Professor L.Rudenko

Institute of Experimental Medicine, Institute of Influenza, “Microgen”, PATH

LAIV H7N3

Placebo

n 30 10 Age (years) Mean (SE) 30.1 (1.88) 38.5 (3.05)

Median 26,5 43,5

Sex [n(%)] Female 15 (50) 4 (40) Male 15 (50) 6 (60)

DEMOGRAPHIC CHARACTERISTICS

40 healhty adult volunteers were recruited for this Phase 1 study

Percentage of Subjects with Solicited Local and Systemic Reactions Occurring up to Day 7 of Administration of Dose One

Intent-To-Treat (ITT) Population

LAIV H7N3 (N=30) Placebo (N=10) n (%) 95% CI n (%) 95% CI Any Solicited Local and Systemic 11* (36.7) 19.9 - 56.1 4*(40.0) 12.2 - 73.8 Reaction Any Solicited Local Reaction 2* (6.7) 0.8 - 22.1 1* (10.0) 0.3 - 44.5 Any Solicited Systemic Reaction 11* (36.7) 19.9 - 56.1 4* (40.0) 12.2 - 73.8 Any Concomitant Medications Given 1 (10.0) 0.3 - 44.5 * all mild

Percentage of Subjects with Solicited Local and Systemic Reactions Occurring up to Day 7 of Administration of Dose Two

Intent-To-Treat (ITT) Population

LAIV H7N3 (N=30) Placebo (N=10) n (%) 95% CI n (%) 95% CI Any Solicited Local and Systemic 5* (17.2) 5.8 – 35.8 1* (10.0) 0.3 – 44.5 Reaction Any Solicited Local Reaction 1* (3.4) 0.1 – 17.8 Any Solicited Systemic Reaction 5* (17.2) 5.8 – 37.8 1* (10.0) 0.3 - 44.5 Any Concomitant Medications Given * all mild

Vaccination Preparation*

Virus isolation confirmed by RT–

PCR Total No**

(D1–D7) D1 D2 D3 D4–D7

Vaccination Vaccine (30) 18/30 3/30 1/30 0/30 18/30

Placebo (10) 0/10 0/10 0/10 0/10 0/10

Revaccination Vaccine (29) 15/29 2/29 1/29 0/29 15/29

Placebo (10) 0/10 0/10 0/10 0/10 0/10

*All volunteers were negative before vaccination.

**Total number of positive volunteers: 18 and 15 after vaccination and revaccination,

correspondingly.

H7N3 LAIV VIRUS ISOLATION FROM NASAL SWABS

(RT–PCR data)

H7N3 LAIV VIRUS ISOLATION FROM NASAL SWABS

(data obtained in embryonated chicken eggs)

Vaccination Total No*

(D1–D7)

Vaccination

4/30

0/10

Revaccination

0/29

0/10

*Four total isolates were obtained from 4 volunteers.

Isolate/virus Virus titer at 32C,

log10 EID50/ml

Mean log10 reduction of

virus titer* (EID50/ml) at: Phenotype

32C/40C 32C/26C

#1 8.70 8.70 2.60 ts, ca

#2 9.95 9.95 2.50 ts, ca

#3 9.20 9.20 2.00 ts, ca

#4 8.20 8.20 2.25 ts, ca

MDV** 9.00 9.00 2.50 ts, ca

RESTRICTION OF GROWTH OF H7N3 LAIV ISOLATES FROM VOLUNTEERS AT DIFFERENT TEMPERATURES

(in ovo data)

*From titer at permissive temperature (32C). **A/Len/134/17/57 (H2N2) MDV was used as a control of ts/ca markers.

Confirmation of vaccine genotype of clinical isolates by partial sequencing: All nucleotides associated with attenuating mutations are not changed .

SUMMARY: H7N3 LAIV virus detection and isolation

• 18 out of 30 vaccinees after the first vaccine dose and 15 out of 29 - after the second dose had detectable virus in nasal swabs by RT–PCR

• Vaccine virus could be isolated in eggs from nasal swabs obtained from 4 out of 30 vaccinees after the first vaccine dose

• No placebo recipients were found to be positive for the virus (by RT–PCR and isolation in eggs data). Our data suggest the absence of transmission of vaccine virus from vaccinees to non-vaccinees.

• Vaccine virus isolates retained their attenuating phenotype (cold–adaptation and temperature sensitivity).

• Vaccine virus isolates did not revert and did not lose nucleotides known to confer an attenuating phenotype on the molecular level. Our data suggest high level of vaccine virus genetic stability after replication in humans.

Methods Groups N

Number of Ab

conversions

Ab GMT**

(reverse values)

GMT fold

changes

After 1st

vaccination

(II – I )

After 2nd

vaccination

(III –II )

I*** II*** III*** II/I III/I

HAI (4HAU *) – serum

Ab

LAIV 29 3 (10,3 %) 9 (31,0 %) 2,8 3,5 4,7 1,3 1,7

Placebo 10 0 0 3,3 3,3 3,5 1,0 1,1

HAI (2HAU *) – serum

Ab

LAIV 29 7 (24,1 %) 13 (44,8 %) 3,0 5,5 7,0 1,8 2,3

Placebo 10 0 0 4,1 4,1 4,7 1,0 1,1

Microneutralization test

– serum Ab

LAIV 29 5 (17,2 %) 12 (41,4 %) 4,2 6,2 12,4 1,5 3,0

Placebo 10 0 1 (10,0 %) 4,4 4,4 5,7 1,0 1,3

ELISA – serum IgA LAIV 29 3 (10,3 %) 8 (27,6 %) 11,7 13,9 17,6 1,2 1,5

Placebo 10 0 0 18,4 16,0 18,4 0,9 1,0

ELISA – serum IgG LAIV 29 1 (3,4 %) 3 (10,3 %) 12,0 12,9 13,9 1,1 1,2

Placebo 10 0 0 13,0 14,9 13,9 1,1 1,1

ELISA – local IgA

(nasal swabs)

LAIV 29 12 (41,4 %) 12 (41,4 %) 7,6 14,9 12,6 2,0 1,7

Placebo 10 1 (10,0 %) 1 (10,0 %) 10,6 9,8 9,2 0,9 0,9

Antibody (Ab) immune response in volunteers vaccinated with А (H7N3) LAIV

Ab conversions summary P

ers

on

s w

ith

Ab

co

nv

ersi

on

s

HAI (4 HAU)

HAI (2 HAU)

Local IgA ELISA

Serum IgG ELISA

Serum IgA ELISA

Micro-neut.

TOTAL

9 + 4 + 4 + 1 + 6 24

Basic test Additional information

TOTAL

after 2 vaccinations (Day 56)

Markers

(T-cell

phenotype)

Cell type Cell functions Monoclonal Ab

brand

CD4+IFNγ+* Т-helpers Regulate antibody and

cellular immune responses Beckton Dickinson

CD8+IFNγ+ Cytotoxic T-cells Kill virus-infected cells Beckman Coulter,

Beckton Dickinson

CCR7+CD45R

A–

Central memory

T-cells

Both subsets are long-living

memory T-cells, which

provide faster and stronger

immune response at a

second contact with antigen.

The ways of mobilization are

different.

Beckman Coulter,

Beckton Dickinson

CCR7–

CD45RA–

Effector memory

T-cells

Beckman Coulter,

Beckton Dickinson

Cell markers for flow cytometry studies

*IFNγ – interferon gamma is necessary for revealing virus-activated

cells

Production of virus-specific memory T-cells in volunteers twice vaccinated with А (H7N3) LAIV

Vacci-nation

Group

Number of persons with significant increases in cell levels (≥3 SD of placebo mean value)

CD4 CD8

T cm T em T cm T em

1st

LAIV 5 (17%) 1 (3 %) 1 (3 %) 0

Placebo 0 0 0 0

2nd

LAIV 6 (21 %) 3 (10 %) 6 (21 %) 5 (17 %)

Placebo 0 0 0 0

Cells which produced IFNγ in response to in vitro stimulation with vaccine strain А/17/Mallard/ Netherlands/00/95 (H7N3) were considered as virus-specific cells. PBMC collection was performed at day 28 after vaccination or revaccination. The following cell phenotypes were analyzed: T cm – central memory T cells (CD4+IFNγ+CCR7+CD45RA– and CD8+IFNγ+CCR7+CD45RA–) T em – effector memory T cells (CD4+IFNγ+CCR7–CD45RA– and CD8+IFNγ+CCR7–CD45RA–)

Percent of persons with significant increases in memory T cm and T em cell levels after two-dose vaccination with A (H7N3) LAIV

0

5

10

15

20

25

Tcm Tem Tcm Tem

21

10

21

17

0 0 0 0

% CD4 CD8

– LAIV – Placebo

Individual fold changes in CD4 and CD8 memory T cell levels after two-dose vaccination with A (H7N3) LAIV

–mean FC

– significant increases (≥ 3 Standard Deviations of placebo mean value)

T cm – central memory T cells (CD4+IFNγ+CCR7+CD45RA– and

CD8+IFNγ+CCR7+CD45RA–)

T em – effector memory T cells (CD4+IFNγ+CCR7–CD45RA– and

CD8+IFNγ+CCR7–CD45RA–) CD4 CD8 T cm T em

LAIV LAIV Placebo Placebo

(67,0)

n=6

n=3

LAIV LAIV Placebo Placebo

n=6

n=5

T cm T em

#

Significant antibody responses Significant cellular responses Total

Ab +

Cells HAI

4 HAU

HAI

2 HAU MN

Serum

IgG

ELISA

Serum

IgА

ELISA

Local

IgА

ELISA

Total CD4

T CM

CD4

T EM

CD8

T CM

CD8

T EM Total

1 + + + + + +

2

3 + + + + + + +

4 + + +

5 + + + + + + + +

6 + + + + + + + +

7 + + + + + + + +

8 + + + + + +

9 + + + + + + +

10

11 + + + +

12 + + +

13 + + + + +

14 + + + + + + + +

15 + + + + + + + + + +

16 + + + +

17 + + +

18 + + + + +

19 + + + + + + +

20 + + + + + +

21 + + +

22 + + + +

23 + + + + + + +

24 + + + +

25

26 + + + + + +

27 + + + + +

28 + + + + +

29 + + + + +

Tot

al 9 (31%) 13 (45%) 12 (41%) 3 (10%) 8 (28%) 12 (41%) 24 (83%) 6 (21%) 3 (10%) 9 (31%) 7 (24%) 15 (52%) 26 (90%)

Summary data after two-dose vaccination with A (H7N3) LAIV

31

45 41

10

28

41

83

21

10

31 24

52

90

0

10

20

30

40

50

60

70

80

90

100

Immune responses in volunteers with significant increases after two-dose vaccination with A (H7N3) LAIV

% Antibody responses Cellular

responses

Total

SUMMARY

1. The vaccine was generally well tolerated. No serious adverse events occurred during clinical trial.

2. - The vaccine appeared to replicate well after first and second dose. - At the molecular level, vaccine virus isolates did not revert at

nucleotides known to confer an attenuated phenotype, which confirmed the genetic stability LAIV after replication in humans.

- There was no evidence of vaccine strain transmission from vaccinees to non-vaccinees.

3. 90% of the vaccine-receiving subjects developed an immune response as measured by any assay: HA, MN, ELISA IgA or CD4 and CD8 T-cell production.

4. Two-dose immunization appears more beneficial for immune response than single-dose vaccination

Institute of

Experimental Medicine

Institute of Influenza

“Microgen” PATH

L. Rudenko O.Kiselev A.Nikiforova K. Neuzil

I.Kiseleva M.Erofeeva J.C. Victor

N.Larionova M.Stukova V. Tsvetnitsky

A.Naykhin V. Krivitskaya J. Flores

S. Donina E.Oxankina Tang Yuxiao

G. Petukhova

D. Korenkov

I. Isakova-Sivak

A.Rekstin

E. Grigorieva

LIST OF PARTICIPANTS

Adrian Reber (CDC)

Jackie Katz (CDC)

This study was supported by PATH

ACKNOWLEDGEMENT