Chemo and Immunoprophylaxis of infant macaques against SIV'

1
Chemo- and Immunoprophylaxis of infant macaques
against SIV.
Koen Van Rompay, D.VM, Ph.D.
California National Primate Research Center
Dec. 12-13, 2002 Gent, Belgium
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Animal model of pediatric AIDS
Newborns/infants
Simian Immunodeficiency Virus (SIV)
- SIV closely related to HIV - Similar
physiology - Similar route ORAL - SIV causes
similar disease
3
Animal model of perinatal infection
Virulent SIVmac251

- High virus levels - Rapid disease (lt 3-4
months)
1 or 2 doses
lt 3 days of age
100 ANIMAL INFECTIOUS DOSE
4
Animal model of perinatal infection
Virulent SIVmac251

- High virus levels - Rapid disease (lt 3-4
months)
1 or 2 doses
prophylaxis
5
PROPHYLAXIS OF PERINATAL TRANSMISSION
Prevention of SIV infection in rhesus neonates
Antiviral drugs AZT, tenofovir Passive
Active Immunization
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• Prevention of HIV transmission
• from mother to infant
• IDEAL drug
• High antiviral activity
• Transplacental transfer
• No or low toxicity
• Relatively low cost
• No/ slow development of resistance

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PREVENTION OF SIV INFECTION AZT
AZT (3x/day) - 2 h to 6 weeks
n 8
SIVmac
5/8 INFECTED 3/ 8 UNINFECTED
(Van Rompay, Antimicrob. Agents Chemotherapy
1992, 1995)
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Tenofovir (PMPA) 9-2-(phosphonomethoxy)-propyl
adenine

• - Acyclic nucleoside phosphonate
• RT inhibitor
• Active against HIV-1 and HIV-2
• Active against Hep B
• - FDA approved
• Gilead Sciences (Foster City, CA)

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Tenofovir (PMPA) Pharmacokinetics

• - Tenofovir DF (disoproxil fumarate)
• prodrug for oral administration
• Active diphosphate form
• long intracellular half-life gt 12-50 h -
  dosage regimen once daily
• Single-dose PMPA (30 mg/kg) to lactating
  macaques
• PMPA secreted in breast-milk
• AUC in breast milk 20 of serum AUC
• unlikely to be absorbed by nursing infant.

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Untreated control newborns
all 16 controls infected
(n 16)
SIVmac251 orally
1 high dose 105 TCID50 , 109 RNA per ml
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POST-EXPOSURE PMPA PROPHYLAXIS
Immediate high-dose PMPA treatment for 2 weeks
(30mg/kg subQ SID)
n 4
1/4 SIVmac251-INFECTED 3/ 4 UNINFECTED
(Van Rompay et al., 1998, AIDS Res. Hum.
Retrovir., v14761)
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2 high doses of PMPA
1 dose of PMPA (30mg/kg) 4 hours before virus
inoculation
n 4
1 dose of PMPA (30mg/kg) 24 hours later
Oral SIVmac251
4/ 4 UNINFECTED ( 2 years of age)
(Van Rompay et al., 1998, AIDS, v12F79-F83)
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2 low doses of PMPA
PMPA 4 mg/kg SQ
1 dose of PMPA 4 hours before virus inoculation
n 4
1 dose of PMPA 24 hours later
3/ 4 UNINFECTED ( gt18 months of age) 1/4
infected
(Van Rompay et al., 2001, J.Infect.Dis, v184429)
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PROPHYLAXIS OF PERINATAL TRANSMISSION
1 dose PMPA (TDF) before delivery
PACTG 394 Phase I (USA) Chair Dr. Pat
Flynn
PMPA
1 dose of PMPA (TDF) next day
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2 DOSES OF PMPA POST-EXPOSURE
PMPA 4 mg/kg SQ
n 4
1 dose of PMPA 1 h after virus inoculation
1 dose of PMPA 24 h later
2/ 4 UNINFECTED ( gt18 months of age)
(Van Rompay et al., 2001, J.Infect.Dis., v184429)
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PREVENTION OF INTRA-PARTUM TRANSMISSION
1 dose tenofovir immediately at birth
1 dose of tenofovir next day
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• Systemic tenofovir levels
• Effective against single high-dose oral
  SIVmac251 infection in newborn macaques.
• Effective against intravenous, intravaginal SIV
  infection of juvenile adult macaques (Tsai
  1995 Otten 2000).

Should also be effective against repeated
low-dose oral exposure in breast-feeding infants!
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Tenofovir prophylaxis systemic levels 4 mg/kg
works in infant macaques! Can we use even
100-fold less?
Topical tenofovir prodrug?
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Topical Tenofovir DF
In vitro EC50 for prodrug 0.005 µM Topical
prodrug conc. 5,000 x EC50 25 µM 5 ml of 25
µM TDF per day 0.037 mg per day
100-fold less than 4 mg/kg SQ!) (or 1
gram lasts 74 years!)
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Topical PMPA prodrug for prevention of oral SIV
infection

• In vitro Strong antiviral effects
• TDF enters cells within a few seconds!
• In vivo
• Topical TDF failed to prevent SIVmac251
  infection of infant macaques
• (Van Rompay et al, 2002 J. Infect. Diseases, v.
  186, 1508-1513)
• Future studies other prodrugs that target
  lymphoid cells

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Tenofovir (PMPA) resistance

• K65R mutation in reverse transcriptase of HIV
  SIV.
• Tenofovir prophylaxis partially effective to
  protect newborn macaques against infection with
  tenofovir-resistant K65R mutants of SIVmac251
• Even if infected with tenofovir-resistant SIV
  mutants, prolonged treatment offers therapeutic
  benefits (survival of SIVmac251-infected macaques
  7 years!!).

(Van Rompay et al, 2000 J. Virol., v.74
1767-1774 1999 Antimicrob. Agents Chemother., v.
802 802-812 unpublished data)
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Tenofovir (PMPA) resistance in HIV-1

• Genotypic and phenotypic resistance emerges
  infrequently slowly after prolonged tenofovir
  treatment of HIV-infected adults.
• (Margot et al, 2002 AIDS, v. 16, 1227-1235).

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Prolonged PMPA (tenofovir) safety?

• (Tarantal, J.AIDS, 1999 2002)
• Prolonged high dose (30 mg/kg) to gravid
  macaques
• Fetal neonatal growth retardation
• - bone lesions
• - hypophosphatemia in mother and fetus
• - fetal drug levels 10 of maternal levels
• Mechanism??

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Short-term tenofovir - 39 newborn infant
macaques - doses ranging from 4-30 mg/kg SQ -
Duration 1 day to 8 weeks - Follow-up 12
animals for 2 years

• No toxicity!

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Prolonged high-dose PMPA (30 mg/kg)

• gt 8 months of treatment of infant macaques
• Growth retardation, bone abnormalities
• Proximal renal tubular disorder - FANCONI
  SYNDROME
• Reduced tubular absorption increased urinary
  loss of
• Phosphate hypophosphatemic rickets
• glucose
• amino acids
• .
• Upon withdrawal/dosage reduction quite
  REVERSIBLE

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Prolonged tenofovir safety for infants?

• (Tarantal, J.AIDS, 19992002)
• High dose (30 mg/kg) to gravid macaques
• Fetal neonatal growth retardation
• - bone lesions
• - hypophosphatemia in mother and fetus
• - fetal drug levels 10 of maternal levels
• Alternative explanation
• Prolonged high-dose tenofovir is more toxic
• for adult animals than for infants! !
• Severe renal toxicity of mothers resulting in
  maternal hypophosphatemia instead of direct
  effect on fetus!

!
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Chronic low-dose PMPA administration

• 2 newborns started on PMPA 10 mg/kg per day
• PK 18-fold (at birth) to 3-fold (juvenile
  animal) higher than currently approved 300 mg TDF
  in adults
• - Normal weight gain
• - Normal bone radiographs, DXA, bone histology
• - Serum chemistry values all in normal range
• 24h urines phosphate, calcium, potassium
• - 4.5 years of treatment (into adulthood!)
• No significant evidence of toxicity!
• (Manuscript in progress copy available upon
  request)

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• Prevention of vertical HIV transmission
• Tenofovir (PMPA)
• High antiviral activity (therapy prophylaxis)
• Transplacental transfer
• Low chronic toxicity
• Relatively low cost
• No/ slow development of resistance
• Active against HIV-1 and HIV-2
• Efficacy against Hep B prophylaxis?

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PROPHYLAXIS OF PERINATAL TRANSMISSION
Prevention of mucosal SIV infection in rhesus
neonates

• Antiviral drugs
• AZT, tenofovir
• Passive Active Immunization
• 1-2 high-dose SIVmac251
• multiple low-dose SIVmac251

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Animal model of perinatal infection
Virulent SIVmac251
Orally

- High virus levels - Rapid disease (lt 3-4
months)
1 or 2 doses
lt 3 days of age
100 ANIMAL INFECTIOUS DOSE
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Passive immunization
Anti-viral IgG can protect newborn macaques
against oral SIVmac251 infection
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Active immunization of mother
ACTIVE IMMUNIZATION
- Live-attenuated SIVmac1A11 (2nd trimester) -
Whole inactivated SIV (early 3rd trimester)
SIV IgG
n3
2/3 Uninfected !!
(Van Rompay, J. Infect. Diseases, 1996
1731327-1335)
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Active immunization of mother
ACTIVE IMMUNIZATION
Whole inactivated SIV (2nd 3rd trimester)
SIV IgG
n11
6/11 Uninfected
(Marthas et al., unpublished data)
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Passive immunization
SIV-negative female
SIV hyperimmune serum
SIV IgG
6/6 PROTECTION
Control serum no protection
(Van Rompay et al,J. Infect. Dis.,1998 177
1247-59)
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ACTIVE IMMUNIZATION to prevent HIV breast-milk
transmission
Active immunization of infant rhesus macaques
against oral SIV challenge

• An effective neonatal vaccine must elicit
  protective anti-viral immune responses
• in the first weeks of life
• that persist for months to years
• in the presence of maternal antibodies

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Oral SIVmac251 (2 doses of 100,000 TCID50
4/4 infected
3
0
4
MVA-SIVgag-pol-env
Maternal Ab
4/4 infected
SIVmac1A11
4/4 infected
Weeks of age
(2003, Van Rompay et al, J. Virol., in press)
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Infant SIV vaccines high dose oral SIV challenge

SIV vaccines administered at 0 3 weeks of
age all vaccinates controls infected but
vaccinated animals - better control of
viremia - more sustained SIV antibody responses
(better CD4 help?) - longer
survival SIV-gag T cell responses detected
rarely by IFNg elispot assay in vaccinated
animals Maternal antibody did not affect
antibody responses (2003, Van Rompay et al, J.
Virol., in press)



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Active immunization of infant rhesus
macaques against oral SIV challenge
SIVmac251 challenge at 4 weeks

• 1999-2000
• 2 high doses (100,000 TCID50 per dose),
• ketamine anesthesia
• 2001-2002
• 15 low doses (10,000 TCID50 per dose)
• while awake

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Animal model of breast-milk transmission
4 weeks of age
SIVmac251
ORAL
Multiple low-dose exposure (5 days x 3 times/day
15 inoculations) Each dose 2 ml _at_ 10,000
TCID50/ml with sucrose (for palatability)
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Step 1 Animals are immunized at birth and 2, 3
weeks of age.
immunization
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Step 2 Animals are trained to drink a 11
mixture of sucrose and medium.
sucrose/medium
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Step 3 At 4 weeks of age, animals are given 2
ml of diluted SIVmac251 in sucrose/medium, 3 x
daily for 5 days.
SIV
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Infant SIV Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) 17(11) MVA-SIVgpe (3x) 8
(4) ALVAC-SIVgpe (3x) 8 (2) 8 (4) 16
(6) ALVAC vector (3x) 4 (2)
animals(infected)
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Infant Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) MVA-SIVgpe (3x) 8 (4) 17
(11) ALVAC-SIVgpe (3x) 8 (2) 8 (4) 16
(6) ALVAC vector (3x) 4 (2) ALVAC-SIVgpe
(3x) 8 (6) ?????CD8 ab (pre-challenge) Mater
nal SIV ab only 4 (1) 7 (1) Mat. SIV ab
1A11 (2x) 3 (0)
animals(infected)
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Infant Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) MVA-SIVgpe (3x) 8 (4) 17
(11) ALVAC-SIVgpe (3x) 8 (2) 8 (4) ALVAC
vector (3x) 4 (2) ALVAC-SIVgpe (3x) 8
(6) anti-CD8 ab (pre-challenge) Maternal SIV ab
only 4 (1) Mat. SIV ab 1A11 (2x) 3
(0)
p0.13
animals(infected)
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Infant Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) MVA-SIVgpe (3x) 8 (4) ALVAC-SIVgpe
(3x) 8 (2) 8 (4) 16 (6) ALVAC vector
(3x) 4 (2) ALVAC-SIVgpe (3x) 8 (6)
anti?CD8 ab (pre-challenge) Maternal SIV ab
only 4 (1) Mat. SIV ab 1A11 (2x) 3
(0)
p0.005
animals(infected)
47
Infant Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) MVA-SIVgpe (3x) 8 (4) 17
(11) ALVAC-SIVgpe (3x) 8 (2) 8 (4) 16
(6) ALVAC vector (3x) ALVAC-SIVgpe (3x)
8 (6) anti CD8 ab (pre-challenge) Maternal SIV
ab only 4 (1) 7 (1) Mat. SIV ab 1A11
(2x) 3 (0)
p0.096
animals(infected)
48
Infant Vaccine Studies, 2001 - 2002
Vaccine 2001 2002 Total None 8
(7) 8 (7) 16 (14) MVA-SIVgpe (2x)
9 (7) MVA-SIVgpe (3x) 8 (4) 17
(11) ALVAC-SIVgpe (3x) 8 (2) 8 (4) 16
(6) ALVAC vector (3x) 4 (2) ALVAC-SIVgpe
(3x) anti?CD8 ab Maternal SIV ab only 4
(1) 7 (1) Mat. SIV ab 1A11 (2x) 3
(0)
p0.01
animals(infected)
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Correlates of Protection antibodies?
ACTIVE
PASSIVE
50
Correlates of Protection antibodies?
No Neutralizing antibodies against challenge
virus!
ACTIVE
PASSIVE
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Correlates of Protection CMI?
(2001 only)
ACTIVE
PASSIVE
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Outcome of poxvirus vaccines in infants
SIV vaccines given at 0, 2, 3 weeks of
age SIV-specific immunity MVA-SIV gt
ALVAC-SIV binding antibody (whole SIV
ELISA) SIVgag IFN-? Elispot Protection (no
viremia) ALVAC-SIV gt MVA-SIV 6/17 MVA-SIVgpe
vaccinates 10/16 ALVAC-SIVgpe vaccinates No
immune correlate of vaccine protection
identified no SIV neutralizing abs to
SIVmac251-5/98 rare SIV gag IFN gamma responses
(MVA) Innate immune responses induced by
poxvirus??



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Outcome of poxvirus vaccines in infants
SIV vaccines given at 0, 2, 3 weeks of age For
infants which became SIV-infected correlation
between - virus levels - SIV-specific immune
responses - survival

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Future studies

• - Duration of protective immunity?
• Efficacy of these vaccines in presence of
• maternal antibodies?
• - Efficacy of single dose administered at birth?

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Acknowledgements (I)
University of California, Davis California
National Primate Research Center
Marta Marthas Mike McChesney Kimberly Schmidt
Chris Miller Jonathan Lawson Bapi Pahar Raman
Singh Kristen Bost Laurie Brignolo Lara
Compton Abbie Spinner Phil Allen Veterinary
Colony Services
Gilead Sciences Norbert Bischofberger John
Desjardins Denny Meyer Ray Bendele Marta
Hamilton Julie Cherrington Michael Miller
Skeletech Inc. Chris Jerome
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Acknowledgements (II)
UCD-School of Medicine
Thomas Evans
NIH Patricia Earl, Bernard Moss Genoveffa
Franchini Jorge Flores
Beth Israel Deaconess Keith Reimann
Duke University David Montefiori
Aventis Pasteur, Toronto James Tartaglia Raphael
El-Habib
Bayer Diagnostic Lab Casey Wingfield Jenny Booth
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Research support from
NCRR NIH E. Glaser Pediatric AIDS
Foundation Gilead Sciences
California National Primate Research
Center University of California, Davis