Live-attenuated pathogens as vaccine

Grow the pathogen in non-human cells.

Polio vaccine (Sabin): poliovirus grown in monkey cells

Measles vaccine: rubella virus grown in duck embryo cells
TB vaccine (BCG): Mycobacterium bovis grown in media with bile.

Attenuated pathogen induce immunity without causing disease.

May regain virulence through mutation.

May cause disease in immunodeficient patients.

 Mucosal vaccine

Mucosal
IgA

Systemic immunization give poor induction of mucosal IgA.

Systemic IgG Plasma cells induced in lymph nodes do not express 47 integrins and CCR10.

Problems of mucosal vaccine:

Degradation, dilution
Tolerance
 HIV

HIV (R5 strain) infects memory CD4 T cells.

receptor
CD4

gp120/gp41
CCR5
co-receptor

gp120 is covered with glycans. gp120

Recessed CD4 CD4

binding site

gp120 trimer
 Co-receptor binding site on gp120 is formed after CD4 binding.

gp41

gp120

Co-receptor binding CD4 binding site

gp41

CD4

CCR5 CD4 T cell

Co-receptor binding CD4 binding site

gp120 HIV vaccine trial (2003)

Vaxgen
 Antibody therapy

immunization

Isolate individual B cells

Activation of B cells that recognize

different epitopes.

Monoclonal antibody
Polyclonal antibody in serum
 Hybridoma

Immunized mouse
spleen Myeloma (mouse tumor derived from plasma cells)

Ig production No Ig production
Ig secretion apparatus
Short-lived in culture
Immortal

Fusion with PEG (polyethylene glycol)

Hybridoma

Ig production
Ig secretion apparatus
Immortal
 Hybridoma screening
De novo pathway
Plate cells into 96-well plates (inhibited by HAT)
Nucleotide synthesis
Salvage pathway
(require HPRT)
Splenocytes hybridoma myeloma

de novo de novo de novo

Salavage Salavage
HPRT+ HPRT+ HPRT-

HAT inhibits de novo

pathway

hybridoma
Splenocytes

de novo de novo
Most wells contain a single hybridoma. Salavage
Salavage
HPRT+ HPRT+

Screen culture supernatant with ELISA Short-lived

in culture

hybridoma

de novo
Salavage
HPRT+
 Monoclonal antibody in cancer therapy

Monoclonal antibody
Tumor antigen Immunize mice
Against tumor antigen
Cancer cell

ADCC Cancer patient

Macrophages
NK cells Complement
activation

Cancer cell

BCG-CFA lymphoma

mice
Anti-TNP IgG

51
Cr TNP-conjugated lymphoma

Activated M

ADCC
Lysis
 Anti-tumor function of mAb requires FcR.

melanoma 14-17 days

Count lung
metastasis

+/- mAb against

melanoma Ag
UPC10: control mAb
TA99: anti-melanoma mAb

Deletion of inhibitory FcRIIB enhances anti-tumor

activity of mAb.

Human breast
Cancer carcinoma
Follow tumor
development

Herceptin Nude mice

(anti-breast cancer) No thymus Loss of FcRIIB enables
No graft rejection mAb to inhibit tumor
at low dose.
 Humanizing mAb for clinical application
Chimeric antibodies

Tumor antigen hybridoma

Mice

mVH mCH

Mouse Ig gene mVL mCL Mouse mAb

Replace mouse Ig constand region

with human Ig constant region

mVH hCH (C1, C3)

Chimeric Ig gene mVL hCL chimeric mAb

The same antigen binding

specificity

Transfect chimeric Ig gene Human IgG1 and IgG3

into myeloma. for ADCC and complement
activation
 Humanized mAb

Tumor antigen hybridoma

Mice

mVH mCH

Mouse Ig gene mVL mCL Mouse mAb

Introduce CDRs from mouse Ig

gene into human Ig gene

hVH hCH

Humanized Ig gene hVL hCL

Humanized mAb

The same antigen-binding

specificity as mouse mAb

Transfect humanized Ig gene

into myeloma.
 Human mAb
Transgenic mice with human Ig genes.

Human IgH locus

Mouse IgH knockout

Human IgL locus Mouse IgL knockout

breeding

Immunize with hybridoma

tumor antigen

Human IgH, IgL Human mAb

 Phase III clinical trial of trastuzumab (Herceptin)

Humanized mAb against HER2/NEU (epidermal growth factor receptor).

HER2 is over-expressed in 25-30% of breast cancer.

Complete response: disappearance of detectable tumor; 2 mg/kg weekly iv infusion of mAb

Partial response: > 50% decrease in the sum all lesions;

Disease progression: > 25% increase in lesion Kaplan-Meier plot

Phase III clinical study of combined trastuzumab and chemotherapy
Rituximab: anti-CD20 chimeric mAb for B cell lymphoma.
Improving the efficacy of mAb
 Cancer vaccine
Clinical trials in National Cancer Institute (1995-2004)
 Outside of NCI

Objective response rate: 3.8%

 Immune evasion by tumors
Tumors induce Treg cells.

Tumor free mouse Tumor free mouse

Analyze HA-T cells
HA-T cells

CD4+ T cells
specific for
Immunize with HA
tumor antigen
(HA)

Mouse with tumor Analyze HA-T cells

Mouse with tumor
HA-T cells

+Tumor -Tumor
Thy1.1

CD4
Cell #

CFSE

Less cell division in the presence

of tumor
 HA-T cells stimulated in vitro with
HA + APC.

+ tumor HA-T cells from tumor+ mice do not

proliferate and produce IFN-.
- tumor

-tumor +tumor

HA-T cells express FoxP3 and IL-10.

Tumor-induced Treg (TMTreg)

TMTreg suppress the activation of

Th1 effector cells.
-tumor +tumor
TMTreg are induced from CD4+CD25-GITR- T cells
by tumor.

CD4+CD25-GITR- T cells

tumor

Th1

Tumor-induced Treg

Suppress anti-tumor
effector T cells
 Treg number increases in tumor.

Ovarian carcinoma

Treg number in tumor correlates inversely with survival

of patients with ovarian carcinoma.

Low: < 131 per field

Medium: 131-346 per field

High: > 346 per field

 DCs may induce T cell tolerance to tumors.

Tumor cells
infection
Tumor cells
IL-10 IL-10, VEGF inflammation
Lack of inflammation

IL-10R

Tolerogenic DC
STAT3

Down regulation of co-stimulation

Tumor-induced
Treg effector T cells
 Defective DC maturation in cancer patients

DCs from control or patients

Activation of normal T cells

through allogenic MHC

Increased immature DCs in cancer patients

Reduction of immature DCs after
removal of tumor.
 Tumor cells could lose class I MHC to evade CTL.

Tumor cells secrete soluble MICA to evade NK cells and CTL.

Tumor cell NK
NKG2D

Soluble MICA induces endocytosis

and degradation of NKG2D.

NKG2D

CD8 T