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HIV-1 envelope glycoproteins isolated from
Viremic non-progressors HIV-infected
individuals are fully functional and cytophatic
Silvia Marfil1, Veronique Hebman2, Sara Marrero-Hernandez3, Daniel
Márquez-Arce3, Romina Cabrera-Rodríguez3, Maria Soledad Varela3,
Concepción Casado4, Cecilia Cabrera1, Víctor Urrea1, María Pernas4,
Bonaventura Clotet1, Cecilio López-Galíndez4, Martine Biard-
Piechazyck2, Agustín Valenzuela3, Julià Blanco1,5
2017/11/30
1 Inst. de Recerca
de la Sida IrsiCaixa,
Inst. de Recerca
Germans Trias i
Pujol (IGTP),
Badalona, Spain.
2 CNRS, Centre
d’études d’agents
Pathogènes et
Biotechnologies
pour la Santé,
Montpellier, France
3 Lab. Inmunología
Celular y Viral, Fac.
Ciencias de la
Salud, Univ. de La
Laguna (ULL),
Tenerife, Spain
4 Instituto de Salud
Carlos III, Madrid
Spain.
5 Universitat de
ViC-UCC,
Barcelona, Spain
G E S I D A 2 0 1 7
VIREMIC NON PROGRESSION
HIV-1 disease progression
RP, CD4<350 // <3 year
SP
LTNP, VL>2x103
LTNP-EC, VL<50
VNP, VL>104 // CD4>500 //>2 year
G E S I D A 2 0 1 7
HYPOTHESIS AND OBJECTIVES
Viruses isolated from VNP show low replicative fitness. Weber et al. Impaired human immunodeficiency virus type 1 replicative fitness in atypical viremic
non-progressor individuals. AIDS Res Ther 14, 15. 2017
Viruses isolated from VNP show preserved nef function. Heigele et al. Viremic long-term nonprogressive HIV-1 infection is not associated with
abnormalities in known Nef functions. RETROVIROLOGY 11, 13. 2014
Viral fitness is determined by Env function. Rangel et al. Role of the human immunodeficiency virus type 1 envelope gene in viral fitness. J Virol
77:9069-73. 2003
To analyze the role of Env in the VNP phenotype
To compare Env functions betweenVNP and RP
G E S I D A 2 0 1 7
SAMPLES
VNP(n=4) RP(n=5) P-value
CD4Abs(cells/uL) 840 262 *
VL(copies/mL) 78953 77000 ns
Timefromseroconversion(years)
5
0.7
*
HAART 0 0 ns
Gender(%ofmale) 100 100 ns
Curriu et al. Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins
Show Anti-gp41 Antibodies with Unique Specificity and Function. PLoS One 7: e30330. 2013
G E S I D A 2 0 1 7
ENV CLONING AND SELECTION
Selected clones show similar activity and heterogeneity than
full set.
VNP8VNP9
VNP11VNP16
RP2RP6
RP7RP8
RP10
0
100
200
300
400
500
p=0.0065 M-W test
p=0.030 K-W test
Rela
tive F
usio
n
(% o
f B
aL
)
VNP RP0
100
200
300
400
500p=0.0065
Rela
tive F
usio
n(%
of
BaL
)
164
(n=44)
236
(n=56)
VNP RP0
100
200
300
400
500ns
Rela
tive F
usio
n(%
of
BaL
)
172
(n=15)
220
(n=17)
G E S I D A 2 0 1 7
EXPRESSION AND FUSION (Cell-to-cell fusion)
Env clones from VNP and RP show similar expression and
specific fusogenicity.
0
5
10
15
20
VNP RPF
US
OG
EN
ICIT
Y
(FU
SIO
N/E
XP
RE
SS
ION
)
0
10
20
30
40
VNP RP
EX
PR
ES
SIO
N(%
of E
nv+
ve c
ells
)
env
293T cells
TZM-bl cells
tat
+
Light
Env Expression
6 h
G E S I D A 2 0 1 7
INFECTIVITY (Cell-free virus infection)
0
2
4
6
INF
EC
TIV
ITY
(Lo
g T
TC
ID50/m
L)
VNP RP
0 2 4 60
100
200
300
400
500
VNP
RP
r = 0.3601
p= 0.0364
INFECTIVITY (Log TCID50/mL)
RE
LA
TIV
E F
US
ION
(%
of B
aL
)
0 2 4 60
10
20
30
40
VNP
RP
r = 0,4728
p= 0,0072
INFECTIVITY (Log TCID50/mL)
En
v +
ve 2
93T
cells (%
)
Env clones from VNP and RP show similar infectivity.
env
TZM-bl cells
pSG3
Light 293T cells
titer
48 h
G E S I D A 2 0 1 7
MULTIVARIATE ANALYSIS OF INFECTIVITY
INFECTIVITY vs
ESTIMATE SD Z-score P-value
Expression 0.797330 0.281987 2.8275 0.0035**
Fusogenicity 0.002769 0.003123 0.8865 0.367
Group (VNP or RP) -0.196258 0.582594 -0.3369 0.7171
Infectivity of Env clones is related to expression but not to
fusogenicity or their origin (VNP or RP).
G E S I D A 2 0 1 7
CD4 BINDING AND SIGNALING CAPACITY
VNP RP0
10
20
30
40
HIV
TR
AN
SF
ER
(p24+
cells, %
)
VNP RP0
1
2
3
4
Rela
tive
CD
4 B
ind
ing
VNP RP0
10
20
30
40
EX
PR
ES
SIO
N(%
of
En
v+
ve c
ells)
env pSG3
+ 293T cells
Primary CD4 T cells
CEM.NKR-CCR5 cells
CD4 binding and signaling are similar in VNP and RP.
Blanco et al. High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells..
J Biol Chem 279: 51305-14, 15. 2004
Valenzuela et al. HDAC 6 regulates HIV-1 infection. Mol Biol Cell 16:11, 5445-54. 2005
2 h
1 h WB
G E S I D A 2 0 1 7
CYTOPATHICITY (Autophagy induction)
Autophagosomes identified by puncta using
anti LC3 antibody.
No information on primary isolates or
CCR5-using viruses
G E S I D A 2 0 1 7
CYTOPATHICITY (Autophagy induction)
env
+ 293T cells
HUT CCR5+ T cells
Env isolated from VNP are cytophatic
Cytopathicity is associated with fusogenic activity.
% o
f d
ead
cells
5
10
15
20
25
30
35
40
NK RIS
Analysis of cell death by Trypan blue exclusion
LC3
pu
nct
a/ce
ll
NK RIS
5
6
7
8
9
10
11
Analysis of autophagy by endogenousLC3 labelling
r = 0.731
p = 0.025
0 200 400 600 8005
6
7
8
9
10
FUSOGENICITY (%)
AU
TO
PH
AG
Y
(LC
3 p
un
cta
/ce
ll)VNP
72 h
RP
G E S I D A 2 0 1 7
CONCUSIONS
Small differences in fusogenicity hardly justify the
VNP phenotype
Infectivity, CD4 binding and signaling are similar in
both groups.
Cytopathicity as measured by autophagy induction
is similar in both groups. (First description of autophagy
induction by primary Env)
IMMUNOLOGICAL FACTORS??
G E S I D A 2 0 1 7
Silvia Marfil
Victor Urrea
Elisabet García
Jorge Carrillo
Marta Curriu
Bonaventura Clotet
Julià Blanco
Elisabet Gómez
Cecilia Cabrera
THANKS
Sara Marrero-Hernandez
Daniel Márquez-Arce
Romina Cabrera-Rodríguez
Maria Soledad Varela
Agustín Valenzuela
Concha Casado
María Pernas
Cecilio López-G.
Veronique Hebman
Martine Biard-P.
G E S I D A 2 0 1 7
