Transcript Document

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
Rationale
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.
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:
Cytokines
Costimulatory molecules
Inhibitors of negative regulation
Enhanced epitopes--sequences modified to increase
binding to MHC molecules
Targeting of vaccines to mucosal sites
CD8 + Cytotoxic T cells recognize
endogenous antigenic proteins even if not
expressed intact on the cell surface
+
CD8 Cytotoxic T cell
Antibody
Cell surface protein
MHC
Class I
Endogenous
protein
Golgi
ro
te
as
e
Transporter
P
Endoplasmic
Reticulum
Peptide Fragments of Viral Proteins Bind Specifically in the Groove
of Major Histocompatibility Molecules such as HLA-A, B, C
Sendai Virus Peptide Bound to H-2Kb
From DH Fremont, M. Matsumura, EA Stura, PA Peterson,
& IA Wilson. Science 257: 919-926, 1992
Strategy: Epitope Enhancement by Sequence Modification to
Increase Peptide Affinity for the MHC Molecule
IL-2 & IL-15: SHARED FUNCTIONS IN IMMUNE SYSTEM
(ADOPTIVE IMMUNITY)
Macrophage
Epithelium
DC
BM Stroma
IL-2
IL-15
B
CTL
Proliferation Effector
Function
IgA
CD4
CD8
Proliferation
Activated
CD4
IL-2 & IL-15: DISTINCT FUNCTIONS
IL-15
Mast cell
proliferation
NK cell
development
IL-2
Activated
T cells
Memory
T cells
Maintenance
AntigenInduced
Cell Death
5
Boosted
% of IFN-gamma producing
CD8+ T cells
IL-15 expression by a vaccine vector induced longer-lived
memory CD8+ CTL: IFN-gamma-producing cells
4
Unimmunized
vPE16
vPE16/VV-IL-15
vPE16/VV-IL-2
3
2
1
0
Time after Booster Immunization
Oh et al., PNAS 2003
Explained by 1. Higher IL-15Ra expression
2. Greater homeostatic proliferation
% of maximum lysis
Immunization with antigen + IL-15 induces higher
avidity memory CD8+ CTL
120
vPE16
vPE16/IL-15
100
80
60
40
20
0
Target cells pulsed with [P18-I10], mM
Oh et al., PNAS 2004
2 months after immunization
High Avidity CTL Clear Virus Infection in SCID Mice
More Effectively Than Low Avidity CTL
Improved viral clearance by high
avidity CTL
High
avidity
CTL
Derby et al., J. Immunol. 2001
Low
avidity
CTL
Role of IL-15 and costimulation in CTL Avidity Maturation
APC
Signal 1
Signal 2
IL-15 in vaccine
CD8 T cells
High
Avidity
CTL
Selection
Increased survival
and
Homeostatic proliferation
Selection at
population level
IL-15
Increased
IL-15Ra
Induction
Increased
CD8 ab
Higher
functional avidity
Induction at
individual cell level
Oh et al. J. Immunol. , 2003; and Oh et al. PNAS, 2004
CD4+ T-cell Help for CD8+ CTL Mediated Through Activation of Dendritic Cell
IL-15 with vaccine
+
CD8 Cytotoxic T cell
CD4+ Helper T cell
TCR
CD28
IL-15
IL-12
MHC
Class I
CD40L
TCR
CD40
B7 costimulator
MHC
Class II
Dendritic Cell
Immunotherapy of cancer or HIV is still not an
established modality of treatment.
WHY?
Immune suppression
by tumor or HIV
Immune suppression
by immune cells
Myeloid suppressor cells (MSC)
CD4+CD25+ T regulatory cells (Treg)
Immune evasion
M2 macrophages
or tumor associated macrophages (TAM)
Natural Killer (NK) T cells
Regulatory/Suppressor T cells
CD4
Contact inhibition
TReg
MHC class II-restricted
CD25 (IL-2Ra)
Th3
abTCR
Tr1
TGF-b
MHC class II-restricted
IL-10 (& TGF-b) MHC class II-restricted
CD4
NKT
IFN-g, IL-4, IL-13
CD1d-restricted
?
Qa-1-restricted
NK1.1
CD8
Ts
abTCR
NKT cells and IL-13 suppress CTL tumor immune
surveillance though the IL-4R-STAT6 pathway
to induce TGF-b production by CD11b+Gr-1+ cells
CTL
Tumor cells
tumor lysis
suppression of CTL activation
APC
glycolipid
TGF-b
IL-13
CD1d
CD1d-restricted
CD4+NKT
IL-13Ra1
IL-4Ra
STAT6
CD11b+ Gr1+
Terabe et al., Nat Immunol, 2000., Terabe et al., J Exp Med, 2003.
Absence of CD1d-restricted NKT cells unmasks CD8+ Celldependent immunosurveillance and not improved
by deletion of CD4+ Cells
Number of Nodules
>250
Wild-type BALB/c CD1- KO
200
150
100
Lungs stained with India ink
for contrast
50
0
CD1-KO
Park et al., Internat. J. Cancer, 2005.
Prevention of lung tumor metastases of CT26
by anti-TGF-b antibody
Number of nodules/lung
250
200
150
100
50
0
Terabe et al., J Exp Med , 2003
Conclusions: CT26 model
CTL
Tumor cells
tumor lysis
suppression of CTL activation
APC
glycolipid
CD1d
CD1d-restricted
CD4+NKT
TGF-b
IL-13
IL-13Ra1
IL-4Ra
STAT6
CD11b+ Gr1+
• 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.
% specific lysis
Enhancement of Vaccine Elicited CD8+ CTL response by
in vivo treatment with an IL-13 inhibitor
Immunization: PCLUS6.1-P18 +GM-CSF+CD40L
100
Peptide-im munized
+IL-13 inhibitor
80
+anti-CD4
60
40
20
0
1:1
Ahlers et al.
3:1
6:1 12:1 25:1 50:1 100:1
E:T ratio
PNAS, Oct. 2002
INTRARECTAL IMMUNIZATION WITH SYNTHETIC PEPTIDE
HIV VACCINE INDUCES BOTH SYSTEMIC AND MUCOSAL CTL
Intrarectal
Immunization
Subcutaneous
Immunization
SPLEEN
TARGET CELLS
PEYER'S PATCH
+ peptide
- peptide
LAMINA
PROPRIA
0
10
20
30
40
500
10
20
% SPECIFIC LYSIS AT 50:1
Belyakov et al. PNAS 1998
30
40
50
PROTECTION INDUCED BY MUCOSAL IMMUNIZATION WITH
HIV PEPTIDE IS DEPENDENT ON CD8 POSITIVE T CELLS
VACCINIA VIRUS TITER (log10)
1E+09
1E+08
1E+07
1E+06
1E+05
1E+04
IMMUNIZATION
NONE
IR
TREATMENT
NONE
NONE
Belyakov et al., JCI 1998
IR
ANTI-CD8
MUCOSAL IMMUNIZATION WITH HIV-1 PEPTIDE INDUCES
PROTECTIVE IMMUNITY AGAINST INTRARECTAL RECOMBINANT
HIV-VACCINIA CHALLENGE
VACCINIA VIRUS TITER (log10)
1E+09
1E+08
1E+07
1E+06
1E+05
1E+04
IMMUNIZATION
NONE
CHALLENGE
vPE16
Belyakov et al., JCI 1998
SC
vPE16
IR
vPE16
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
Prime
Group
Peptide
Vaccine
Boost
NYVAC
Poxviral vector
vaccine
1
2
3
4
+
+
_
_
_
+
+
_
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 from
Mucosal Site of Transmission
mRNA copies
1E+06
1E+05
Peptide Vaccine
Peptide Prime- NYVAC boost
1E+04
Recombinant NYVAC
1E+03
1E+02
Cytokine + CpG ODN alone
10 20 30 40 50
Days After Viral Challenge
Belyakov et al., Blood, in press 2006
Strong inverse correlation between vaccine-induced Tetramer+
CD8+ T cells in colon before challenge and viral load in blood
after challenge
Viral Load (log10)
7
6
r = -0.84; p<0.00001
5
4
3
2
1
0
0
1
2
3
% CL10-Tetramer+CD8+
Belyakov et al., Blood, in press 2006
4
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
Viral Load (log10)
7-
6543210-
0
0.5
1
1.5
% CL10-Tetramer+CD8+
Belyakov et al., Blood, in press 2006
2
Inverse Relationship with Viral Load is greater for high avidity CTL
Day 17 after Challenge
Targets with 1 mM peptide
Targets with 10 mM peptide
7
7
Viral load (log10)
Viral Load (log10)
Low avidity
r= -0.70 (p=0.006)
r= -0.61(p=0.03)
6
6
5
5
4
4
3
3
2
2
0
20
40
0
60
40
60
Targets with 0.01 mM peptide
Targets with 0.1 mM peptide
7
20
7
r= -0.78 (p=0.001)
6
6
5
5
4
4
3
3
2
r= -0.82 (p<0.001)
High avidity
2
0
20
40
Belyakov et al., Blood, in press 2006
60
0
% Specific Lysis
20
40
60
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