1

Giuseppe Del Giudice, MD, PhD Global Head Translational Medicine Novartis Vaccines, Siena, Italy WHO, Geneva, 5 May 2014

Influenza-specific CD4+ICOS1+IL-21+ T cells as predictors of specific antibody responses

MF59® induces priming of Th0-Th1-type memory cells in adults (H5N1) and in young children (seasonal TIV)

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 2

Galli et al, P

roc Natl A

cad Sci U

SA 2009

submitted

Adults vaccinated with MF59®-adjuvanted H5N1 exhibit early expansion of IL-2+ and TNF-α+ CD4 cells

Identical quality of response is observed in 6-35 months-old children vaccinated with MF59®-adjuvanted TIV

No Th2-type responses were ever detectable in subjects receiving MF59®-adjuvanted flu vaccine at any age

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Figure 2 | Target population for vaccines in the twenty-first century. a | The most important vaccines for each age group are reported. b | Special target groups for vaccination in the twenty-first century. The most important vaccines for each target group are reported. The lists of vaccines reported are indicative

and they are not intended to be exhaustive. %�|FKHHKEKNG, %NQUVTKFKWO�FKHHKEKNG; '�|EQNK, 'UEJGTKEJKC�EQNK��'8����GPVGTQXKTWU|����*�|KPHNWGP\CG, *CGOQRJKNWU�KPHNWGP\CG; -�|RPGWOQPKCG,�-NGDUKGNNC RPGWOQPKCG; 2�|CGTWIKPQUC,�2UGWFQOQPCU�CGTWIKPQUC;�5�|CWTGWU, 5VCRJ[NQEQEEWU�CWTGWU; SARS, severe acute respiratory syndrome.

(discussed in more detail below). However, an immunization schedule should be established for adults and should include vaccination against emerging strains of influenza virus and periodic boosts to maintain immunity to diphtheria, tetanus, pertussis, hepatitis B virus, respiratory syn-cytial virus and meningococcus (groups A, B, C, Y and W135).

Elderly individuals. An important target group for the development of new vac-cines will be the elderly, a demographic group for which vaccines have not been fully exploited yet. This population has

several new medical needs. First, the aging immune system makes them more vulner-able to many infections against which they were previously immune. Susceptibility to infections, such as influenza virus, meningococcus, group B streptococcus, pneumococcus, respiratory syncytial virus and varicella zoster virus, becomes higher in this age group; as such, they will need more-frequent booster vaccinations, in many cases with vaccines potentiated by adjuvants that are specifically designed to stimulate the aging immune system to respond better to vaccination. One exam-ple of a licensed adjuvant that has been

efficiently used to boost immune responses in the elderly is the oil-in-water emulsion MF59 (Novartis), which is licensed for use as an adjuvanted seasonal influenza vac-cine in European countries and in several other countries and has been shown to reduce hospitalization in the elderly11. Other licensed or experimental adjuvants are good candidates for novel vaccines for elderly patients in the future (TABLE 1).

The second medical need for the elderly is immunity to antibiotic-resistant bacteria that are acquired during hospitalization — these infections are most frequent in this age group. Therefore, it would be useful to

PERSPECT IVES

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 867

© 2012 Macmillan Publishers Limited. All rights reserved

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Figure 2 | Target population for vaccines in the twenty-first century. a | The most important vaccines for each age group are reported. b | Special target groups for vaccination in the twenty-first century. The most important vaccines for each target group are reported. The lists of vaccines reported are indicative

and they are not intended to be exhaustive. %�|FKHHKEKNG, %NQUVTKFKWO�FKHHKEKNG; '�|EQNK, 'UEJGTKEJKC�EQNK��'8����GPVGTQXKTWU|����*�|KPHNWGP\CG, *CGOQRJKNWU�KPHNWGP\CG; -�|RPGWOQPKCG,�-NGDUKGNNC RPGWOQPKCG; 2�|CGTWIKPQUC,�2UGWFQOQPCU�CGTWIKPQUC;�5�|CWTGWU, 5VCRJ[NQEQEEWU�CWTGWU; SARS, severe acute respiratory syndrome.

(discussed in more detail below). However, an immunization schedule should be established for adults and should include vaccination against emerging strains of influenza virus and periodic boosts to maintain immunity to diphtheria, tetanus, pertussis, hepatitis B virus, respiratory syn-cytial virus and meningococcus (groups A, B, C, Y and W135).

Elderly individuals. An important target group for the development of new vac-cines will be the elderly, a demographic group for which vaccines have not been fully exploited yet. This population has

several new medical needs. First, the aging immune system makes them more vulner-able to many infections against which they were previously immune. Susceptibility to infections, such as influenza virus, meningococcus, group B streptococcus, pneumococcus, respiratory syncytial virus and varicella zoster virus, becomes higher in this age group; as such, they will need more-frequent booster vaccinations, in many cases with vaccines potentiated by adjuvants that are specifically designed to stimulate the aging immune system to respond better to vaccination. One exam-ple of a licensed adjuvant that has been

efficiently used to boost immune responses in the elderly is the oil-in-water emulsion MF59 (Novartis), which is licensed for use as an adjuvanted seasonal influenza vac-cine in European countries and in several other countries and has been shown to reduce hospitalization in the elderly11. Other licensed or experimental adjuvants are good candidates for novel vaccines for elderly patients in the future (TABLE 1).

The second medical need for the elderly is immunity to antibiotic-resistant bacteria that are acquired during hospitalization — these infections are most frequent in this age group. Therefore, it would be useful to

PERSPECT IVES

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 867

© 2012 Macmillan Publishers Limited. All rights reserved

The early CD4+ T cell response to H5N1 predicts the antibody response at late boost (6 months) and persistence

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 3

Galli et al, Proc Natl Acad Sci USA 2009

Which is the function of these cells?

T follicular helper (Tfh) cells

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 4

TFH Ag-primed B cell

McHeyzer-Williams et al, Nature Rev Immunol 12: 24-34, 2011

Which is the T cell population that better predicts antibody response to vaccination?

§  Are antigen specific CD4+IL-21+ cells detectable in human peripheral blood ?

§  Is their frequency modulated by vaccination ?

§  Is there a correlation between frequency of antigen-specific CD4+IL-21+ cells and antibody response to vaccination

§  Do blood IL-21+ T cells help B cell differentiation in vitro ?

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 5

H5N1 A/Vietnam/1194/2004 (7.5 µg) H1N1 A/Solomon Islands/3/2006 (15µg) H3N2 A/Wisconsin/67/2005 (15µg) B/Malaysia/2506/2004 (15µg)

ClinicalTrials.gov NCT00620815

As expected, two vaccinations are required to increase HI antibody titers to H5N1, one for H3N2

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 6

H5N1 H3N2

Pre

Post 1

Post 2

0

200

400

600

800 ***

HI t

iters

PrePost

10

500

1000

1500***

HI t

iters

Monovalent

Tetravalent

2

One vaccination is sufficient to induce the expansion of total cytokine+ CD4+ T cells specific for either H5N1 or H3N2

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 7

4% 14%

28%

54% 6%

18%

30%

46%

CD4+ CTK+

Pre

Post 1

Post 2

Pre

Post 1

0

500

1000

1500

20002000250030003500

*** ***

CD

4+ CTK

+ /106 to

tal C

D4

(n°

of c

ells

)

3x!

4x!

2x!1x!

H5N1 H3N2

One vaccination is sufficient to induce the expansion of IL-21+ CD4+ T cells specific for either H5N1 or H3N2

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 8

18%

37% 30%

15% 25%

42%

23% 10%

CD4+ IL-21+

Pre

Post 1

Post 2

Pre

Post 1

0

100

200

300

400

500500

1000** ****

CD

4+ IL-2

1+ /106

tota

l CD

4(n

° of

cel

ls)

H5N1 H3N2

3x!

4x!

2x!1x!

Blood influenza-specific IL-21+ CD4+ T cells accumulate in the CXCR5- ICOS1+ T cell population

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 9

Spensieri et al Proc Natl Acad Sci USA 110: 14330, 2013

H3N2 CD4+IL21+ !

Post 1 !H5N1 CD4+IL21+ !

Post 1!

CXCR5 − ICOS1−!

CXCR5+ ICOS1−!

CXCR5+ ICOS1+!

CXCR5− ICOS1+!

64% 4%

1%

31%

TFH Ag-primed B cell

77%

7%

1% 15%

Levels of post-2 antibodies are predicted by the frequency of CXCR5-ICOS1+CD4+ T cells which exert cognate help for Ag-specific Ab production in vitro

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 10

Spensieri et al Proc Natl Acad Sci USA 110: 14330, 2013

Antigen-specific in vitro helper assay

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 11

CD4 enrichment

Sorting

PBMC +seasonal FLU antigen (H3N2 Brisbane)

18 h

+ autologus B cells

+ H3N2 10 days

Ig quantification as read-out of the help activity

Buffy coats from healthy donors

T resting for 10 days

CD4+

CXCR5

ICO

S1

Peripheral blood CXCR5-ICOS1+ CD4+ T cells help autologous B cells to secrete antibodies in vitro

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 12

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

100

200

300

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

20

40

60

80

sam

ple

lostTo

tal I

gG(M

FI)

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

2

4

6

8

10Total IgG sbj 1 Total IgG sbj 2 Total IgG sbj 3

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

1

2

3

4

5

sam

ple

lost

Tota

l IgM

(MFI

)

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

1

2

3

4

5

IC+C

X-

IC+C

X5+IC

-CX-

B Only

0

50

100

150

200Total IgM sbj 1 Total IgM sbj 2 Total IgM sbj 3

3

CXCR5-ICOS1+CD4+ T cells exert cognate help for antigen-specific antibody production in vitro

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 13

0

1

2

3

H3N

2 Ig

G(M

FI)

0.0

0.5

1.0

1.5

H3N

2 Ig

M(M

FI)

0.0

0.2

0.4

0.6

0.8

1.0

DT

CR

M Ig

G(M

FI)

H3N2- IgG sbj 3 H3N2- IgM sbj 3

DT (CRM 197)- IgG sbj 3

Spensieri et al Proc Natl Acad Sci USA 110: 14330, 2013

T follicular helper cells (Tfh) and affinity maturation of high affinity germinal center B cells

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Tangye et al, Nat Rev Immunol 13: 412, 2013

MF59®-driven enhancement of the avidity of HA-specific antibodies, which are directed against the HA1 moiety

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 15

Khurana S et al. Sci Transl Med 2011; 3: 85ra48-85ra48

Khurana S et al. PLoS ONE 2014; 9: e95496

In conclusion

§  ICOS1+IL-21+ T cells found in the peripheral blood may represent Tfh cells that have modulated the expression of the CXCR5 after antigen re-stimulation

§  These cells retain a strong antigen-specific helper function for the production of high avidity antibodies, especially after immunization with MF59®-adjuvanted vaccines

§  Their measurement at early time points post-vaccination predicts the raise of HI antibodies at later time points

§  Extend the analysis to other vaccines to understand the ability of this early parameter to predict persistence of Abs responses, generation of memory B cells, affinity maturation, and evaluate the impact of antigen formulation and age on the development of this T cell subset

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 16

Acknowledgements

§ Novartis Vaccines • Flora Castellino, Oretta Finco & Co (Translational Medicine) • Clinical Development

§ Hana Golding & Co (FDA/CBER)

§ Karl Nicholson & Co (University of Leicester, UK)

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 17