Russian-backbone LAIV history and clinical development. L.Rudenko Institute of Experimental Medicine, St. Petersburg, Russia

Transcription

1 Russian-backbone LAIV history and clinical development L.Rudenko Institute of Experimental Medicine, St. Petersburg, Russia

2 EFFECTIVENESS OF LIVE COLD-ADAPTED INFLUENZA VACCINE FOR ADULTS. SUMMARY FROM 126 TRIALS (500 to 45,000 adults per trial; total > 500,000 adults) Efficacy was confirmed for each trial. Mean effectiveness for 126 trials was = 45.0%.

3 EFFECTIVENESS OF LIVE COLD-ADAPTED INFLUENZA VACCINE DURING THE CIRCULATION OF DRIFTED VARIANT OF INFLUENZA Year Age of vaccines No. of vaccinated children Circulated epidemic strain Vaccine components Effectiveness (%) (95% CI) Alma-Ata, Kazakhstan 3-14 Vaccine Placebo A/Taiwan/1/86 (H1N1) A/Brazil/11/78 (H1N1) A/Philippines/2/82 (H3N2) 27.6 ( ) Alma-Ata, Kazakhstan 3-14 Vaccine Placebo A/Taiwan/1/86 (H1N1) B/Victoria/2/87 A/Taiwan/1/86 (H1N1) A/Philippines/2/82 (H3N2) 34.7 ( ) Velikij Novgorod 7-14 Vaccine 4693 Placebo 4168 A/Shanghai/16/89 (H3N2) A/Taiwan/1/86 (H1N1) A/Sichuan/2/87 (H3N2) B/USSR/3/ ( ) Velikij Novgorod 7-14 Vaccine 5288 Placebo 6385 A/Taiwan/1/86 (H1N1) B/Victoria/2/87 B/Yamagata/16/88 A/Taiwan/1/86 (H1N1) A/Shanghai/11/87 (H3N2) B/USSR/3/ ( ) Velikij Novgorod 7-14 Vaccine 3403 Placebo 3022 A/Beijing/32/92 (H3N2) A/Taiwan/1/86 (H1N1) A/Shanghai/11/87 (H3N2) B/Beijing/203/ ( ) St.Petersburg Region 7-14 Vaccine 554 Placebo 1265 A/New Caledonia/20/99 (H1N1) A/Beijing/262/95 (H1N1) A/Sydney/5/97 (H3N2) B/St.Petersburg/95/ ( ) St.Petersburg Vaccine 539 Placebo 297 A/Wyoming/3/03 (H3N2) A/New Caledonia/20/99 (H1N1) A/Panama/99/242 (H3N2) B/Victoria/2/ ( )

4 EFFICACY OF LIVE ATTENUATED INFLUENZA VACCINE (LAIV) IN CHILDREN 7-14 YEARS OLD (SEROLOGICAL AND VIROLOGICAL CONFIRMATION) LAIV Infuenza morbidity in vaccinees - 5.3% Influenza morbidity in placebo group % Placebo Coefficient of efficacy %

5 Vaccine efficacy for completely vaccinated children, 1 January through 4 March 1990 Vaccine group No. of vaccinated Morbidity in epidemic season (No. of cases / %) Efficacy % (95% confidence interval) Live / 13.0 Inactivated / ( ) 29.6 ( ) Placebo / 27.0

6 Efficacy live influenza vaccine against school absence due to physician-diagnosed influenza or acute respiratory disease and against 4-fold rise in antibody titer to influenza A (H3N2), Vologda, Russia, 1 January to 2 February 1992 Absence No./Total (%) Efficacy measure Placebo Live Influenza Vaccine 18/187 (9.6) 10/196 (5.1) Relative risk/vaccine efficacy (%) */47 4-fold antibody rise No./Total (%) 37/163 (23) 10/160 (6) Relative risk/vaccine efficacy (%) **/72 * P=0.06 vs. placebo (Fisher's exact test, one-tailed) ** P<0.001 vs. placebo Vaccine strains: A/Leningrad/92/89(H1N1), A/Zakharpatije/354/89(H3N2), B/Yamagata/16/98 Circulated epidemic strain: A/Beijing/353/89 (H3N2) Khan AS, Polezhaev F, Vasiljeva R, Drinevsky V, Buffington J, Gary H, Sominina A, Keitel W, Regnery H, Lonskaya NL, Doroshenko E, Gavrilov A, Ivakhov I, Arden N, Schonberger LB, Couch R, Kendal A, Cox N. Comparison of US inactivated split-virus and Russian live attenuated, cold-adapted trivalent influenza vaccines in Russian schoolchildren. The Journal of Infectious Diseases. 1996;173(2):453-6.

7 LONG-TERM PROTECTION AGAINST INFLUENZA AMONG CHILDREN 16 MONTHS POST-VACCINATION BY LAIV ( ) No. of City Velikij Novgorod vaccinated children Vaccine Placebo Effectiveness % 42.1 Vaccine strain - A/Shanghai/11/87 (H3N2)-like Circulated epidemic strain - A/Beijing/32/92 (H3N2)

8 INDIRECT PROTECTION I. Kaliningrad clinical trial children 3-6 years old received 2 doses of vaccine or placebo. The high level of vaccination is required to induce indirect protection. Percent of Vaccinated Illness, % 20,0 30,0 67,6 8,0 81,6 2,5 II % of protection of Smolensk region from were twice vaccinated with LIV. Two nearby control towns Kaluga and Vitebsk were not vaccinated. Thus the 5 year study showed that large scale vaccination with LIV is an effective, economical and expedient means of influenza control. III. As shown by the Novgorod school study, in which it was observed that influenza morbidity was significantly lower amoong non-vaccinated pupils and staff, where more than 50% pupils were vaccinated, compared with those schools, where pupils received inactivated influenza vaccine.

9 PERCENT OF CHILDREN WHO RECEIVED LIVE VACCINE IN EACH SCHOOL VS. PERCENT OF STAFF WHO WERE ILL IN THAT SCHOOL, Percent by school of children vaccinated with live vaccine Percent ill in staff Rudenko LG, Slepushkin AN, Monto AS, Kendal AP, Grigorieva EP, Burtseva EP, Rekstin AR, Beljaev AL, Bragina VE, Cox N, Ghendon YZ, Alexandrova GI. Efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in Novgorod, Russia. The Journal of Infectious Diseases. 1993;168(4):881-7.

10 REGRESSION ANALYSIS RELATING VACCINATION RATE IN SCHOOL IN WHICH CHILDREN RECEIVED VACCINE OR PLACEBO TO PROPORTION ILL IN UNVACCINATED CHILDREN OR STAFF Vaccine group β (95% confidence interval) r 2 % of ill staff vs. % of students vaccinated Live (-1.82, -1.15) 0.86 Inactivated (-0.72, 0.26) 0.11 Placebo (-0.65, 0.25) 0.07 % of ill nonvaccinated children vs. % of students vaccinated Live (-1.15, -1.63) 0.51 Inactivated 0.04 (-0.38, 0.46) 0.00 Placebo (-0.91, 0.41) 0.06 Rudenko LG, Slepushkin AN, Monto AS, Kendal AP, Grigorieva EP, Burtseva EP, Rekstin AR, Beljaev AL, Bragina VE, Cox N, Ghendon YZ, Alexandrova GI. Efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in Novgorod, Russia. The Journal of Infectious Diseases. 1993;168(4):881-7.

11 LIST OF PANDEMIC LAIVs PREPARED ON A/LENINGRAD/134/17/57 (H2N2) BACKBONE Vaccine strain Subtype Wild-type parental virus The stage of study A/17/duck/Potsdam/86/92 H5N2 A/duck/Potsdam/1402-6/86 (H5N2)** Phases I and II clinical trials completed The vaccine is registered in Russia А/17/California/2009/38 H1N1 A/California/07/2009(H1N1)* Phases I and II clinical trials completed The vaccine is registered in Russia, Thailand, India А/17/mallard/Netherlands/ 00/95 H7N3 А/mallard/Netherlands/12/ 2000 (H7N3)* Phase I clinical trial completed А/17/turkey/Turkey/05/133 H5N2 A/turkey/Turkey/1/2005 (H5N1), clade 2.2** Phase I clinical trial completed A/17/Vietnam/04/65107 H5N2 A/Vietnam/1203/2004 (H5N1) clade 1** Studied in animal models cavn1203-len17rg H5N1 A/Egypt/321/2007 (H5N1) clade 2.2* Studied in animal models caeg321-len17rg H5N1 A/Vietnam/1203/2004 (H5N1) clade 1* Studied in animal models А/17/California/66/395 H2N2 А/California/1/66 (H2N2)* Phase I clinical trial completed A/17/Tokyo/67/326 H2N2 A/Tokyo/3/67/326 (H2N2)* Studied in animal models А/17/Anhui/2013/61 H7N9 А/Anhui/1/2013 (H7N9)* Phase I clinical trial completed *Vaccine strain inherited HA and NA genes from wild-type parental virus and remaining six genes from master donor virus, i.e., 6:2 genetic formula. **Vaccine strain inherited only HA gene from wild-type parental virus and remaining seven genes from master donor virus, that is, 7:1 genetic formula.

12 Methods for study immunogenicity of LAIV Serum hemagglutinating antibodies in HAI test Serum neutralizing antibodies in MN test IgA, IgG serum specific (ELISA) Secretory IgA in nasal swabs (ELISA) Is highly desirable study of CMI by methods of flow cytometry and ELISPOT

13 SERUM ANTIBODY IMMUNE RESPONSES BEFORE AND AFTER ADMINISTRATION OF H7N9 LAIV OR PLACEBO Reciprocal geometric mean titers Haemagglutinin inhibition (95% CI) Placebo (n=10) Vaccine (n=29) Baseline P value Microneutralization (95% CI) Placebo (n=10) Vaccine (n=29) P value 3.3 ( ) 3.0 ( ) ( ) 5.2 ( ) 0.88 Participants with titers 1/40 0% (0-31) 0% (0-12) - 0% (0-31) 0% (0-31) - Reciprocal geometric mean titer Participants with seroconversion Reciprocal geometric mean titer in participants with seroconversion 28 days after dose ( ) 5.1 ( ) ( ) 17.7 ( ) % (0-31) 10% (2-27) % (0-31) 48% (29-68) NA NA 44.2 ( ) - Participants with titers 1/40 0% (0-31) 0% (0-12) - 0% (0-31) 38% (21-58) Reciprocal geometric mean titer Participants with seroconversion Reciprocal geometric mean titer in participants with seroconversion 28 days after dose ( ) 10.2 ( ) ( ) 27.3 ( ) % (0-31) 66% (46-82) % (0-31) 72% (53-87) NA 17.3 ( ) - NA 45.6 ( ) - Participants with titers 1/40 0% (0-31) 14% (4-32) % (0-31) 59% (39-77) 0.002

14 Antibody immune responses to H5N1 IIV among individuals previously primed with H5N2 LAIV (compared to unprimed control) Antigen % seroconverted LAIV primed (n=19) Unprimed control (n=24) Day 7 Day 28 Day 7 Day 28 GMT % seroconverted HAI assay GMT % seroconverted GMT % seroconverted t/turkey (H5N2) LAIV NIBRG-23 (H5N1) A/Indonesia/5/05 (PR8- based) A/duck/Potsdam (H5N2) LAIV Microneutralization assay t/turkey (H5N2) LAIV NIBRG-23 (H5N1) GMT H5N2 LAIV primed for a robust recall response after a later alum-adjuvanted H5N1 IIV vaccination

15 Comparison of H1N1pdm LAIVs This slide summarizes some features of LAIV reassortants prepared on the Len/17 and Ann Arbor backbones. Some key amino acids in HA and virus titers in eggs, MDCK cells and mouse URT are shown. Original LAIV based on California strain had good growth characteristics, but the virus had Glutamic acid (E) at position 47, and this made the virus unstable. Our colleagues from Medimmune made mutagenesis of HA to improve virus growth characteristics, and the key positions were 222, 223. Although more recent Bolivia strain was more stable due to K-47 residue, the LAIV based on this virus grew poorly in MDCK cells and didn t replicate in mouse nasal turbinates. We introduced two mutations at positions 222/223 (as was done by MedImmune) but the virus still grew poorly in mammalian cells. Only the LAIVs prepared from two recent strains (South Africa and New York) replicated well in cells and mouse tissues. We suggest that the key position might be E-127. This mutation was seen by other researchers when they adapted California virus to mice, and the D-127- E mutation enhanced virus growth in mouse tissues. Surface genes of LAIV strain A/California/7/ 2009 (Len17: IEM) A/California/7/ 2009 (AA: mut- MedImmune* A/Bolivia/559/ 3013 (Len17: IEM) A/Bolivia/559/ 3013 (Len17: mut-iem) RG A/South Africa/3626/20 13 (Len17: CDC) A/New York/61/2015 (Len17: IEM) clade HA2 47 HA1 127 HA1 222 HA1 223 Titer of LAIV strain (ml -1 ) eggs MDCK cells mouse NT - E D D R E D G Q 7.4? 4.0 6b K D D R <1.1 6b K D G Q <1.1 6b K E D Q b.2 K E N Q n.d. *enhanced growth in eggs (Chen et al, JVI 2010) D127E mutation enhanced H1N1 viral growth in mouse tissues (Sakabe et al, Virus Res 2011 E47K (HA2) mutations enhances temperature and ph stability of HA (Cotter et al, PLoS PATH, 2014)

16 NP gene major target for CTL response CTLs targeted to some NP epitopes of Len/17 MDV do not recognize NP of currently circulated influenza A viruses Inclusion of NP from currently circulated influenza A viruses into LAIV formulation would bring advantages in terms of CTL response wt NP is the best candidate for inclusion into LAIV 5:3 reassortants Rimmelzwaan GF, Kreijtz JH, Bodewes R, Fouchier RA, Osterhaus AD. Influenza virus CTL epitopes, remarkably conserved and remarkably variable. Vaccine Oct 23;27(45):

17 In vivo killing activity of CTLs induced by 6:2 or 5:3 LAIVs This slide shows the in vivo killing activity of mouse CTLs induced after immunization with 6:2 or 5:3 LAIV reassortants. We prepared two pairs of reassortants of H7N9 (left graph) and H1N1 subtype (right graph). Each dot shows the proportion of survived target cells (mouse splenocytes) loaded with wild-type virus (or specific NP epitope of wild-type virus) after intravenous inoculation into immunized mice. For H7N9 viruses, only in 5:3-immunized mice these target cells loaded with NP epitope of wt virus were effectively killed by CTLs, whereas no difference were seen for 6:2 LAIV. For H1N1 viruses, where the target cells were loaded with whole wild-type virus, the effect was the same: only CTLs of 5:3-immunized mice could effectively kill these target cells. These data confirm that 5:3 reassortant LAIV induce more relevant CTL immunity, compared to 6:2 LAIV.

18 Cross-protection afforded by 6:2 or 5:3 LAIVs Both LAIV pairs protected animals from homologous challenge. But consistent with the previous slide, the 5:3 LAIV reassortants protected immunized mice better against heterologous challenge. For H7N9 LAIVs we used two viruses for challenge experiments: avian H7N3 virus and PR8-based reassortant with NP gene from H7N9 virus. For both viruses viral titers in lungs were reduced, compared to controls, only in mice immunized with 5:3 LAIV. For H1N1 viruses, a virus A/New York/2014 and PR8-based reassortant with NP gene from South Africa/2013 virus were used as heterologous challenge. For New York challenge viral titers in lungs were reduced compared to control only for 5:3 LAIV (but not 6:2). For PR8 challenge both vaccines reduced pulmonary titers, but titers in 5:3 LAIV were significantly lower than in 6:2 LAIV group. These data suggest that the wild-type NP protein incorporated into LAIV genome has significant impact on the induction of cell-mediated immunity, and as a consequence on the vaccine s cross-protection.

19 Summary Overall, it is clear that we need to make the LAIV better, and to improve the performance of current LAIVs we should do the following: Pay more attention to the selection of influenza viruses for generating LAIV reassortants; perform thorough analysis of the LAIV candidates, i.e. study their growth properties in cell cultures and in animal models. Use standardized immunological assays to measure LAIV immunogenicity, i.e. include measuring siga antibody and cellmediated immunity. Modify genome structures of LAIV reassortant viruses, i.e. include additional wild-type gene (NP) into LAIV viruses: from 6:2 genome composition towards 5:3. Develop better stabilizers used for LAIV manufacturing, to improve LAIV stability during storage, shipment and application. Use the most recent advances in the development of influenza vaccines inducing cross-reactive and long-lasting immunity (towards the universal LAIV).