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Collaborating Institutions:
University of Nairobi
Seattle/Kenya Collaboration- MTCT
Julie Overbaugh
Fred Hutchinson Cancer Research Center
University of Washington
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Target cell (e.g. T Cell)
Antibody
Designing an effective vaccine: -> Identifying immune correlates
• A major focus of HIV vaccine efforts is trying to elicit HIV-
specific NAbs.
• Most successful vaccines are thought to work through
antibody-mediated protection.
• Do NAbs protect against HIV?
-
(Mascola et al. Nat. Med. 2000)
NAbs can block HIV (SHIV) infection in macaques
Controls Given HIV NAb (HIVIG/2F5/2G12)V
irus leve
ls
• High levels of antibodies were used, much higher than in HIV infection.• Challenge dose was high• Antibody was matched to the virus (SHIV89.6PD), but would not have ‘matched’ most viruses in the ‘real world’
There is surprisingly little evidence that NAbs contribute
to protection from HIV in humans
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HIV is both genetically and antigenically diverse
Neutr
aliz
atio
n s
ensitiv
ity
Easy to neutralize
Hard to neutralize
•At any given time, most people harbor a mixture of neutralization sensitive and neutralization resistant viruses due to the dynamic process of immune escape.
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Studies of HIV-exposed cohorts: MTCT
� MTCT occurs in the presence of a HIV-specific NAb
response in the mother.� HIV-specific NAbs are present in the infant due to
passive transfer, especially near the time of delivery and
during breastfeeding
MTCT
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Nairobi breastfeeding clinical trial 1992-1998:
Ruth Nduati
University of Nairobi
• Randomized clinical trial comparing infant HIV-1 infection between breast-fed and formula-fed groups (N=425) to determine the frequency and timing of breast milk HIV-1 transmission from a mother to her infant.
• In 1992: Risk was unclear and ARVs were not available.
Joan
KreissUniversity of Washington
Dorothy Mbori-Ngacha
Grace John-Stewart
Barb Richardson
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Child age0%
10%
20%
30%
40%
6 wks
14 wks
6 months12 months
24 months
HIV Infection rate-Breastfeeding
HIV Infection rate-Formula feeding
Nduati, et al. Effect of Breastfeeding and Formula Feeding on Transmission of HIV-1.
JAMA 2000:283:1167-1174
Nairobi Breastfeeding Clinical Trial (1992-1998)
Breast feeding doubles the risk of infant infectionThe majority of breastfeeding transmission occurs in the first 6 weeks postpartum
% infe
cte
d
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~ Sixty % of infants do not get infected despite
exposure in utero, intrapartum and through
breastfeeding
100%
80%
60%
40%
20%
0%
Do NAb contribute to protection?
uninfected
-
Do NAb play a role in protection of infants?
100%
80%
60%
40%
20%
0%
N=32
N=68
Select 100 infants in the
breastfeeding arm who
were HIV negative at birth
Examine the breadth of the NAb response
near delivery: mom and baby
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Hypothesis: NAb contribute to protection
If a broad and potent NAb response
provides protection, then these infants are
less likely to be infected
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To measure breadth and potency of the NAb
response, we use a panel of circulating HIV variants
Neutr
aliz
atio
n s
ensitiv
ity
This heterologous panel gives a measure or breadth and potency of the NAb
response
Easy to neutralize
Hard to neutralize
• A range of NAb sensitivities
Pla
sm
a R
NA
• Variants from early in infection representing circulating viruses
-
IC50s are defined as the reciprocal dilution of plasma or MAb required to inhibit infection by 50% (based on the dose response curve, log function).
Higher IC50 means more potent neutralization
Virus 1 IC50 = 620Virus 2 IC50
-
20 55
148
403
1096
IC50
Lynch et al. J. Virol. 2011
100 infa
nt pla
sm
a a
t birth
-
There was no difference in the breadth or potency of HIV NAb in infants who became infected versus those who remained
uninfected
Lynch et al., JV 2011
Similar results were obtained when we examined the breadth of the
maternal Nab response in relation to infant infection. Majiwa, submitted. J. Lynch
M. Majiwa
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100%
80%
60%
40%
20%
0%
Do NAbs protect against the most neutralization sensitive viruses?
} Infected
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Baby virus (week 6)
Mom virus(week 0)
Isolate maternal and infant envelope variants and
test neutralization sensitivity (N= 12 pairs, 96 envs)
NAbS
NAbR
NAbR?
Maternal antibodies (wk 0)
-
10
100
1000
Neutr
aliz
atio
n s
ensitiv
ity
IC50
va
lue
s v
ers
us a
uto
logou
s A
b
ne
ar
the
tim
e o
f in
fection
Transmitting
mothers
Infected
infants
Infant viruses variants are poorly neutralized by maternal NAbs.
Viruses
neutralized by
maternal
plasma
No detectable
neutralization
�p=0.02
Wu, JV, 2006X. Wu
-
10
100
1000
Neutr
aliz
atio
n s
ensitiv
ity
IC50
va
lue
s v
ers
us a
uto
logou
s A
b
ne
ar
the
tim
e o
f in
fection
Transmitting
mothers
Infected
infants
Infant viruses variants are poorly neutralized by maternal NAbs.
Sensitive variants are
not transmittedWu, JV 2006
-
Easy to neutraliz
e
Hard to neutraliz
e
Nab elicited by HIV infection may have adequate potency to block the most neutralization sensitive variants
Ab n
eed
ed t
o n
eutr
aliz
e
10
100
1000
Transmitting
mothers
Infected
infants
-
A broad and potent Nab response per se does not correlate with transmission because the mothers harbor a mix of neutralization sensitive and resistant viruses and the Nab are only able to get the ‘low hanging fruit”
Ab n
eed
ed t
o n
eutr
aliz
e
Mom’s virus
How does this fit with the first study, showing a lack of association
between breadth and potency and risk of infant infection?
-
(Mascola et al. Nat. Med. 2000)
NAbs can block HIV (SHIV) infection in macaques
Controls Given HIV NAb (HIVIG/2F5/2G12)V
irus leve
ls
• Antibody was matched to the virus (SHIV89.6PD), which was a very neutralization sensitive virus.
-
Easy to neutraliz
e
Hard to neutraliz
e
These data suggest together with animal model studies, suggest that Nab perhaps can protect against the more neutralization sensitive viruses
> The Nab breadth/potency needed for protection remains unclearA
b n
eed
ed t
o n
eutr
aliz
e
10
100
1000
Transmitting
mothers
Infected
infants
Current
SHIVs
-
Perhaps the protective antibodies are acting
locally, e.g. in breast milk to reduce
infectiousness?
Courtesy, Grace John-Stewart
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Breastmilk NAbs are low
• Low level neutralizing activity is detected in BMS,
but much of this is non-specific.
• NAbs are only rarely detected in purified antibody
fractions (IgG, IgA).
Mabuka, J et.al PloS Path 2012J. Mabuka
-
1.11
1.67
Rousseau, JID 2004
Transmitting mothers have higher levels of cell-associated
breast milk HIV than non transmitting mothers.
Non transmitting Transmitting
Cell-
associa
ted B
M levels
(Log
10
BM
cell
DN
A/1
06 c
ells
)
Adjusted
p =0.002
C. Rousseau
Cell-free virus
RNA form
Cell-associated virus
DNA form
Infected
cell
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Fc Receptor
ADCC : Antibody-dependent cell cytotoxicityLeads to elimination of infected cells
Antibody neutralizationPrevents new rounds of infection
Target cell (e.g. T Cell)
Effector cell (e.g. NK cell)
granzymes, perforin
Infected cell
-
ADCC assay measures killing of HIV coated cells in the presence of Ab.
HIV Envelope protein
(subtype A)
Target cell (CEM.NKr cells)
CFSE
PKH-26Effector cell
(PBMCs
Envelope Coated
Target cells
Uncoated
Target cells
CFSE
PK
H-2
6
BMS 1:100
*ADCC % Killing= %PKH+ cells, CFSE- cells - 2x background
42.8 57.24.0 96
+ +
BM Abs
-
Breast Milk ADCC is correlated with
risk of infant infection
J. Mabuka
Non transmitting Transmitting
p =0.039
Mabuka, J et.al PloS Path 2012
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• NAb can protect against highly sensitive variants in MTCT, but the amount needed and the breadth to achieve a
protective Nab responses is unclear.
• Suggest the breadth and potency needed to protect against diverse HIV variants circulating globally is likely higher than elicited by chronic Hiv infection.
• Antibodies that act through ADCC may contribute to protection, especially in settings where cell-associated virus is contributing to transmission.
Take home