vector mediated immunoprophylaxis dr. bruce schnepp · image by the national cancer institute 11...

Vector Mediated ImmunoprophylaxisDr. Bruce Schnepp

1The screen versions of these slides have full details of copyright and acknowledgements

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Bruce Schnepp, Ph.D.

Senior Research Scientist

Children’s Hospital of Philadelphia

Vector Mediated Immunoprophylaxis

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Lecture outline

1. Introduction to HIV and past vaccine strategies

2. New vaccine strategy: antibody gene transfer

3. Introduce gene transfer viral vector

4. Case study: antibody gene transfer for HIV

5. Limitations and other applications for antibody gene transfer

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Morbidity Mortality Weekly Report Centers for Disease Control



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THE VAST MAJORITY OF

PEOPLE LIVING WITH HIV

ARE IN LOW- AND MIDDLE-

INCOME COUNTRIES,

PARTICULARLY IN SUB-

SAHARAN AFRICA

THE VAST MAJORITY OF

PEOPLE LIVING WITH HIV

ARE IN LOW- AND MIDDLE-

INCOME COUNTRIES,

PARTICULARLY IN SUB-

SAHARAN AFRICA

The global HIV/AIDS epidemic

www.aids.gov

19 MILLION DO NOT KNOW THAT

THEY HAVE THE VIRUS.

35 MILLION PEOPLE WORLDWIDE

ARE CURRENTLY

LIVING WITH

HIV/AIDS.

3.2 MILLION CHILDREN WORLDWIDE ARE LIVING WITH HIV.

MOST OF THESE CHILDREN WERE

INFECTED BY THEIR HIV-POSITIVE

MOTHERS DURING PREGNANCY,

CHILDBIRTH OR BREASTFEEDING

19 MILLION DO NOT KNOW THAT

THEY HAVE THE VIRUS.

35 MILLION PEOPLE WORLDWIDE

ARE CURRENTLY

LIVING WITH

HIV/AIDS.

3.2 MILLION CHILDREN WORLDWIDE ARE LIVING WITH HIV.

MOST OF THESE CHILDREN WERE

INFECTED BY THEIR HIV-POSITIVE

MOTHERS DURING PREGNANCY,

CHILDBIRTH OR BREASTFEEDING

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- Dr. Larry Corey, Director HIV Vaccine Trials Network



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HIV vaccine history

• VAX003 and VAX004 (2005)

– Subunit vaccine with no efficacy

• Step Study (2008)

– Viral vector vaccine with no efficacy

• RV144 (2009)

– Virus/protein prime/boost with modest efficacy

• HVTN505 (2013)

– DNA/virus prime/boost halted early due to lack of efficacy

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So, why don’t we have an HIV vaccine in 2015?

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What is HIV?

4 millionths of an inch

Electron microscope picture

Human Immunodeficiency Virus

Schematic diagram



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How does HIV cause AIDS?

HIV infects/destroys immune cells needed to fight infection

Image by the National Cancer Institute

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Difficult to block HIV entry

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Simplified model of HIV envelope



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HIV infection (in most humans) does not elicit neutralizing antibodies

with broad specificity

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Bone marrow

B cells

Antibody

genes

Monoclonal antibodiesthat neutralize HIV

Needle in a haystack

HIV +

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>12MAbs

>25

2

>25

>5

1

Binding sites of anti-HIV human monoclonal Ab’s

>12MAbs

>25

2

>25

>5

1



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Can we use these rare human antibodies to prevent HIV infection?

T-lymphocyte

AntibodiesHIV

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• Immunity produced by injecting

antibodies derived from animal

or human serum (or monoclonal

antibodies) – Synagis (RSV)

• Administered by injections

into muscle or a vein

• No interaction with the adaptive

immune system (passive)

• Immunity is short-lived;

might require multiple injections

Traditional immunization strategies

“Active” “Passive”

• Vaccine produces immunity,

therefore protecting the body

from the disease

• Administered through needle

injections (measles), by mouth

(polio), or by aerosol (influenza)

• Requires effective engagement

(active) of the adaptive

immune system

• Immunity can be long-lived

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Classic passive immunization



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Transfer the gene that represents the antibody of choice

to the person to be immunized

New idea…

Transfer the gene that represents the antibody of choice

to the person to be immunized

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Short half lifeRepeated injections


Organs

CirculatorySystem

Mucosa

Vein

Traditional passive immunization

ProduceproteinProduceprotein

Insert geneinto vector

Anti-HIVmolecule

Vein

Inject vector

Muscle


Immunoprophylaxis by gene transfer

Organs

CirculatorySystem

Mucosa

Single injection Continuous production

Antibody gene transfer

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Plasma cell Myofibers

Can we turn muscle into an antibody factory?



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Antibody gene transfer

AAV

Produceprotein

Insert geneinto vector

Anti-HIVmolecule

Vein

Inject vector

Muscle


Immunoprophylaxis by gene transfer

Organs

CirculatorySystem

Mucosa


Single injection Continuous production

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What is AAV?(Adeno-Associated Virus)

• Parvovirus, replication incompetent,

needs a helper virus (adenovirus)

• Non-enveloped, icosahedral capsid

• Single-strand DNA genome (4.7 kb)

• Multiple species, multiple “serotypes”

• Non-pathogenic in all species

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In vivo human biology

Inoculum

Dissemination

LungLiver

SpleenHeart

IntestineBone marrow

Brain

Tonsils/adenoids:

episomal DNA

Schnepp et al., 2005, J Virol 79: 14793-803Schnepp et al., 2009, J Virol 83: 1456-64

Infectious DNA clones

1o replicationNasopharynx



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ITR ITR

rep cap

wtAAV genome

A.

B. Antibody LCPromoter 2Antibody HCPromoter 1 pA 1 pA 2 ITR

ITR ITRDual promoter rAAV genome

VH

VL

CL

CH1

CH2

CH3

Native antibody

AAV gene transfer vectors

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AAV is a DNA delivery vehicle

27Ambient Ultraviolet

Transduction of muscle

ITR

Promoter pA

ITR

GFP



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Using rAAV vectors to deliver anti-HIV antibody genes(vector mediated immunoprophylaxis)

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Bone marrow

B cells

Antibody

genes

Monoclonal antibodiesthat neutralize HIV

HIV +

Source of anti-HIV antibodies

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Evolution of anti-HIV human monoclonal antibodies

b12



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A

(200X)

A

(200X)

Antibody gene transfer in mice

B

In vivo production of human b12

ITR ITR

HIV antibody gene (b12)

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b12 produced for > 6 months

0

1

2

3

4

5

6

7

8

9

0 6 8 12 20 24

RF1 RF2

RC1 RC2

RC3 RD2

RD3 RA1

RA2 RA3

RB1 RB2

RB3

1

Weeks after injection

Hu

man

IgG

1(m

g/m

l)

9

8

7

6

5

4

3

2

1

0

0 6 8 12 20 24

Lewis et al., 2002, J Virol 76: 8769-8775

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Can we express antibodies in monkeys and prevent infection?

From mice to monkeys



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Simian Immunodeficiency Virus

SIV Rhesus monkey

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ITR ITR

SIV antibody gene

Rhesus monkey

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Blood tests

Check antibody

levels

Blood tests

Check antibody

levels

“Immunize”

Inject SIV antibody genes

into muscle

“Immunize”

Inject SIV antibody genes

into muscle

Challenge

Inject live SIV into the blood

Challenge

Inject live SIV into the blood

Experimental protocol



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“Immunized” animals were protected

Control 4L6

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Antibody gene transfer in monkeys

6+ years

0 50 100 150 200 250 3000

50

10

20

30

40

Weeks

Co

nce

ntr

atio

n m

g/m

L

Monkey ID

05C020

05C066

05D014

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Proof of concept in humans

Positive data from animal studies

led to the initiation of a clinical trial in humans



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b12


PG9

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rAAV1-PG9DP vaccine vector

Promoter 2 pA 2PG9 LCPG9 HCPromoter 1 pA 1

ITR ITR

VH

VL

CL

CH1

CH2

CH3

PG9 antibody

rAAV Vector Genome

Antibody gene insiderAAV vector

i.m. injection PG9 secretion



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A Phase 1, randomized, blinded, dose-escalation study

of rAAV1-PG9DP (recombinant AAV vector coding for PG9 antibody)

in healthy male adults

– Sponsor - International AIDS Vaccine Initiative

– Collaboration with DAIDS/NIAID/NIH

– Site - University of Surrey CRC

– Subjects - healthy male volunteers

Protocol A003

(ClinicalTrials.gov)

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A003 study design

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A003 clinical trial progress

1. First subject injected Feb 10, 2014

2. Groups A (low dose) and B (mid dose) fully enrolled

3. Group C (high dose) ready to start

(ClinicalTrials.gov)



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Primary

To evaluate the safety of rAAV1-PG9DP in healthy male adults

(first in human study)

Secondary

1. Evaluate the level and duration of PG9 in serum

and HIV neutralization activity in serum

2. Monitor the development of anti-PG9 antibodies,

anti-AAV1 antibodies, and AAV1-specific T cell responses

3. Measure the distribution and persistence of AAV1 vector

in the blood

A003 objectives

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• More potent antibodies

• Combination vaccine

Vector 1 Vector 2

What’s next?

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PGT121 PGDM1400b12 PG9



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Other antibody gene transfer applications

Application Antibody Reference

HIV vaccineSIV immunoadhesins, 4E10, 2G12,

2F5, b12, VRC01, eCD4-Ig, PG9a

Lewis 2002, Johnson 2009, Balazs 2012, Gardner 2015

HIV therapy 10-1074 Horwitz 2013

RSV vaccine Palivizumab Skaricic 2008

HCV vaccine FI6, F10, CR6261 de Jong 2014

Malaria vaccine NIC9D9, GNC92H2 Deal 2014

Influenza vaccine FI6, F10, CR6261 Limberis 2013, Balazs 2013

Drug addiction NIC9D9, GNC92H2 Hicks 2012, Rosenberg 2012

Cancer therapy DC101 Fang 2005

a The first clinical trial using rAAV vector mediated PG9 antibody gene transfer began in 2014 as a result of collaboration between The Children’s Hospital of Philadelphia, The International AIDS Vaccine Initiative, and DAIDS

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Y

Y

1. Pre-existing immunity blocks rAAV injection

2. Immune response to anti-HIV antibody

3. Can’t turn off expression if serious side effect

Antibody gene transfer limitations

Y

Y

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Summary

1. Gene transfer can bypass adaptive immunity

2. rAAV vectors mediate durable transgene expression

in pre-clinical models

3. Limitations may reduce effectiveness

4. Opportunity for designer anti-infective molecules

(beyond HIV – malaria, HCV)



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vector mediated immunoprophylaxis dr. bruce schnepp · image by the national cancer institute 11...

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