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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
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