 25, no. 20, pp. 4056–4063. PMID: 17412461. doi: 10.1016/j.vaccine.2007.02.039
20. Stephenson I., Wood J.M., Nicholson K.G., Zambon M.C. Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin. J. Med. Virol., 2003, vol. 70, pp. 391–398. doi: 10.1002/jmv.10408
21. Trombetta C.M., Montomoli E. Influenza immunology evaluation and correlates of protection: a focus on vaccines. Expert. Rev. Vaccines, 2016, vol. 15, no. 8, pp. 967–976. doi: 10.1586/14760584.2016.1164046
22. Tsang T.K., Cauchemez S., Perera R.A., Freeman G., Fang V.J., Ip D.K., Leung G.M., Malik Peiris J.S, Cowling B.J. Association between antibody titers and protection against influenza virus infection within households. J. Infect. Dis., 2014, vol. 210, no. 5, pp. 684–692. doi: 10.1093/infdis/jiu186
23. Verschoor C.P., Singh P., Russell M.L., Bowdish D.M., Brewer A., Cyr L., Ward B.J., Loeb M. Correction: Microneutralization assay titres correlate with protection against seasonal influenza H1N1 and H3N2 in children. PLoS One, 2015, vol. 10, no. 6: e0131531. doi: 10.1371/journal.pone.0131531
24. Zhu H., Ding X., Chen X., Yao P., Xu F., Xie R., Yang Z., Liang W., Zhang Y., Li Y., Shen J., He P., Guo Z., Su B., Sun S., Zhu Z. Neutralizing antibody but not hemagglutination antibody provides accurate evaluation for protective immune response to H5N1 avian influenza virus in vaccinated rabbits. Vaccine, 2011, vol. 29, no. 33, pp. 5421–5423. doi: 10.1016/j.vaccine.2011.05.067
