1 Immune Responses of Healthy Subjects to a Single Dose of Intramuscular Inactivated Influenza A/Vietnam/1203/2004 (H5N1) Vaccine After Priming With an Antigenic Variant. Nega Ali Goji, M.D., Carrie Nolan, N.P., Heather Hill, M.S., CCRC, Mark Wolf, Ph.D., Thomas Rowe, M.S., John J Treanor, M.D. University of Rochester Medical Center, Rochester, NY, EMMES Corporation, Rockville, MD, Southern Research Institute, Birmingham, AL

2 Background Non-adjuvanted, inactivated subvirion H5N1 vaccine required two 90 mcg doses to elicit neutralizing antibody in more than half of subjects In one study, a third dose of inactivated A/Duck/Singapore/97 (H5N3) vaccine 16 months after a priming series resulted in significant boosting (Stephenson, 2003) Pre-priming might generate better immunity, allowing a single dose strategy in face of emerging pandemic. However, any emergent pandemic virus will likely represent antigenic variant from the priming virus We took advantage of a previous study evaluating a baculovirus recombinant H5 A/HK/156/97 (clade 3) vaccine performed in 1998 to evaluate effect of boosting with a single dose of subvirion A/VN/1203/04 (clade 1) vaccine.

3 Neutralizing antibody responses to rH5 A/HK/156 vaccine Treanor Vaccine 19:1732, 2001 Neutralizing GMT Vaccine administered at visit S1 and S

4 Objectives Determine the ability of a clade 3 H5 recombinant vaccine to prime for immune responses to a subsequent clade 1 H5 subvirion vaccine in healthy adults –Comparison of responses in H5 primed subjects to those of H5 naïve subjects Determine the safety of revaccination with a clade 1 vaccine in primed subjects

5 Methods Subjects: participants in 1998 study who received a clade 3 rH5 vaccine baculovirus-expressed recombinant H5 vaccine (A/HK/156/97, Clade 3) Vaccine: single 90 mcg dose of subvirion rgA/Vietnam/1203/04 x PR8 (clade 1) vaccine Safety diary card x 7 days, all adverse events recorded over 56 days Serum HAI and MN antibody tested at days 0, 28, and 56 Results compared to responses to one (primary analysis) or two (secondary analysis) 90 mcg doses of clade 1 vaccine in naïve subjects

6 Evaluation of priming with an antigenic variant: schematic of study design Placebo 25 ug x 2 45 ug x 2 90 ug x 2 90 ug x ug x 1 UR A/HK/156/97 rH5 147 subjects 1998 DMID rgA/VN/1203/04 90 ug x 1 37 subjects DMID rgA/VN/1203/04 90 ug x subjects Healthy recipients of any rH5 Placebo excluded CLADE 1CLADE 3 NEJM 354:1343, 2006 Vaccine 19:1732, 2001 H5- PRIMED H5- NAIVE 2005

7 Demographics H5 NAIVE (DMID ) n=103 White Black Asian Pacific Islander Multiracial Hispanic Female Age (median, range) 84 (82%) 11 (11%) 8 (8%) 0 (0%) 13 (13%) 55 (54%) 38 (18-64) H5 PRIMED (DMID ) n=37 35 (95%) 2 (5%) 0 (0%) 23 (62%) 42 (33-51)

8 Rates and severity of side effects after 1 (D1) or 2 doses (D2) in naïve subjects and after one dose in H5 vaccine-primed (PR) subjects

9 Serum hemagglutination-inhibition (HAI) titers following one or two doses of H5 vaccine in naïve subjects or following a single dose in H5 vaccine-primed subjects H5 NAIVE H5 PRIMED DMID DMID GMT HAI Antibody 90 mcg

10 Serum neutralizing (NT) antibody responses following one or two doses of H5 vaccine in naïve subjects or following a single dose in H5 vaccine-primed subjects H5 NAIVE H5 PRIMED DMID DMID GMT NT Antibody 90 mcg

11 Rates of serum HAI and NT antibody responses and proportion achieving a titer of 1:40 or greater after one or two doses of H5 vaccine in naïve subjects or after one dose in H5 vaccine-primed subjects Result 28 days after: Dose 1 Dose 2 Dose 1 HAI 23 (15, 33) 43 (33, 54) 68 (50, 82) NT 10 (5, 18) 41 (32, 52) 76 (59, 88) Percent responding* (95% CI) HAI 24 (16, 34) 44 (34, 55) 70 (53, 84) NT 11 (6, 19) 42 (33, 53) 76 (59, 88) Percent >1:40 (95% CI) Group H5 naïve H5 primed * Response defined as 4-fold or greater increase in titer from baseline. For the HAI assay, a response also must achieve a titer of 1:40 or greater

12 Effect of the priming dose of recombinant H5 vaccine administered in 1998 on responses to 90 mcg of H5 vaccine in Priming Dose 25 ug x 2 45 ug x 2 90 ug x 2 90 ug/10 ug N GMT Response* n (%) 7 (58%) 6 (86%) 5 (63%) 7 (70%) GMT Response* n (%) 10 (83%) 6 (86%) 7 (88%) 9 (90%) HAI NT * Response defined as 4-fold or greater increase in titer from baseline. For the HAI assay, a response also must achieve a titer of 1:40 or greater Response to a single dose of H5 vaccine in 2006

13 Relationship between response in 1998 and response in 2005 Response to rH5 in 1998^ No Yes GMT Response* n (%) 17 (63%) 8 (80%) GMT Response* n (%) 22 (81%) 10 (100%) HAI NT Response to a single dose of H5 vaccine in 2006 N * Response defined as 4-fold or greater increase in titer from baseline. For the HAI assay, a response also must achieve a titer of 1:40 or greater ^ Response in 1998 was defined as 4-fold or greater increase in MN titer to a titer of 1:80 or greater, accompanied by positive WB

14 Memory B cell responses of primed subjects in study DMID

15 Conclusions The antibody responses to a single dose of non- adjuvanted vaccine support the hypothesis that previous vaccination with a clade 3 H5 vaccine primed for responses to a clade 1 H5 vaccine Antibody responses to a single booster dose exceeded those seen after two doses in naïve subjects, and were better than those seen with the original recombinant vaccine. The reasons for such vigorous responses to revaccination are unclear: Revaccination was well tolerated, with a side effect profile similar to vaccination of naïve subjects.

16 Conclusions Further studies to evaluate different priming schedules and to verify priming between clade 1 and clade 2 viruses are needed If the results were confirmed in larger studies, then pre- pandemic vaccination programs could be considered for some populations (first responders, HCW, military)

17 Acknowledgements University of Rochester Carrie Nolan Diane O’Brien Mhorag Hay Dave Topham John Treanor DMID Roland Levandowski Linda Lambert Shy Shorer EMMES Corporation Mark Wolff Ken Wilkins Heather Hill Southern Research Institute Tom Rowe

18 Response of healthy adults to a third dose of subvirion H5 vaccine (study DMID ) GMT HAI antibody Study day Vaccine

19 Alignment of A/Hong Kong/156/97 and A/Vietnam/1203/04 H5 Hemagglutinins Clade 3Clade 2Clade 1´Clade 1H3 No.Functional significance N45*DDD54†Antigenic site C S84NNN92Antigenic site E A86AAV93Antigenic site E N94DDD (1)101Near Y91; receptor binding? N124DSS129Antigenic site B S129SLL133aReceptor binding L138QQQ142Antigenic site A S155–N155–159Antigenic site B T156‡AAT160N154 glycosylation motif L175LLL (2)179Near H179; receptor binding? T188TTT (3)192Near L190; receptor binding? K189RRK193Adjacent to receptor binding, antigenic site B E212KKR216Antigenic site D S223–N223§– (4)227Receptor binding T263AAT266Antigenic site E 325R¶Absent–– HA cleavage efficiency EID 11: , 2005

20 Antigenic relatedness of clade 3 and clade 1 viruses Table 2. Antigenic analysis of H5N1 isolates from Asia Virus antigenClade Reference ferret antisera* HK156NCVD8HK213VN1203VN04xPR8-rgVN78VN4207VN14VN32321 A/Hong Kong/156/9731, A/ck/Vietnam/NCVD8/ < A/Hong Kong/213/031´1,280 2, A/Vietnam/1203/ < A/Vietnam/1203/04xPR8-rg180< A/Vietnam/1194/ A/Vietnam/JP178/ <101, A/Vietnam/JP4207/ , A/Vietnam/JP14/05120< A/Vietnam/JP30321/ <104010< *Homologous HI titers are in boldface. EID 11: , 2005