Strategies for Vaccine Design

Jay A. Berzofsky, M.D., Ph.D.

Chief, Vaccine Branch, CCR, NCI

Preparing for Biothreats: Emerging and Re-emerging Infectious Diseases

Boston University, Boston, MA, December 14, 2005

For viruses causing acute, self-limited infections, the most widely used strategy is to mimic the natural infection with an attenuated, inactivated, or subunit vaccine. However, for viruses causing chronic infection, such as HIV or hepatitis C virus, or for cancer, the natural disease does not induce sufficient immunity to eradicate the infection or tumor. A vaccine must elicit better immunity than the virus or cancer itself.

Rationale

Vaccine strategies to elicit cytotoxic T lymphocytes (CTL) need to focus on

several properties:• Quantity of CTL.• Avidity of CTL ==> Greater efficacy at clearing

virus or killing tumor• Longevity or memory of CTL.

Strategies based on use of:CytokinesCostimulatory moleculesInhibitors of negative regulationEnhanced epitopes--sequences modified to increase

binding to MHC moleculesTargeting of vaccines to mucosal sites

CD8 Cytotoxic T cell

Antibody

Cell surface protein

Endogenous protein

TransporterEndoplasmic Reticulum

Golgi

MHC Class I

+

CD8 Cytotoxic T cells recognize endogenous antigenic proteins even if not

expressed intact on the cell surface

+

From DH Fremont, M. Matsumura, EA Stura, PA Peterson, & IA Wilson. Science 257: 919-926, 1992

Sendai Virus Peptide Bound to H-2Kb

Strategy: Epitope Enhancement by Sequence Modification to Increase Peptide Affinity for the MHC Molecule

Peptide Fragments of Viral Proteins Bind Specifically in the Grooveof Major Histocompatibility Molecules such as HLA-A, B, C

IL-2 & IL-15: SHARED FUNCTIONS IN IMMUNE SYSTEM (ADOPTIVE IMMUNITY)

Epithelium

BM Stroma

Macrophage DC

IL-15

B CTL CD4

ProliferationIgA

EffectorFunction

CD8

Proliferation

ActivatedCD4

IL-2

IL-2 & IL-15: DISTINCT FUNCTIONS

IL-15 IL-2

Mast cell proliferation

NK cell development

ActivatedT cells

MemoryT cells Antigen-

InducedCell DeathMaintenance

IL-15 expression by a vaccine vector induced longer-lived memory CD8+ CTL: IFN-gamma-producing cells

% o

f I

FN

-gam

ma

pro

du

cin

g

CD

8+ T

cel

ls

3 wks

2 month

s

4 month

s

6 month

s

8 month

s

14 month

s

Time after Booster Immunization

Boo

sted

0

1

2

3

4

5UnimmunizedvPE16vPE16/VV-IL-15vPE16/VV-IL-2

Oh et al., PNAS 2003

Explained by 1. Higher IL-15R expression2. Greater homeostatic proliferation

0

20

40

60

80

100

120

10 1 0.1

0.01

0.00

1

0.00

01

1E-0

5

Control

Target cells pulsed with [P18-I10], M

% o

f m

ax

imu

m ly

sis

vPE16

vPE16/IL-15

Immunization with antigen + IL-15 induces higher avidity memory CD8+ CTL

2 months after immunizationOh et al., PNAS 2004

High Avidity CTL Clear Virus Infection in SCID MiceMore Effectively Than Low Avidity CTL

Alexander-Miller et al., PNAS 1996

Improved viral clearance by high avidity CTL

Low avidityCTL

HighavidityCTL

Derby et al., J. Immunol. 2001

HighAvidity

CTL

IL-15

IL-15 in vaccine

IncreasedIL-15R

APC

CD8 T cells

Signal 2Signal 1

Strong Signal 2

Increased survivalandHomeostatic proliferation

Selection

Selection at population level

Induction

Increased CD8

Higherfunctional avidity

Induction at individual cell level

Role of IL-15 and costimulation in CTL Avidity Maturation

Oh et al. J. Immunol. , 2003; and Oh et al. PNAS, 2004

CD8 Cytotoxic T cell

MHC Class I

+

CD28

MHC Class II

Dendritic Cell

CD4+ T-cell Help for CD8+ CTL Mediated Through Activation of Dendritic Cell

CD40

CD4+ Helper T cell

CD40L

TCR

TCR

B7 costimulator

IL-15IL-12

IL-15 with vaccine

Immunotherapy of cancer or HIV is still not an established modality of treatment.

WHY?

Immune suppression by tumor or HIV

Immune evasionImmune suppressionby immune cells

M2 macrophages or tumor associated macrophages (TAM)

CD4+CD25+ T regulatory cells (Treg)

Myeloid suppressor cells (MSC)

Natural Killer (NK) T cells

Th3

Tr1

CD4

TGF- MHC class II-restricted

IL-10 (& TGF-MHC class II-restricted

TCR

Regulatory/Suppressor T cells

TReg

CD25 (IL-2R)

Ts

CD8? Qa-1-restricted

TCR

IFN-IL-4, IL-13CD1d-restrictedNKTNK1.1

Contact inhibition MHC class II-restricted

CD4

CD1d-restrictedCD4+NKT

CTL

IL-4RIL-13R1

CD11b+ Gr1+

STAT6

NKT cells and IL-13 suppress CTL tumor immune surveillance though the IL-4R-STAT6 pathway

to induce TGF- production by CD11b+Gr-1+ cells

Tumor cells

IL-13

TGF-

tumor lysis

APC

CD1dglycolipid

suppression of CTL activation

Terabe et al., Nat Immunol, 2000., Terabe et al., J Exp Med, 2003.

0

50

100

150

200

>250

Num

ber o

f Nod

ules

BALB

/cUn

treat

edan

ti-CD

4an

ti-CD

8an

ti-CD

4,CD

8Ra

tIgG

CD1-KO

Absence of CD1d-restricted NKT cells unmasks CD8+ Cell-dependent immunosurveillance and not improved

by deletion of CD4+ Cells

Park et al., Internat. J. Cancer, 2005.

Wild-type BALB/c CD1- KO

Lungs stained with India inkfor contrast

0

50

100

150

200

250

Prevention of lung tumor metastases of CT26 by anti-TGF- antibody

contr

ol

contr

ol

mAb(13C4)

anti-T

GF-

(1D11)

Num

ber o

f nod

ules

/lun

g

Terabe et al., J Exp Med , 2003

CD1d-restrictedCD4+NKT

CTL

IL-4RIL-13R1

CD11b+ Gr1+

STAT6

Tumor cells

IL-13

TGF-

tumor lysis

APC

CD1dglycolipid

suppression of CTL activation

• Even in a non-regressor tumor model, this new immunoregulatory circuit plays a role in suppressing CD8+ CTL-mediated immunosurveillance.

• Abrogation of this pathway unmasks otherwise inapparent spontaneous natural tumor immunosurveillance and reduces tumor growth even in the absence of any vaccine or other immunotherapy.

Conclusions: CT26 model

Enhancement of Vaccine Elicited CD8+ CTL response by in vivo treatment with an IL-13 inhibitor

% s

pec

ific

lys

is

0

20

40

60

80

100

1:1 3:1 6:1 12:1 25:1 50:1 100:1

+anti-CD4

+IL-13 inhibitor

Peptide-immunized

E:T ratio

Immunization: PCLUS6.1-P18 +GM-CSF+CD40L

Ahlers et al. PNAS, Oct. 2002

0 10 20 30 40 50

% SPECIFIC LYSIS AT 50:1

0 10 20 30 40 50

LAMINA PROPRIA

PEYER'S PATCH

SPLEEN

Intrarectal Immunization

Subcutaneous Immunization

- peptide

+ peptide

TARGET CELLS

INTRARECTAL IMMUNIZATION WITH SYNTHETIC PEPTIDEHIV VACCINE INDUCES BOTH SYSTEMIC AND MUCOSAL CTL

Belyakov et al. PNAS 1998

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09V

AC

CIN

IA V

IRU

S T

ITE

R (

log

10

)

PROTECTION INDUCED BY MUCOSAL IMMUNIZATION WITHHIV PEPTIDE IS DEPENDENT ON CD8 POSITIVE T CELLS

IMMUNIZATION

TREATMENT

NONE IR IR

NONE NONE ANTI-CD8

Belyakov et al., JCI 1998

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09V

AC

CIN

IA V

IRU

S T

ITE

R (

log

10

)

MUCOSAL IMMUNIZATION WITH HIV-1 PEPTIDE INDUCESPROTECTIVE IMMUNITY AGAINST INTRARECTAL RECOMBINANT

HIV-VACCINIA CHALLENGE

IMMUNIZATION NONE SC IR

CHALLENGE vPE16 vPE16 vPE16

Belyakov et al., JCI 1998

Berzofsky et al., Nature Reviews Immunology 2001;Belyakov et al., Nature Medicine, 2001.

Belyakov IM, Berzofsky JA, Immunity, 2004, Vol.20, 247

Comparison of Mucosal Peptide or Poxviral Vaccine with Peptide-Prime, Poxviral Boost

in Rhesus Macaques

Group Peptide Vaccine

NYVAC

Poxviral vector vaccine

1 + _

2 + +

3 _ +

4 _ _

Prime Boost

All animals received GM-CSF, IL-12, CpG, and LT(R192G)In DOTAP with or without peptide at the times of peptide priming.

Peptide-Prime/NYVAC Boost Mucosal Vaccine Delays Acute Peak Viremia, Suggesting Delayed Dissemination fromMucosal Site of Transmission

1E+02

1E+03

1E+04

1E+05

1E+06

mR

NA

co

pie

s

10 20 30 40 50Days After Viral Challenge

Cytokine + CpG ODN alone

Recombinant NYVAC

Peptide Prime- NYVAC boost

Peptide Vaccine

Belyakov et al., Blood, in press 2006

0

1

2

3

4

5

6

7

0 1 2 3 4

Vir

al L

oad

(lo

g10

)

% CL10-Tetramer+CD8+

Strong inverse correlation between vaccine-induced Tetramer+ CD8+ T cells in colon before challenge and viral load in blood

after challenge

r = -0.84; p<0.00001

Belyakov et al., Blood, in press 2006

0 0.5 1 1.5 2

% CL10-Tetramer+CD8+

Vir

al L

oad

(lo

g10

)7-

6-

5-

4-

3-

2-

1-

0-

No correlation between vaccine-induced Tetramer-binding T cells in blood before challenge and viral load in blood after challenge

r = 0.007, p > 0.7

Belyakov et al., Blood, in press 2006

0 20 40 602

3

4

5

6

7

0 20 40 602

3

4

5

6

7

0 20 40 60

Vir

al L

oa

d (

log 1

0)

2

3

4

5

6

7

0 20 40 602

3

4

5

6

7

r= -0.61(p=0.03) r= -0.70 (p=0.006)

r= -0.78 (p=0.001) r= -0.82 (p<0.001)

Targets with 10 M peptide Targets with 1 M peptide

Targets with 0.1 M peptide Targets with 0.01 M peptide

% Specific Lysis

Vir

al l

oad

(lo

g10

)

Inverse Relationship with Viral Load is greater for high avidity CTLDay 17 after Challenge

Low avidity

High avidity

Belyakov et al., Blood, in press 2006

Conclusions• Natural transmission of many viruses is through mucosal

surfaces. High avidity mucosal CTL may prevent or reduce dissemination from this site and abort the infection.

• Mucosal immunization is most effective at inducing mucosal CTL.

• CTL avidity is critical in clearing virus infections.• CTL avidity & longevity can be increased by immunization

in the presence of IL-15 or costimulatory molecules.• Immunogenicity can be increased by

– Use of appropriate cytokines– Epitope enhancement by sequence modification to increase

binding to MHC molecules– Blockade of negative regulatory pathways, including NKT cells

and T reg cells, IL-13 and TGF-beta