Neutralizing Abs to HIV1 Env from Hyperimmune Bovine Colostrum as a Mucosal Microbicide Strategy for

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Neutralizing Abs to HIV-1 Env from Hyperimmune
Bovine Colostrum as a Mucosal Microbicide
Strategy for Preventing Virus Transmission
Marit Kramski, Robert Center, Grant Rawlin,
Carly Siebentritt and Damian Purcell.
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Aims

• vaccinate cows with HIV-1 Env gp140 oligomers
• produce large quantities of bovine colostrum
  polyclonal Abs
• test breadth and potency of HIV-1 neutralizing
  activity

Longer term

• show efficacy safety of H2BC Ab based
  microbicide gel
• application of Abs to macaques (SHIV vaginal
  challenge model)

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Microbicides to prevent HIV Transmission
AIM easy to use, no requirement of cooperation,
consent or even knowledge of the partner
Balzarini J. and L. Van Damme et al., 2005,
Canadian Medical Association Journal
http//www.who.int/hiv/topics/microbicides
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Bovine Colostrum (BC)

• first milk or immune milk
• produced in mammals in late pregnancy
• nutrition (vitamins and minerals)
• passive immunization for newborn

• BC quite similar to human colostrum
• BUT higher protein concentration
• 4 weeks before birth, Abs pumped into BC

http.//www.colostrumresearch.org/generateditems/co
lostrum_info/colostrum_how.html
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1. Antigen HIV-1 Env gp140 oligomers

• Cell lines engineered to express HIV-1 Env gp140
  oligomers in supernatant
• stable expression of gp140 Env UG8 clade A,
  AD8 clade B, MW clade C
• purify soluble Env gp140 oligo using lentil
  lectin and gel filtration

virus
SN
gp120
gp41
Ectodomain
Cytoplasmic domain
TM
sEnv gp140
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2. Vaccination
Intramuscular vaccination of cows with 100µg of
Env gp140 in adjuvant
1. group 2 cows only AD8 clade B (3318, 7004)
autum calvers
3318
spring calvers
7004
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2. Vaccination
Intramuscular vaccination of cows with 100µg of
Env gp140 in adjuvant
1. group 2 cows only AD8 clade B (3318, 7004)
autum calvers
3318
6055
spring calvers
7004
7008
2. group 2 cows trimix UG8, AD8 and MW clade
A/B/C (6055, 7008)
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3. Vaccination regimen
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4. Serum IgG to Env
determined by ELISA with gp140
2w after 1. boost
2w after 2. boost



32
24
15
12
9
7
pre-bleed
no blood for 6055 and 3318
weeks p.vac.


titer below 1100 for 6055
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5. Serum IgG - broad specificity for HIV Env
IgG in sera are HIV-1 gp140 specific, no
cross-reactivity against human proteins
3318 clade B
6055 trimix
Ad8/ pNI/ HeLa UG8/MW/ pN1/ 293T
Ad8/ pNI/ HeLa UG8/MW/ pN1/ 293T
gp140
gp140
gp140
gp140
autumn calvers
t2 bleed 1100
Transfection with gp140 env in HeLa and 293T
cells / SDS page of SN and Western Blot with
bovine sera and a-bovine IgG-HRP
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6. Good titres of IgG for HIV gp140 in H2BC
n3
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7. Env-pseudoviruses (psv) for Nab assay

• primary isolates
• R5 and X4 tropic
• acute infection/early stage of seroconversion
• PSV using Env from different clades (NIH
  reference panel of Env clones)

• clade A/E 966, 967
• clade B NIH HIV-1 Env reference panel 10/12
  clones
• AD8, NL4.3, SF162, 89.6
• clade C NIH HIV-1 Env reference panel 11/11
  clones

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7. Env-pseudoviruses (psv) for Nab assay

• primary isolates
• R5 and X4 tropic
• acute infection/early stage of seroconversion
• PSV using Env from different clades (NIH
  reference panel of Env clones)

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8. Neutralisation assay
3 colostrum samples H2BC 6055 and 3318, 10 non
immune BC
Pseudotyped HIV-GFP equal to 10-20 infectivity


2 days
neutralisation
Colostrum dilution (116-11024)
CF23 Cf2th-CD4/CCR5/CXCR4 carnine thymocites
Gray, L. et al. 2005, Virology
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9. Neutralising activity of H2BC
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9a. Neutralisation of B clade Env panel
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9b. Neutralisation of C and A/E clade Env
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Next experiments

• Transcytosis assay effects beyond virus
  inactivation
• Neutralising activity of pasteurized and purified
  IgG
• Neutralising activity across the pH range (eg.
  under pH 4)
• Neutralising activity in seminal plasma and
  vaginal secretions
• Induction of inflammation markers - epithelial
  cells (HEC-1) with H2BC --gt (cytokine
  expression/secretion)
• Growth effects on typical bacteria flora in
  vagina and rectum (Lactobacillus acidophilus,
  corynebacteria etc.) in presence of H2BC
• is growth promoted or inhibited?
• Protection in a macaque challenge experiment

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Potential for scale up

• Commercial experience with hyperimmune colostrum
  products
• Each cow produces on average 850 gram of Ab per
  year
• Cost of active product including profit _at_ 600
  per Kg 0.3 cents / dose

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Potential for scale up

• Commercial experience with hyperimmune colostrum
  products
• Each cow produces on average 850 gram of Ab per
  year
• Cost of active product including profit _at_ 600
  per Kg 0.3 cents / dose

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Thank you.....
ACH2 for funding
Marit Kramski Rob Center Adriana Gaeguta Sharmila
M. Reddy Jane Howard Adam Wheatley Carly
Siebentritt
Immuron Limited Grant Rawlin Brian Muller Victor
Wong
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Thank you.....
ACH2 for funding
Marit Kramski Rob Center Adriana Gaeguta Sharmila
M. Reddy Jane Howard Adam Wheatley Carly
Siebentritt
Immuron Limited Grant Rawlin Brian Muller Victor
Wong