Embed Size (px)
Citation preview
A Vaccine Targeting Protease Cleavage Sites Protects Cynomolgus Monkeys Against SIVMa Luo, David Tang, Rupert Capina, Xin-Yang Yuan, Jorge F Correia-Pinto, Cecilia Prego, So-Yon Lim, Christina Barry, Richard Pilon, Christina Daniuk, Mikaela Nykoluk, Stephane Pillet, David La, Tomasz Bielawny,
Jeffrey Tuff, Chris Czarnecki, Philip Lacap, Gary Wong, Shaun Tyler, Binhua Liang, Zhe Yuan, Qingsheng Li, Terry B Ball, James Whitney, Maria J Alonso, Paul Sandstrom, Gary Kobinger, Francis A. PlummerNational Microbiology Laboratory, Winnipeg, Manitoba, Canada; University of Manitoba, Winnipeg, Manitoba, Canada; University of Santiago de Compostela, Spain; University of Nebraska-Lincoln, Nebraska, USA
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
ABSTRACTBackground: With the sobering results of the STEP and HVTN 505 HIV vaccine clinical trials, novel approaches to HIV vac-cine development must be explored. The HIV protease is a 99-amino acid aspartic enzyme that mediates the cleavage of Gag, Gag-Pol and Nef precursor polyproteins. This process is a highly specific, temporally regulated and essential for the production of infectious viral particles. A total of 12 proteolytic reactions are required to generate an infectious virion, and a single impaired cleavage reaction can render the virus noninfectious. Thus, HIV vaccine-elicited responses targeting the protease cleavage sites (PCS) could be highly efficacious.Methods: We assess vaccine immunogenicity elicited against PCS immunogens using a modifiedVesicular stomatitis virus (VSV) vector combined with a nanodelivery system. We then evaluate protective efficacy to disrupt SIV acquisition and disease progression using a Cynomolgus macaque (Macaca facicularis, Philippines) and SIVMAC239 intrarectal chal-lenge model. To examine whether the immune driven viral mutations surrounding the PCS were detrimental to the virus, we amplified and sequenced the plasma viruses by 454-pyrosequencing and correlated the amino acid mutations sur-rounding PCS with alterations in viral load and CD4 count.Results: PCS peptides expressed by rVSVs and packaged in nanoparticles are able to generate both antibody and T cell responses in macaques. The ability of macaques to withstand high dose SIVMAC239 challenge was significantly correlated with the antibody and antibody/T cell responses to the number of PCS peptides(p=0.0005, R=0.8005). This combination imparted resistance to all but the higher SIV doses that were required to infect vaccinnees (p=0.01). the vaccine group maintains significantly higher CD4 counts (p=0.0002). Amino acid mutations (SN or NS) surrounding PCS correlated sig-nificantly with reduced viral RNA levels (p<0.0001).Conclusions: We show here in a Cynomolgus macaque/SIV model that a candidate HIV vaccine focusing immunologic re-sponses to the regions surrounding the SIV protease cleavage sites can force viral mutation resulting in impaired SIV fit-ness. Focused immune response to the PCS region enables the macaques to withstand multiple high dose pathogenic SIVMAC239 intrarectal challenges. Virus recovered from vaccinnees harbored mutations surrounding the protease cleavage region that correlated significantly with reduced viral load, and the maintenance of CD4+ T cells in vivo.
Acknowledgement
DISCUSSION
Public HealthAgency of Canada
Agence de santpublique du Canada
é
Gag
Pol
Nef
HIV-1 gag, Gag-Pol and Nef translation
Pr55Gag
Pr160Gag-Pol
Nef
MA CA p2 NC p1 p6gag MA CA P2 NC
TFP p6pol PR RTp51 RTp66 IN NEF
GagPol Cleavage Process
p6gagp1NCp2MA CA
TFP p6pol PR RTp51 RTp66 IN NEF
1 2 3 4 5 6 7 8 9 10 11 12
Pr55 Gag Cleavage process Pr160 Gag-Pol Cleavage process
AmpR N P M
MCS
G
T7P
Mlu1 Bln1
The nucleotide sequences encoding 20 amino acidsoverlapping each of the 12 protease cleavage sites, Gag or Env were cloned into pATX VSV-G vector at Mlu1-Bln1 site.
0 5 10 12 16 18 20week
VSV-peptidesIM
VSV-peptides (IM)nanopeptides (IN)
nanopeptides (IN)
accelerated SIVmac239intrarectal challenge
necropsy of controls
necropsy of vaccine group
29 32 33
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
OD40
5nm
.
IgG
IgM
Sampling points (week)
0
50
100
150
200
250
300
2 3 4 5 6 7 8 10 11 12 13weeks after first immunization
boost 1 boost 2
SFU/
milli
on
0
1
2
3
4
5
6
7
8
9
10
C930
98F
C910
32F
C910
69F
C871
29F
C930
48F
C910
28F
C871
14F
C930
53F
C900
38F
C920
78F
C880
66F
C910
51F
Antib
ody
resp
onse
s to
no.
of P
CS
0
1
2
3
4
5
6
7
C930
98F
C910
32F
C910
69F
C871
29F
C930
48F
C910
28F
C871
14F
C930
53F
C900
38F
C920
78F
C880
66F
C910
51F
T ce
ll re
spon
se to
No.
of P
CS s
ites
A
B C
D E
SIV infection after 7000 TCID50 challenge
0 1000 2000 3000 4000 5000 6000 7000 80000
10
20
30
40
50
60
70
80
90
100 with ab/T cell response=> 6 PCS
with ab/T cell response<6 PCS
p = 0.017Harzard ratio: 0.357, 95%CI:0.0398-0.0731
SIVmac239 TCID50 dosage
Perc
ent r
emai
n un
infe
cted
0 2500 5000 7500 10000 12500 15000 175000
1
2
3
4
5
6
7
8
9
SIVmac239 Challenge (TCID50)
IgG
to N
o. o
f PC
S si
tes
p = 0.0097R = 0.372
0 2500 5000 7500 10000 12500 15000 175000
1
2
3
4
5
6
7
8
9
SIVmac239 Challenge (TCID50)
T ce
ll &
ant
ibod
y to
No.
of P
CS
site
s
p = 0.005R = 0.422
A B
C Dp = 0 .0 1
V a c c in e g ro u p c o n tro l g ro u p0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 2 0 0 0
1 4 0 0 0
1 6 0 0 0
Acc
um
ula
ted
SIV
mac
23
9 c
hal
len
ge
do
sag
e (T
CID
50
)
P =0 .0 0 0 2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
3250Vac c ineC ontrol
We e k s P o s t In fe c tio n
Ab
solu
te C
D4
cel
ls/m
m3
P = 0 .3 1 6
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170
1000
2000
3000
4000
5000
6000
Vac c ineC ontrol
We e k s P o s t In fe c tio n
Ab
solu
te C
D8
cel
ls/m
m3
p = 0 .0 0 0 2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Vac c ineC ontrol
We e k s P o s t In fe c tio n
CD
4/C
D8
Rat
io
p = 0 .0 0 0 2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170
50
100
150
200
250
300
Vac c ineC ontrol
We e k s P o s t In fe c tio n
% B
asel
ine
CD
4 D
eclin
e
A B
C D
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12 14 16 18
C 87-129F
C 90-038F
C 91-028F
C 91-032F
C 93-048F
C 91-069F
C 92-078F
C 87-114F
C 88-066F
C 91-051F
C 93-053F
V accin e M ean
weeks after infection
Log
Vir
al L
oad
2
3
4
5
6
7
0 2 4 6 8 10 12 14 16
C 87-133F
C 88-128F
C 93-055F
C 94-076F
C 97-007F
C o n trol M ean
weeks after infection
Log
Vir
al L
oad
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170
1
2
3
4
5
6
7
8P = 0.3851 Vaccine
Control
Weeks after Infection
Log
vira
l loa
d
2
3
4
5
6
7
8
p = 0 .0 0 9 2
p = 0 .5 0
Lo
g V
ira
l Lo
ad
control controlvaccine vaccine
peak viral load week 9 after infection
A B
C D
p < 0.0001R2: 0.18
0 1 2 3 4 5 6 7 8 90.00.10.20.30.40.50.60.70.80.91.01.1
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS1
p = 0.003R2: 0.07
2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS3
p = 0.03R2: 0.04
2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS4
p < 0.0001R2: 0.15
2 3 4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS5
a b c d
p = 0.02R2: 0.04
2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS6
e p < 0.0001R2: 0.13
2 3 4 5 6 7 8 90.00
0.03
0.06
0.09
0.12
0.15
0.18
0.21
0.24
0.27
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS7
f p = 0.003R2: 0.07
0 1 2 3 4 5 6 7 8 9 100.000.020.040.060.080.100.120.140.160.180.200.220.240.26
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS9
g p < 0.0001R2: 0.19
0 1 2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
Log Viral Load
amin
o ac
id m
utat
ions
arou
nd P
CS10
h
p < 0.0001R2: 0.14
0 1 2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
Log Viral Load
amino
acid
mutat
ions a
roun
d PCS
11
i p = 0 .0009R2 = 0.08
1 2 3 4 5 6 7 8 90.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
amino
acid
mutat
ions a
djeca
nt to
PCS1
j
Log Viral Load
p = 0.005R2 = 0.06
2 3 4 5 6 7 8 90.000.020.040.060.080.100.120.140.160.180.200.220.240.260.28
amino
acid
mutat
ions a
djeca
nt to
PCS2
Log Viral Load
k
2 3 4 5 6 7 8 90.000.020.040.060.080.100.120.140.160.180.200.220.240.260.28
p = 0.03R2 : 0 .04
amino
acid
mutat
ions a
djeca
nt to
PCS
4
l
Log Viral Loadp = 0.02
R2 = 0 .04
2 3 4 5 6 7 8 90.00
0.03
0.06
0.09
0.12
0.15
0.18
0.21
0.24
0.27
0.30
amino
acid
mutat
ions a
djeca
nt to
PCS
7
Log Viral Load
n p = 0.002R2 = 0.07
1 2 3 4 5 6 7 8 90.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
Log Viral Load
amino
acid
mutat
ions a
djeca
nt to
PCS
11
op = 0.02R2 = 0.04
2 3 4 5 6 7 8 90.000.020.040.060.080.100.120.140.160.180.200.220.240.260.28
Log Viral Load
amino
acid
mutat
ions a
djeca
nt to
PCS
6
m
1 2 3 54 6 7 81: 100bp ladder2: negative control3: VSV-L, 357bp4: VSV-M, 419bp5: VSV-N, 423bp6: VSV-P, 476bp7: VSV-G, 820bp8: PSC site 2
Agarose gel electrophoresis of RT-PCR products.The results demonstrate the expression of RNA of peptides overlapping the p27/p2 site of protease cleavage site ofSIVmac239.
week 2 after immunization
PBSPCS1
PCS2PCS3
PCS4PCS5
PCS6PCS7
PCS8PCS9
PCS10
PCS11
PCS120.0
0.5
1.0
1.5
2.0
2.5
protease cleavage sites
IgM
(OD
405
nm
)
IgM antibodies to the peptides overlapping the 12 protease cleavage sites were detected in BALB/c mice 2 weeks after immunization with recombinant vesicular stomatitis virus.
A vaccine expressing PCS peptides generates antibody and T cell responses
Vaccine C ontrol Vaccine C ontrol Vaccine C ontrol Vaccine C ontrol
0
25
50
75
100
% m
ajor
PC
S a
min
o ac
id m
utat
ion
NS NS NS NS
Vaccine C ontrol Vaccine C ontrol Vaccine C ontrol Vaccine C ontrol
0
25
50
75
100
125
% m
ajor
PC
S a
min
o ac
id m
utat
ion
NS NS NS *P=0.02
A. Peak
B. Set-point
P C S 2 (-8 ) P C S 2 (-7 ) P C S 2 (-6 ) P C S 12 (-8)PR QD QE /R GR /E
P C S 2 (-8 ) P C S 2 (-7 ) P C S 2 (-6 ) P C S 12 (-8)PR QD QE /R GR /E
% m
ajor
PC
S am
ino
acid
mut
atio
n%
maj
or P
CS
amin
o ac
id m
utat
ion
C.
METHODS
RESULTS RESULTS
INTRODUCTIONDeveloping an effective preventative vaccine for HIV has proved to be an enormous scientific challenge. The classical approach to HIV vaccine development has shown at best, only modest ef-ficacy. An effective HIV vaccine should not only generate potent immune responses capable of destroying infected cells, but also limit aberrant immune activation that increases the susceptibil-ity of the HIV target cell population to infection. Vaccines that generate focused immune re-sponses to highly conserved regions of HIV protease cleavage regions (PCS) might be able to strike such a balance. The HIV protease is a 99-amino acid aspartic enzyme that mediates the cleavage of Gag, Gag-Pol and Nef precursor polyproteins. This process is highly specific, tempo-rally regulated and essential for the production of infectious viral particles (1-4). Twelve proteo-lytic reactions are required to generate an infectious virion, and a single impaired cleavage reac-tion can render the virus noninfectious (5). Thus, an HIV vaccine eliciting immune responses tar-geting the sequences surrounding these PCS could be highly efficacious.
we assessed vaccine immunogenicity elicited against PCS immunogens using a modified Vesicular stomatitis virus (VSV) vector combined with a nanodeliv-ery system. We then evaluated protective efficacy to disrupt SIV acquisition and disease progression using a Cynomolgus macaque (Macaca facicularis) and SIVMAC239 intrarectal challenge model. To examine whether the immune driven viral mutations surrounding the PCS were detrimental to the virus, we ampli-fied and sequenced the plasma viruses by 454-pyrosequencing and correlated the amino acid mutations surrounding the PCS regions with alterations in viral load and maintenance of CD 4 T cell counts.
PCS specific immune responses protect cynomolgus monkeys against pathogenic SIVMAC239 infection and disease progression
p = 0 .0 3 3R2 = 0 .3 8
2 3 4 5 6 70.110
0.135
0.160
0.185
0.210
0.235
0.260
0.285
0.310
0.335
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S6
C87129Fp = 0 .0 2 3R2 = 0 .3 2
4 5 6 7 80.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
L o g v ira l L o a d
amin
o ac
id m
utat
ions
/PC
S11
C88066Fp = 0 .0 0 0 2R2 = 0 .6 8
5 6 7 80.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S12
C88066Fp = 0 .0 4
R2 = 0 .2 6
4 5 6 7 80.000
0.005
0.010
0.015
L o g V ira l L o a d
mut
atio
ns/a
min
o ac
id a
djac
ent t
o PC
S11
C88066Fp = 0 .0 4 7R2 = 0 .5 1
3 4 5 6 70.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S4
C90038F
p = 0 .0 3 7R2 = 0 .4 9
1 2 3 4 5 6 70
1
2
3
4
5
6
7
8
9
L o g V ira l L o a d
sequ
ence
s w
ith fr
ame
shift
mut
atio
nsat
PC
S12
(%)
C90038FP = 0 .0 3
R2 = 0 .5 1
1 2 3 4 5 6 7 81.1
1.2
1.3
1.4
1.5
1.6
1.7
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS1
2
C90038F
p = 0 .0 8R2 = 0 .6 8
4 5 6 70.00
0.05
0.10
0.15
0.20
0.25
0.30
L o g V ira l L o a d
amin
o a
cid
mu
tati
on
s/P
CS
8
C91069Fp = 0 .0 1 1R2 = 0 .4 1
3 4 5 6 7 80
1
2
3
4
5
6
7
8
9
10
L o g V ira l L o a d
seq
uenc
es w
ith
fram
e sh
ift m
utat
ions
at P
CS1
(%)
C93053F
p = 0 .0 0 6R2 = 0 .8 7
3 4 5 6 7 80.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS1
0 p = 0 .0 0 4R2 = 0 .9
3 4 5 6 7 80.000
0.005
0.010
0.015
L o g V ira l L o a d
mut
atio
ns/a
min
o ac
id a
djac
ent t
o PC
S10
p = 0 .0 3R2 = 0 .4 6
2 3 4 5 6 7 80.000.020.040.060.080.100.120.140.160.180.200.220.240.26
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S11
C92078F
C92078F C92078Fp = 0 .0 5
R2 = 0 .5 6
1 2 3 4 5 6 7 80.000.020.040.060.080.100.120.140.160.180.200.220.240.260.28
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S10
C87114FP = 0 .0 0 2R2 = 0 .5 9
4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS1
1
C91028F
P = 0 .0 3R2 = 0 .7 3
4 5 60.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS7
C91032FP = 0 .0 4 5R2 = 0 .6 8
4 5 6 70.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS6
C91032F
p = 0 .0 0 6R2 = 0 .5 2
4 5 6 7 8 90.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
L o g V ira l L o a d
amin
oa a
cid
mut
atio
ns/P
CS7
C91028F
P = 0 .0 0 7R2 = 0 .5 7
4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS4
C91048Fp = 0 .0 1 2R2 = 0 .5 2
4 5 6 7 8 9
0.10
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.20
0.21
L o g V ira l L o a d
amin
o ac
id m
utat
ions
/PC
S1
C91048FP = 0 .0 4 6R2 = 0 .6 7
4 5 6 70.00
0.02
0.04
0.06
0.08
0.10
0.12
L o g V ira l L o a d
amin
o ac
id m
utat
ions
adj
acen
t to
PCS5
C91032F
p = 0.0005
1 2 3 4 5 6 7 8 9-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Log V iral Load
Co
ns
erv
ed
am
ino
ac
idm
uta
tio
ns
aro
un
d P
CS
1 (
-10
/+1
0)/
PC
S
p < 0 .0001
1 2 3 4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Log V iral Load
No
n-c
on
serv
ed a
min
o a
cid
mu
tatio
ns
aro
un
d P
CS
1 (-
10/+
10)/P
CS
p = 0 .04
1 2 3 4 5 6 7 8 90.00
0.01
0.02
0.03
0.04
0.05
0.06
Log V iral Load
Sto
p c
od
on
s ar
ou
nd
PC
S1
(-10
/+10
)/PC
S
p = 0 .01
2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Log V iral Load
No
n-c
on
se
rve
d a
min
o a
cid
mu
tati
on
s a
rou
nd
PC
S3
(-1
0/+
10
)/P
CS
p = 0 .047
2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Log V iral Load
No
n-c
on
serv
ed a
min
o a
cid
mu
tati
on
s ar
ou
nd
PC
S4
(-10
/+10
)/P
CS
P < 0 .0001
2 3 4 5 6 7 8 90.00
0.03
0.06
0.09
0.12
0.15
0.18
0.21
0.24
Log V iral Load
Non
-con
serv
ed a
min
o ac
idm
utat
ions
aro
und
PC
S5
(-10
/+10
)/PC
S
p = 0 .03
2 3 4 5 6 7 8 90.00
0.03
0.06
0.09
0.12
0.15
0.18
0.21
0.24
0.27
0.30
Log V iral Load
No
n-c
on
serv
ed a
min
o a
cid
mu
tati
on
s ar
ou
nd
PC
S6
(-10
/+10
)/P
CS
P < 0 .0001
2 3 4 5 6 7 8 90.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
Log V iral Load
No
n-c
on
serv
ed a
min
o a
cid
mu
tati
on
s ar
ou
nd
PC
S7
(-10
/+10
)/P
CS
p = 0 .003
1 2 3 4 5 6 7 8 90.00
0.03
0.06
0.09
0.12
0.15
0.18
Log V iral Load
No
n-c
on
serv
ed
am
ino
aci
dm
uta
tio
ns
aro
un
d P
CS
9 (
-10
/+1
0)/
PC
S
p = 0.04
1 2 3 4 5 6 7 8 90.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
Log V iral Load
Co
nse
rve
d a
min
o a
cid
mu
tati
on
s a
rou
nd
PC
S1
0 (
-10
/+1
0)/
PC
S
p < 0.0001
1 2 3 4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Log V iral Load
No
n-c
on
serv
ed
am
ino
aci
dm
uta
tio
ns
aro
un
d P
CS
10
(-1
0/+
10
)/P
CS
p = 0.0004
1 2 3 4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
Log V iral Load
Co
nse
rve
d a
min
o a
cid
mu
tati
on
s a
rou
nd
PC
S1
1 (
-10
/+1
0)/
PC
S
p = 0.0006
1 2 3 4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
Log V iral Load
No
n-c
on
se
rve
d a
min
o a
cid
mu
tati
on
s a
rou
nd
PC
S1
1 (
-10
/+1
0)/
PC
S
a b c d e
f g h i j
k l mp = 0.01R2 =0.84
1 2 3 4 5 6 7 80.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Log V ira l Load
No
n-c
on
serv
ed a
min
o a
cid
sub
stit
uti
on
s ar
ou
nd
PC
S1
2 (-
10
/+1
0) (
%)
n p = 0.0001R2 = 0.68
5.5 6.0 6.5 7.0 7.5 8.00.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Log V ira l Load
No
n-c
on
serv
ed a
min
o a
cid
sbst
itu
tio
ns
aro
un
d P
CS1
2 (-
10/+
10) (
%)
o
p = 0.02R2 = 0.62
3 4 5 6 70.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
No
n-c
on
serv
ed a
min
o a
cid
mu
tati
on
s ar
ou
nd
PC
S6 (-
10/+
10) (
%)
Log V ira l Load
p p = 0.004R2 = 0.55
4 5 6 7 8 90.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Log V ira l Load
No
n-c
on
serv
ed a
min
o a
cid
sub
stit
uti
on
s ar
ou
nd
PC
S1
2 (-
10
/+1
0) (
%)
q p = 0.007R2 = 0.73
4.5 5.0 5.5 6.0 6.50.00
0.01
0.02
0.03
0.04
Log v iral Load
Co
nse
rved
am
ino
aci
dsu
bst
itu
tio
ns
aro
un
d P
CS
1 (-
10/+
10) (
%)
r p = 0.01R2 = 0.52
4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7 .0 7 .5 8 .00 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0
0 .1 2
Log V iral Load
No
n-c
on
serv
ed a
min
o a
cid
su
bst
itu
tio
ns
aro
un
d P
CS
9 (-
10/+
10)
s p = 0.04R2 = 0.48
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.50.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Log V ira l Load
No
n-c
on
serv
ed
am
ino
aci
dsu
bst
itu
tio
ns
aro
un
d P
CS
7 (
-10
/+1
0)
(%)
t
R2 = 0.09 R2 = 0.12 R2 = 0.03 R2 = 0.05 R2 = 0.03
R2 = 0.14 R2 = 0.04 R2 = 0.12 R2 = 0.07 R2 = 0.03
R2 = 0.16 R2 = 0.09 R2 = 0.09
Immune responses to PCS peptides drive viral mutation that is detrimental to the challenge virus
Amino acid mutations surrounding PCS at the peak and set point correlated with reduced peak and set point viremia
2.0
3.0
4.0
5.0
6.0
7.0
8.0
0500
100015002000250030003500400045005000550060006500700075008000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
CD4+ CD8+ Log (VL)
Log
vira
l loa
d
weeks after infection
Q(-7)>R/L (PCS12)@
@ 92%
& P(-8)>R/L (PCS2)
& 43%
& 24%
& 35%
& 23%
& 30%
& 34%
& 26%
# G(-7)>D (PCS2)
# 16%
$ K(+4)>R (PCS11)
* G(-8)>R/E (PCS12)
* 10%
Y(-6)>C (PCS12)
13%
C87114F
CD
4+ o
r CD
8+ T
cel
l cou
nts/
mm
3 Q(-6)>E/R (PCS2)
E(+6)>K/R (PCS12)
# 29%29%
# 17%17%
# 24% 25%
# 17%18%
# 20%20%
16%
& 31%# 31%
28% $ 18%30%29%*
PCS11-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10L V S Q G I R Q V L F L E K I E P A Q E
PCS2-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10G G P G Q K A R L M A E A L K E A L A P
PCS12-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10N Q G Q Y M N T P W R N P A E E R E K L
We thank the staff of VTS of National Microbiology laboratory and Canadian Food Inspection Agency (CFIA) for their dedica-tion in this study; Ms. Michelle Janes for her technical support in viral load analysis; and the professional support from the staff of Genomic Core and Bioinformatics Core of National Microbiology Laboratory for SIV pyrosequencing and data analysis, as well as the project support from Dr. Mike Drebot and Dr. Graham Tipples. The study is supported by a CIHR/CHVI catalyst grant, the National Microbiology Laboratory, Public Health Agency of Canada and a NIH grant R01AI111805.
References
Our proof of concept study has demonstrated the feasibility of this vaccine approach in several ways. 1) it is possible to generate both T cell and antibody responses to the 12 20-amino acid peptides using a modified VSV vector and a Nano deliv-ery system. 2) the low-magnitude, focused immune responses to multiple PCS regions can provide protection against SIVmac239 infection. Studies have shown that higher viral load increases the risk of sexual HIV transmission, and mother-child HIV transmission(6). The increased dose of SIVmac239 intrarectal challenge in our study simulates the high viral load condi-tion in sexual HIV transmission. The resistance of macaques to the higher SIVmac239 challenge dose is significantly correlated with their ability to generate immune responses to the number of PCS. The second measurement of the effectiveness of the vaccine is whether a vaccine targeting PCS can offer protection from disease progression after infection. Our study showed that a vaccine targeting PCS can also protect macaques against disease progression. HIV disease progression is measured by rapid CD4 decline; this is also shown in monkey models. We showed that 1) PCS vaccinated monkeys can maintain significantly higher and healthy CD4 counts. 2) there is a significant inverse correla-tion between mutations surrounding the PCS and the viral load. 3) both conserved and non-conserved amino acid substitu-tions around the PCS correlated significantly with lower viral load. The data demonstrated that protease cleavage of the pathogenic SIVmac239 is extremely vulnerable to any amino acid alternations around PCS, thus the sequences surrounding PCS are good targets for an effective HIV vaccine. Our proof of concept study demonstrated that disrupting HIV maturation process by vaccination against sequences surrounding the PCS is feasible and effective.
1. Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 331:280. 2. Kaplan AH, Zack JA, Knigge M, Paul DA, Kempf DJ, Norbeck DW, et al. Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles. J Virol. 67:4050. 3. Krausslich HG, Ingraham RH, Skoog MT, Wimmer E, Pallai PV, Carter CA. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. PNAS U S A. 86:807. 4. Louis JM, Ishima R, Torchia DA, Weber IT. HIV-1 protease: structure, dynamics, and inhibition. Adv Pharmacol. 55:261. 5. Billich S, Knoop MT, Hansen J, Strop P, Sedlacek J, Mertz R, et al. Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease. J Biol Chem. 263:17905; 6. Chappell CA, Cohn SE. Prevention of perinatal transmission of human immunodeficiency virus. Infect Dis Clin North Am. 28:529
