# Presentation: 453788

## 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

**Notes:**
This page does not require any explanation.  Just show to audience and read title or something like that

## 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.

**Notes:**
In a previous study, we found that a subvirion inactivated influenza vaccine, administered without an adjuvant, required two doses of 90 mcg each to elicit serum neutralizing antibody in more than half of subjects.
Stephenson and colleagues have previously demonstrated that a third dose of a subvirion inactivated H5N3 A/Singapore/97 vaccine administered 16 months after a two-dose priming schedule resulted in antibody titers that were higher than those achieved after the first two doses. 
If boosting were seen with subsequent doses of H5 vaccine, then a pre-priming strategy might induce better immune responses and would allow a single-dose response to an emerging pandemic virus, a significant logistical advantage
However, such a strategy must take into account the continuing antigenic evolution of the H5 viruses, meaning that the emerging pandemic virus is very likely to be a significant antigenic variant from the virus used for priming
In a previous study, conducted in 1998, we evaluated a subunit H5 influenza vaccine comprised of baculovirus-expressed recombinant H5 hemagglutinin antigen of the A/Hong Kong/156/97 virus, a representative of clade 3 H5 viruses. We took advantage of the existence of these subjects to evaluate the effect of priming with clade 3 HA antigen on responses to a single dose of clade 1 H5 antigen

## Neutralizing antibody responses to rH5 A/HK/156 vaccine

- Treanor Vaccine 19:1732, 2001

## 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

**Notes:**
The primary objective of this study was to determine the ability of a clade 3 H5N1 vaccine, represented by the recombinant A/Hong Kong/156/97 vaccine, to prime for responses to subsequent vaccination with a clade 1 H5N1 vaccine, represented by the A/Vietnam/1203/04 vaccine.
In order to test this hypothesis, we compared the responses of the H5 primed individuals in the 1998 study with the responses of unprimed individuals who received A/Vietnam/1203/04 vaccine
We also determined the safety of revaccination of primed subjects

## 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

**Notes:**
The subjects for this study were recruited from participants in the 1998 study of recombinant H5 vaccine who received any active product, that is, placebo recipients were excluded
All subjects received a single dose of non-adjuvanted, subvirion, reverse-genetically derived A/Vietnam/1203/04 x PR8 vaccine in open-label fashion
Subjects completed a diary card in which they recorded local and systemic adverse events for 7 days after vaccination  In addition, subjects were queried at 28 and 56 days for other adverse experiences
Sera were obtained on days 0, 28, and 56 and tested for the presence of serum hemagglutination-inhibition antibody using horse erythrocytes and for neutralizing antibody at the Southern Research Institute, as previously described.
These results were compared to those seen in H5 naïve subjects after a single dose of 90 mcg of the same vaccine from a separate study, referred to as study DMID 04-063
Side effects were captured and recorded in identical fashion in both studies and antibody was tested in the same laboratory in both studies.  Analysis of assay performance over time showed no change in absolute values over the period when both studies were tested, but the two sets of serum samples were not tested concurrently

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

- UR-98012

**A/HK/156/97**
**rH5**

**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 1**

**CLADE 3**

- NEJM 354:1343, 2006

- Vaccine 19:1732, 2001

- H5-PRIMED

- H5-NAIVE

**2005**

**Notes:**
This diagram shows a schematic of the study design.  H5-primed subjects were derived from our original study, denoted as UR 98012, which was conducted in 1998.  In that study, subjects were randomized to receive placebo or rH5 at either two doses of 25 mcg, two doses of 45 mcg, two doses of 90 mcg, or one dose of 90 mcg followed by one dose of 10 mcg, at intervals of 21, 28, or 42 days.
Subjects who received any active vaccine were considered H5-primed and were recruited to participate in the current study (DMID 05-0043).  All subjects received a single dose of 90 mcg of A/VN/1203/04 vaccine in open label fashion.
Their results are compared to those of H5-naïve individuals who had received two doses of 90 mcg of A/Vietnam/1203/04 vaccine in study DMID 04-063

## Demographics

- H5 NAIVE
- (DMID 04-063)
*n=103*

- White
- Black
- Asian
- Pacific Islander
- Multiracial
- Hispanic
- Female
- Age (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)

**Notes:**
This slide shows the demographics of the populations in both studies.  
There was slightly more ethnic diversity in study 063, conducted in 2004.  The male to female ratio was similar in both subject populations.  
As expected, the participants in 043 were slightly older than the comparison group

## 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

**Notes:**
This slide shows the results of 7-day reactogenicity in three groups:  D1 shows the reactions reported in H5 naïve subjects following the first dose of vaccine in study 04-063, while D2 shows the reactions in these subjects after the second dose administered at 28 days.  RV shows the results in subjects who were revaccinated in the current study.
Adverse events were reported on a 4 point scale, where 0 meant absent, 1 meant mild, or just barely noticeable (shown in green), 2 meant moderate, or resulting in some impairment in normal activities (shown in blue), and 3 meant severe, or that the side effect prevented normal daily activities (shown in red).
Vaccination was generally well tolerated in all three groups, and the majority of reported side effects were mild.  There were no differences in the rates or severity of local or systemic side effects after either dose of vaccine in naïve subjects or on revaccination.
No subjects reported fever, and there were no vaccine-attributable serious adverse events in either group

## 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

**Notes:**
This slide shows the geometric mean titer of serum HAI antibody with 95% confidence intervals, after one or two doses in vaccine naïve subjects and after one dose in revaccinated subjects.
As expected, the primed subjects did not have detectable antibody to the A/VN/04 virus at the beginning of the study, consistent with the high degree of antigenic difference between A/HK/97 and A/VN/04
A single 90 mcg dose resulted in HAI antibody titers that were significantly higher than seen after one dose in vaccine naïve subjects and actually higher than seen after two doses in these subjects.  Because the two groups were not randomized, formal statistical testing has not been done, but the 95% confidence intervals for GMT in the two groups do not overlap
Among primed subjects who received a single dose of vaccine, a modest drop in antibody titer was noted by day 56 after vaccination.

## 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

**Notes:**
This slides show the neutralizing antibody responses in H5-naïve subjects shown in the left and the H5-primed subjects shown in the right, with 95% confidence intervals.
Neutralizing titers after a single 90 mcg dose in H5 primed subjects were also higher than after one or even two doses of 90 mcg in vaccine naïve subjects

## 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

**Notes:**
This slide shows the frequency of serum antibody responses after one or two doses of H5 vaccine in naïve subjects, and after one dose in previously H5 primed subjects.  Note that the definition of an HAI response includes the acheivement of a titer of 1:40 or greater, but that this is not included in the definition of a neutralization response.
The slide also shows the proportion of subjects achieving a titer of 1:40 or greater 28 days after either one or two doses in naïve subjects, or 28 days after one dose in primed subjects.

## Effect of the priming dose of recombinant H5 vaccine administered in 1998 on responses to 90 mcg of H5 vaccine in 2006  

- 1998
- Priming
- Dose
- 25 ug x 2
- 45 ug x 2
- 90 ug x 2
- 90 ug/10 ug

- N
- 12
- 7
- 8
- 10

- GMT
- 63.5
- 155.1
- 42.1
- 48.7

- Response*
- n (%)
- 7 (58%)
- 6 (86%)
- 5 (63%)
- 7 (70%)

- GMT
- 195.6
- 304.5
- 174.5
- 134.5

- 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

**Notes:**
The response to a single dose of A/VN/1203/4 vaccine did not depend on the priming experience within the limits of the small numbers of subjects enrolled in this study.
This slide shows the GMT and response frequency by both HAI and neutralization in revaccinated subjects grouped by the dose of recombinant H5 vaccine received in 1998.
Although the numbers of subjects in each group is small, there was no trend towards better priming with higher doses of vaccine administered in 1998.  This is in contrast to the original results of the study, in which neutralizing responses to A/Hong Kong/97 were highly dependent on the dose of vaccine received.
There was also no relationship between the interval between doses in 1998 (21 days, 28 days, or 42 days) and the response to revaccination (those data are not shown on this slide)

## Relationship between response in 1998 and response in 2005

- Response to rH5 in 1998^
- No
- Yes

- GMT
- 44.3
- 172.5

- Response*
- n (%)
- 17 (63%)
- 8 (80%)

- GMT
- 67.7
- 226.3

- Response*
- n (%)
- 22 (81%)
- 10 (100%)

- HAI

- NT

- Response to a single dose of H5 vaccine in 2006

- N
- 27
- 10

- * 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

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

## 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.

**Notes:**
The vigorous serum hemagglutination-inhibition and neutralizing antibody responses to a single dose of vaccine in this study, compared to the modest responses seen to a single dose in immunologically naïve subjects, support the hypothesis that previous vaccination with a clade 3 vaccine can prime for responses to a clade 1 vaccine.
If these results were verified in future studies, then pre-pandemic vaccination programs could be considered, especially in populations of first responders, health care workers, or the military.  Such populations might be able to be effectively and rapidly vaccinated with a single dose of a vaccine specific for an emerging pandemic
This study only evaluated responses in a small number of subjects, and with a limited number of priming and boosting strategies.  Future studies, which could be conducted using the A/Vietnam/04 clade 1 vaccine and vaccines generated against clade 2 viruses, should evaluate a spectrum of priming and boosting strategies

## 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)

## 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

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

- 180

- 208

- 56

- 28

- 0

- 4

- 8

- 16

- 32

- 64

- GMT HAI antibody

- Study day

- Vaccine

## 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

## 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