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.

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Neutralizing antibody responses to rH5 A/HK/156 vaccine

Treanor Vaccine 19:1732, 2001

S1 S2 S3 S4 S5 S6 S7

25 ug

45 ug

90 ug

90 ug / 10 ug

Placebo

Ne

utr

aliz

ing

GM

T

Vaccine administered at visit S1 and S3

8

16

32

64

128

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

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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

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Evaluation of priming with an antigenic variant: schematic of study design

Placebo25 ug x 245 ug x 290 ug x 290 ug x 1

+10 ug x 1

UR-98012

A/HK/156/97rH5

147 subjects

1998

DMID 05-0043

rgA/VN/1203/04

90 ug x 1

37 subjects

DMID 04-063

rgA/VN/1203/04

90 ug x 2

103 subjects

Healthy recipients of any rH5

Placebo excluded

CLADE 1CLADE 3

NEJM 354:1343, 2006

Vaccine 19:1732, 2001

H5-PRIMED

H5-NAIVE

2005

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Demographics

H5 NAIVE(DMID 04-063)

n=103

WhiteBlackAsianPacific IslanderMultiracialHispanicFemaleAge (median, range)

84 (82%)11 (11%) 8 (8%) 0 (0%) 0 (0%)13 (13%)55 (54%)38 (18-64)

H5 PRIMED(DMID 05-0043)

n=37

35 (95%) 2 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)23 (62%)42 (33-51)

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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

0

10

20

30

40

50

60

70

80

90

100

D1 D2 PR D1 D2 PR D1 D2 PR D1 D2 PR D1 D2 PR D1 D2 PR D1 D2 PR

Pain Tenderness Feverish Malaise Myalgia Headache Nausea

Pe

rce

nt

Severe

Moderate

Mild

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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

Day 0 Day 28 Day 56 Day 0 Day 28 Day 56

4

8

16

32

64

128

256

4

8

16

32

64

128

256H5 NAIVE H5 PRIMED

DMID 04-063 DMID 05-0043

GM

T H

AI

Ant

ibod

y

GM

T H

AI

Ant

ibod

y

90 mcg 90 mcg 90 mcg

5.1 13.3

27.7

5.2

64.0

48.2

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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

Day 0 Day 28 Day 56 Day 0 Day 28 Day 56

4

8

16

32

64

128

256

4

8

16

32

64

128

256H5 NAIVE H5 PRIMED

DMID 04-063 DMID 05-0043

GM

T N

T A

ntib

ody

GM

T N

T A

ntib

ody

90 mcg 90 mcg 90 mcg

5.18.3

22.9

6.4

93.868.9

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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 1Dose 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

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Effect of the priming dose of recombinant H5 vaccine administered in 1998 on responses to

90 mcg of H5 vaccine in 2006

1998PrimingDose

25 ug x 245 ug x 290 ug x 290 ug/10 ug

N

1278

10

GMT

63.5155.142.148.7

Response*n (%)

7 (58%)6 (86%)5 (63%)7 (70%)

GMT

195.6304.5174.5134.5

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

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Relationship between response in 1998 and response in 2005

Response to rH5 in 1998^

No Yes

GMT

44.3172.5

Response*n (%)

17 (63%)8 (80%)

GMT

67.7226.3

Response*n (%)

22 (81%)10 (100%)

HAI NT

Response to a single dose of H5 vaccine in 2006

N

2710

* 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

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Memory B cell responses of primed subjects in study DMID 05-043

B

B

B

B

J

J

J J

H

H

H

HF F

F

F

Ñ ÑÑ

ÑÉ É É É0

1

2

3

4

5

0 10 20 30 40 50 60

% H

A/T

ota

l Ig

Days

B H5/VN

J H5 HK

H H1 NC

F H3 WY

Ñ H6 TEAL

É KLH

**

Figure 1: Human memory B cell ELISPOT analysis of H5 specificresponses in H5/97 immune subjects revaccinated with H5/04experimental influenza vaccine. ** H5/97 and H5/04 responsessignificantly increased from day 0, P < 0.01.

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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.

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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)

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Acknowledgements

University of Rochester Carrie Nolan Diane O’Brien Mhorag Hay Dave Topham John TreanorDMID Roland Levandowski Linda Lambert Shy Shorer

EMMES Corporation Mark Wolff Ken Wilkins Heather Hill

Southern Research Institute Tom Rowe

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Response of healthy adults to a third dose of subvirion H5 vaccine (study DMID 04-090)

7.5 mcg (n=84)

15 mcg (n=86)

45 mcg (n=78)

90 mcg (n=85)

180 208562804

8

16

32

64

GM

T H

AI a

ntib

ody

Study day

Vaccine

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Alignment of A/Hong Kong/156/97 and A/Vietnam/1203/04 H5 Hemagglutinins

Clade 3 Clade 2 Clade 1´ Clade 1 H3 No. Functional significance

N45* D D D 54† Antigenic site C

S84 N N N 92 Antigenic site E

A86 A A V 93 Antigenic site E

N94 D D D (1) 101 Near Y91; receptor binding?

N124 D S S 129 Antigenic site B

S129 S L L 133a Receptor binding

L138 Q Q Q 142 Antigenic site A

S155 – N155 – 159 Antigenic site B

T156‡ A A T 160 N154 glycosylation motif

L175 L L L (2) 179 Near H179; receptor binding?

T188 T T T (3) 192 Near L190; receptor binding?

K189 R R K 193 Adjacent to receptor binding, antigenic site B

E212 K K R 216 Antigenic site D

S223 – N223§ – (4) 227 Receptor binding

T263 A A T 266 Antigenic site E

325R¶ Absent – – Absent HA cleavage efficiency

EID 11:1515-1521, 2005

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Antigenic relatedness of clade 3 and clade 1 viruses

Table 2. Antigenic analysis of H5N1 isolates from Asia

Virus antigen Clade

Reference ferret antisera*

HK156 NCVD8 HK213 VN1203 VN04xPR8-rg VN78 VN4207 VN14 VN32321

A/Hong Kong/156/97 3 1,280 320 640 80 320 40 80 80 80

A/ck/Vietnam/NCVD8/03 2 640 160 80 80 160 20 <10 160 40

A/Hong Kong/213/03 1´ 1,280 1,280 2,560 80 640 160 160 640 640

A/Vietnam/1203/04 1 40 20 <10 640 320 40 160 80 40

A/Vietnam/1203/04xPR8-rg 1 80 <10 10 640 320 40 160 160 40

A/Vietnam/1194/04 1 40 20 10 640 320 40 160 160 40

A/Vietnam/JP178/04 1 80 10 <10 1,280 320 80 160 160 80

A/Vietnam/JP4207/05 1 160 40 40 1,280 640 80 320 160 80

A/Vietnam/JP14/05 1 20 <10 10 640 80 20 40 80 40

A/Vietnam/JP30321/05 1 40 40 10 <10 40 10 <10 40 160

*Homologous HI titers are in boldface.

EID 11:1515-1521, 2005