JVI Accepts, published online ahead of print on 2 April 2014 J. Virol. doi:10.1128/JVI.03785-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
3
Pathogenic Features Associated with Increased Virulence upon Simian Immunodeficiency Virus Cross-Species Transmission from Natural Hosts
4
Running head: SIVsab Infection of Pigtailed Macaques
5
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6 7 8 9
Daniel T. Mandell1*, Jan Kristoff2*, Thaidra Gaufin1, Rajeev Gautam1, Dongzhu Ma2, Netanya Sandler3, George Haret-Richter2, Cuiling Xu2, Hadega Aamer2, Jason Dufour4, Anita Trichel5, Daniel C. Douek3, Brandon F. Keele6, Cristian Apetrei1,2,7*¶, and Ivona Pandrea2,8,9*¶
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11 12 13 14 15 16 17 18 19 20 21 22 23 24
Divisions of 1Microbiology, 4Veterinary Medicine and 8Comparative Pathology, Tulane National Primate Research Center (TNPRC), Covington, LA, 70433; 2Center for Vaccine Research, University of Pittsburgh, Pittsburgh PA 15261; 3Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; 5Division of Laboratory Animal Resources and Departments of 7Microbiology and Molecular Genetics and 9Pathology, School of Medicine, University of Pittsburgh, Pittsburgh PA15261; 6AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD 21702
1 2
*equally contributed to this study Key words: Simian Immunodeficiency Virus, African green monkey, pigtailed macaques, animal models, pathogenic infection, viral loads.
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Title character counts: Running head character counts: Word counts: Abstract: Importance Text: Figures:
136 38 238 117 5643 8 (7 color)
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To whom the correspondence should be addressed. Ivona Pandrea, M.D., Ph.D. Center for Vaccine Research, 9045 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15261. Phone: (412) 624-3242; Fax: (412) 624-4440; E-mail:
[email protected] Cristian Apetrei, M.D., Ph.D. Center for Vaccine Research of the University of Pittsburgh, 9044 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15261. Phone: (412) 624-3235; Fax: (412) 624-4440;E-mail:
[email protected] 1
43
Abstract
44
While SIVs are generally nonpathogenic in their natural hosts, dramatic increases in
45
pathogenicity may occur upon cross-species transmission to new hosts. Deciphering
46
the drivers of these increases in virulence is of major interest for understanding the
47
emergence of new HIVs. We transmitted SIVsab from the sabaeus species of African
48
green monkeys (AGMs) to pigtailed macaques (PTMs). High acute viral replication
49
occurred in all SIVsab-infected PTMs, yet the outcome of chronic infection was highly
50
variable, ranging from rapid progression to controlled infection, which was independent
51
of the dynamics of acute viral replication, CD4+ T cell depletion, or preinfection levels of
52
microbial translocation. Infection of seven PTMs with plasma collected at necropsy from
53
a rapid progressor PTM was consistently highly pathogenic, with high acute and chronic
54
viral replication, massive depletion of memory CD4+ T cells and disease progression in
55
all PTMs. Plasma inoculum used for the serial passage did not contain adventitious
56
bacterial or viral contaminants. Single genome amplification showed that this inoculum
57
was significantly more homogenous than the inoculum directly derived from AGMs,
58
pointing to a strain selection in PTMs. In spite of similar peak plasma viral loads
59
between the monkeys in the two passages, immune activation/inflammation levels
60
dramatically increased in PTMs infected with the passaged virus. These results suggest
61
that strain selection and a massive cytokine storm are major factors behind increased
62
pathogenicity of SIV upon serial passage and adaptation of SIVs to new hosts following
63
cross-species transmission.
64
2
65
Importance
66
We report here that upon cross species transmission and serial passage of
67
SIVsab from its natural host, the sabaeus African green monkey (AGM), to a new
68
pigtailed macaque (PTM) host, viral adaptation and increased pathogenicity involves
69
strain selection and a massive cytokine storm. These results permit the design of
70
strategies aimed at preventing cross-species transmission from natural hosts of SIVs to
71
humans in endemic areas. Furthermore, our study describes a new animal model for
72
SIV infection. As the outcome of SIVsab infection in PTMs, African green monkeys, and
73
rhesus macaques is different, the use of these systems enable comparative studies
74
between pathogenic, nonpathogenic and elite-controlled infections, to gain insight into
75
the mechanisms of SIV immunodeficiency and comorbidities.
76
3
77
Introduction
78
Simian immunodeficiency viruses naturally infect African nonhuman primates
79
(NHPs) with virtually no deleterious pathogenic consequences and have a relatively
80
high propensity for cross-species transmission in the wild and in research settings (1-7).
81
Both human immunodeficiency virus (HIV) type 1 and HIV-2, the viruses responsible for
82
the AIDS pandemic, originated by cross-species transmission of SIVs infecting
83
chimpanzees/gorillas (SIVcpz/gor) and sooty mangabeys (SIVsmm), respectively (8).
84
While multiple cross-species transmission events from NHPs to humans have been
85
documented to occur over the last century (8), only a fraction of these resulted in the
86
emergence of pandemic (HIV-1 group M) or epidemic (HIV-1 group O and HIV-2 groups
87
A and B) viruses. The remaining viral forms were apparently contained, with limited
88
spread restricted to the area of emergence (8). Likewise, accidental or experimental SIV
89
cross-species transmission from multiple African NHP hosts to rhesus macaques (RMs)
90
may have a very diverse pattern of pathogenic outcomes, ranging from progression to
91
AIDS (9), to controlled infections (10-14). The mechanism(s) driving the increases in
92
pathogenicity of SIVs in new hosts upon cross-species transmission are unclear, but
93
serial passage appears to be a requirement (9, 15). Altogether, these observations
94
suggest that in addition to exposure to new species, viral adaptation is required for the
95
successful emergence of the virus as a highly virulent pandemic pathogen in the new
96
host species. Identification of factors driving the efficacy of SIV cross-species
97
transmission is paramount for assessing the risks for emergence of new HIV strains in
98
human populations and for designing strategies to effectively control cross-species
99
transmissions, especially considering the massive exposure of humans in sub-Saharan
100
Africa to a plethora of SIVs naturally infecting numerous species of monkeys (8, 16).
101
The RM, which is a widely used model for AIDS pathogenesis and vaccine
102
studies, is not appropriate for the study of the mechanisms of SIV adaptation to a new 4
103
host upon cross-species transmission. With the exception of SIVsmm, which induces
104
progressive infection upon its direct transmission from the sooty mangabey
105
(Cercocebus torquatus atys) (17, 18), cross-species transmitted SIVs are controlled in
106
RMs (19) due to numerous host restriction factors (20). Conversely, pigtailed macaques
107
(PTMs) appear to be a more appropriate animal model for the study of SIV adaptation
108
following cross-species transmission. In most cases, SIV cross-species transmissions to
109
PTMs result in persistent infection and progressive disease, as reported after
110
experimental PTM exposure to SIVsmm (17, 21), SIVsun from Cercopithecus lhoesti
111
solatus (22), SIVlho from C. lhoesti lhoesti (22), SIVver from Chlorocebus pygerythrus
112
(23, 24) and SIVrcm from Cercocebus torquatus torquatus (25). A reason for increased
113
susceptibility to cross-species-transmitted SIV is that TRIM5α restriction (26) is not
114
effective in PTMs due to aberrant mRNA splicing, resulting in TRIM5α isoforms unable
115
to restrict either HIV-1 or SIV infection (27).
116
Here we report that experimental infection of PTMs with SIVsab, the virus that
117
naturally infects Western African green monkey (AGM) species (the sabaeus monkey)
118
(28), has a highly variable outcome, ranging from rapid progression to controlled
119
infection. A single serial passage of the virus, however, leads to a consistently
120
pathogenic infection, with all PTMs infected with the passaged virus replicating SIVsab
121
at high levels during chronic infection and progressing to AIDS within 1-2 years
122
postinfection (pi).
123 124
Materials and Methods
125
Animals and infection. Twelve PTMs (Macaca nemestrina) 4 to 8 years of age
126
were included in this study. All animals were negative for simian T-cell lymphotropic
127
virus (STLV) (Vironostika HTLV-I/II ELISA, BioMerieux, Durham, NC) and SIVsab (29)
5
128
and were housed at the Tulane National Primate Research Center (TNPRC) and the
129
Regional Industrial Development Corporation (RIDC) Park campus of the University of
130
Pittsburgh. Both sites are Association for Assessment and Accreditation of Laboratory
131
Animal Care (AAALAC) International accredited facilities. The animals were fed and
132
housed according to regulations set forth by the Guide for the Care and Use of
133
Laboratory Animals (30) and the Animal Welfare Act. Animal experiments were
134
approved by the Tulane University and University of Pittsburgh Institutional Animal Care
135
and Use Committees (IACUCs).
136
Five PTMs (passage 1-P1) received an intravenous (iv) inoculation of a plasma
137
virus inoculum obtained from an acutely SIVsab92018-infected sabaeus AGM. The
138
inoculum was adjusted to contain 106 vRNA copies, corresponding to 150 tissue culture
139
infectious doses 50% (TCID50) (31). To increase SIVsab pathogenicity in PTMs and to
140
obtain more reproducible infection patterns, seven PTMs (passage 2-P2) received an
141
inoculum of one ml of plasma adjusted to 106 SIVsab RNA copies/ml harvested from the
142
terminally-infected P1 rapid progressor PTM BH66.
143 144
Sampling and cell separation. Blood was collected (10, 31) twice preinfection,
145
twice weekly for 3 weeks pi, weekly thereafter up to the set-point, and every two months
146
during chronic infection (2-36 months pi). Axillary LNs were collected by excisional
147
biopsy (31) at 0, 21, 180 and 360 dpi. Duodenal biopsies were collected by endoscopic
148
guided biopsy prior to infection, during acute infection (10, 21 and 28 dpi) and during
149
chronic SIVsab infection (monthly for 6 months pi and every three months thereafter).
150
Intestinal resections (5-10 cm) were removed surgically (31) at -35, 8, and 225 dpi.
151
Additional LNs and intestinal tissues were obtained at necropsy.
6
152
Plasma, peripheral blood mononuclear cells (PBMCs), and mononuclear cells
153
from LN and intestinal biopsies, as well as from multiple tissues collected at the
154
necropsy were isolated as described (31-33).
155 156
Viral quantification. Plasma VLs were quantified by SIVsab92018/BH66-
157
specific real-time PCR (31). Viral RNA (vRNA) loads were also quantified in
158
mononuclear cells isolated from blood, LNs and intestinal biopsies, as well as from
159
tissues collected at necropsy (33, 34). Simultaneous quantification of RNAse P (RNase
160
P detection kit, Applied Biosystems, CA), a single copy gene with 2 copies per diploid
161
cell, was performed to normalize sample variability and allow accurate quantification of
162
cell equivalents (25). Assay sensitivity was 10 vRNA copies/105 cells.
163 164
Antibodies and flow-cytometry. To assess changes in numbers of major T cell
165
populations and their immune activation status, whole blood, LN and intestinal
166
mononuclear cells were stained for flow cytometry (10) with the following monoclonal
167
antibodies (MAbs): CD3 fluorescein isothiocyanate (FITC), peridinin chlorophyll A
168
protein (PerCP) or phycoerythrin (PE), CD4 allophycocyanin (APC) or PerCP; CD8β PE
169
(Beckman Coulter) or PerCP; CD20 PE; CD95 FITC; CD28 APC; HLADR-PerCP; and
170
Ki67 FITC. All MAbs were from BD Bioscience Pharmingen, San Diego, CA, unless
171
otherwise indicated. All antibodies were previously tested and calibrated for use in
172
PTMs (25, 35, 36). Cells were stained (10, 31, 33) and analyzed with a FACSCalibur
173
flow cytometer (BD Immunocytometry Systems) using CellQuest software (BD). The
174
relative proportion of CD4+ and CD8+ T cells in LN and intestine samples was calculated
175
as the “index” of these cell populations (i.e., the product of the percentage of CD3+ T
176
cells gated on lymphocytes multiplied by the percentage of CD4+ or CD8+ T cells,
177
divided by 100). The index is a function of both total CD3+ T cells and CD4+ or CD8+ T 7
178
cells, and provides a more accurate reflection of target cells (10).
179 180
Immunohistochemical staining (IHC). IHC was performed as described (10,
181
33) on formalin-fixed, paraffin-embedded tissues using an avidin-biotin horseradish
182
peroxidase technique (Vectastain Elite ABC kit; Vector Laboratories) and a mouse
183
monoclonal anti-human CD4 primary antibody (NCL-CD4-1F6, Novocastra, Newcastle,
184
UK). Sections were visualized with 3,3-diamidino-benzidine (Dako Corporation,
185
Carpinteria, CA) and counterstained with hematoxylin.
186 187
Dynamics of inflammatory cytokines and chemokines. Cytokine testing in
188
plasma was done using the Cytokine Monkey Magnetic 28-Plex Panel (Invitrogen,
189
Camarillo, CA), as per manufacturer’s instructions. Results were read by Bio-Plex
190
reader (Bio-Rad Laboratories, Hercules, CA), using Luminex technology (Luminex
191
Corporation, Austin, TX).
192 193
Assessment of microbial translocation. The levels of microbial translocation
194
were assessed as follows: plasma LPS levels were measured with the Limululs
195
Amebocyte Lysate Assay (Lonza, Walkersville, MD) (10). Plasma soluble CD14
196
(sCD14) levels were measured by ELISA (Quantikine Human sCD14 Immunoassay,
197
R&D Systems, Minneapolis, MN).
198 199
Trim5α genotyping. Total genomic DNA was isolated from PTM PBMCs using
200
the DNeasy Blood and Tissue kit (Qiagen, Valencia CA) and PCR was used for TRIM5
201
PCR genotyping (26).
202
8
203
Stock testing. The plasma viral stock collected from the rapid progressor PTM
204
BH66 was tested by Bactec for Gram positive and Gram negative bacteria and for
205
Mycobacteria. Serological and PCR testing of the stock was done for the following viral
206
pathogens: macaque cytomegalovirus, simian cytomegalovirus, rhesus rhadinovirus,
207
SA8, herpesvirus papio 1, simian parvovirus, RM parvovirus and PTM parvovirus
208
(Zoologix, Chatsworth CA).
209 210
Single genome amplification (SGA) of SIVsab strains from PTMs. Viral
211
diversity of the two stocks, as well as the strain selection in PTMs from both the cross-
212
species-transmitted virus group and the serially passaged group were characterized by
213
SGA, sequencing and phylogenetic analyses (32, 37-39). All animals were sampled
214
during acute infection, and PTM BH66 was also sampled at necropsy as challenge
215
stock for passage infected animals. All 146 viral sequences from all animals were
216
deposited in GenBank under accession numbers KJ362078-KJ362223.
217 218
In vitro replication studies. The two plasma stocks employed to infect the P1
219
and P2 PTMs were assessed for in vitro replication, as described previously (40).
220
Briefly, PBMCs collected from four uninfected PTMs were depleted of CD8+ cells
221
employing a positive selection of CD8+ T cells (CD8 microbead kit, Miltenyi Biotech,
222
Auburn, CA). CD8+-depleted cells were then stimulated with 10 μg/ml PHA for 2 days,
223
followed by overnight incubation in IL-2 media. Five hundred thousands CD8+-depleted
224
cells were incubated with one ml of plasma adjusted to 106 vRNA copies collected from
225
either an acutely-infected AGM (EI45-i.e., the viral stock used for the P1 monkeys) or
226
from the terminally-infected, rapid progressor BH66 PTM (i.e., the viral stock used for
227
the P2 monkeys). Cells were incubated with plasma for 4 h, followed by two washes to
228
remove cell-free virus and addition of fresh media. For 4 weeks, one half of the 9
229
supernatant was collected every third day and replaced with fresh IL-2-containing
230
media. Virus production in culture supernatants was monitored by SIV P27 antigen
231
capture assay, as previously described (40).
232 233
Induction of cytokine production in vitro. The two plasma stocks used for the
234
infection of the P1 and P2 PTMs were adjusted to 106 vRNA copies and used to
235
stimulate cryopreserved PBMCs collected from the PTMs included in this study prior to
236
SIVsab infection. After a four-hour incubation, cells were washed twice to remove cell-
237
free virus and then cultured for 24 hours. Culture supernatants were harvested at six
238
and 24 hours poststimulation without the replacement of the media. Control cultures
239
included the same in vitro conditions without virus stimulation. Cytokine and chemokine
240
concentrations in the supernatants were measured using the Cytokine Monkey
241
Magnetic 28-Plex Panel (Life Technologies, Carlsbad, CA) according to manufacturer’s
242
instructions. Mean fluorescence of samples was quantified and analyzed using the
243
Luminex detection system.
244 245
Statistical analysis of data. Data comparisons were done using two-tailed
246
nonparametric tests (Mann-Whitney). Where needed, the Gehan-Beslow-Wilcoxon test
247
was performed. Significance was assessed at the p=0.05 level. Area under the curve of
248
VL was done by numerical integration of a spline interpolation of the logarithm of the
249
data, to avoid over-emphasizing the peak of viral infection (Mathematica 6.0, Wolfram
250
Research Inc, IL). Horizontal bars in figures reflect medians. Vertical bars in figures
251
reflect standard error of means. All statistics were performed using Prism 5.0 software.
252 253
10
254
Results
255
Clinical follow-up. To ascertain the factors associated with viral adaptation to a
256
new host, we used two PTM groups. The “cross-species transmitted virus group”
257
received SIVsab92018 directly derived from an acutely-infected Caribbean sabaeus
258
monkey. The derivation of this plasma virus stock is described elsewhere (31). The
259
“serially passaged virus group” received terminal virus collected from a rapid progressor
260
PTM (BH66) from the first group of infected animals. All SIVsab-inoculated PTMs
261
became infected, as documented by plasma vRNA quantification. Infection outcome
262
was highly variable in the cross-species transmitted virus group: one PTM (BH66)
263
experienced rapid disease progression and died with AIDS (weight loss and diarrhea)
264
within 180 dpi; two PTMs progressed to AIDS (weight loss, diarrhea, pneumonia and
265
lymphadenopathy) after 900 dpi (EC10) and 1,080 dpi (CF76); PTM CC35 was still alive
266
at the end of the follow-up (1,250 dpi), and PTM BK42 died 186 dpi after an accidental
267
surgical perforation of the intestine with no signs of viral pathogenesis (Figure 1).
268
Conversely, the PTMs from the serially passaged virus group experienced a
269
significantly more rapid disease progression (p=0.0441) with 3/7 rapid progressor PTMs
270
developing AIDS within 300 dpi and the remaining 4/7 PTMs progressing to AIDS within
271
500 dpi (Figure 1).
272 273
Natural
history
of
SIVsab
infection
in
PTMs
upon
cross-species
274
transmission and serial passage. To assess SIVsab adaptation to the new PTM host,
275
we closely monitored viral replication in plasma and PBMCs throughout infection, as
276
well as in numerous intestine and LN samples collected at necropsy from selected
277
animals (Figure 2). Acute viral replication was similar between the two groups, with
278
plasma VLs peaking at 9-10 dpi, at very high levels (1x109±2.27x108 vRNA copies/ml vs
279
1.16x109± 3.76x108 copies/ml, for the PTMs infected with the cross-species transmitted 11
280
virus and those infected with the serially-passaged virus, respectively, p=0.2043)
281
(Figure 2a). However, post-peak decline of plasma VL was drastically different between
282
the two groups. In the cross-species transmitted group, VLs showed a sharp decline in
283
all but one (BH66) PTM. Conversely, in the serially passaged virus group, control of
284
viral replication was very poor (Figure 2a). Viral set-points were reached in both groups
285
by day 42 p.i., being low and highly variable in the PTMs from the first group (average
286
1.44x106±1.2x106 vRNA copies/ml) and homogenously high in the animals infected with
287
the PTM-passaged virus (average 4.2x107±3.3x107 vRNA copies/ml) (Figure 2a,
288
p50% of the PTMs progressing to AIDS during a 3.5-year follow-up.
541
The remaining PTMs controlled SIVsab during the follow-up. A previous study reported
542
that in SIVsmm-infected RMs the differences in viral replication and disease progression
543
can be attributed to Trim5α genotypes (26). Therefore, as PTMs were reported to carry
544
two Trim isoforms, we first characterized the Trim genotypes in PTMs. All PTMs in both
545
study groups harbored the same permissive Trim genotype and thus, the differences in
546
the rates of disease progression were not due to Trim genotypes.
547
PTM BH66 in the cross-species transmitted group was a rapid progesssor
548
showing virtually no virus control and presenting with AIDS within 6 months of infection.
549
Terminal plasma from this PTM was used to infect a second group of PTMs. All the
550
animals in this group progressed to AIDS in less than two years. Set-point VLs were
551
consistently high in this group, and the natural history of SIVsabBH66 was highly
552
consistent between the infected animals, in agreement with previous studies showing
553
that SIVver, which naturally infects the vervet monkey, is pathogenic in only a fraction of
554
PTMs upon direct cross-species transmission and consistently pathogenic after serial
555
passage (23, 24).
556
To understand the reasons for the observed differences in pathogenicity between
557
the PTMs infected with the cross-species transmitted virus and those infected with the
558
passaged SIVsabBH66 strain, we thoroughly compared the viral and immune
559
parameters between the two groups and showed that SIVsabBH66 infection induced a
560
higher level of acute immune activation and a cytokine storm in the serially passaged
561
group. We reasoned that these are critical factors for SIVsab adaptation to the new 22
562
PTM host, because increased acute immune activation/inflammation will result in an
563
increased availability of SIV target cells (which have an activated phenotype) and thus
564
favors persistent high viral replication. Therefore, we focused on understanding the
565
reason(s) for which passaged virus infection was associated with increased levels of
566
immune activation/inflammation.
567
The differences in acute immune activation/inflammation between the two
568
passages were not due to differences in the levels of acute viral replication, which were
569
similar between the two groups.
570
As PTMs in the second group were infected with a plasma virus collected at the
571
time of AIDS, we further hypothesized that presence of adventitious agents in this stock
572
may
573
activation/inflammation between the two groups. Consequently, we tested the plasma
574
stock
575
immunosuppression and AIDS. The stock did not test positive for any of these
576
opportunistic agents.
be
for
responsible
multiple
for
bacterial
the
observed
and
viral
differences
opportunistic
in
levels
agents
of
immune
associated
with
577
We next assessed the in vitro characteristics of the two viral stocks and showed
578
that, in spite of similar ability to replicate in vitro on CD4-enriched PBMCs, the PTM-
579
derived SIVsabBH66 stock induced significantly higher levels of cytokines in cell
580
cultures than the AGM-derived SIVsab92018. This observation suggests that the
581
increased pathogenicity of infection in the second passage can be directly related to the
582
virus stock. These two sets of experiments were performed in different conditions
583
because, while in vitro viral replication is strictly dependent on CD4+ T cells, the
584
production of cytokines and chemokines that might shape the outcome of SIV infection
585
involves multiple cellular subsets. One may argue that the observed differences in
586
cytokine/chemokine production between the two stocks may not necessarily rely on
23
587
intrinsic differences in virus virulence, as the two plasma stocks were collected from
588
monkeys at different stages of SIV infection (acute infection for P1 vs terminal infection
589
for P2). As such, the P2 stock would contain both infectious and soluble factors that
590
could have been responsible for the more aggressive outcome of the in vitro stimulation
591
assays. Yet, as discussed above, we could not detect major viral or bacterial
592
contaminants in the stock. Furthermore, while the AIDS stage is associated with
593
dramatic increases in soluble factors, the massive viral replication characteristic of
594
acute SIV infection is also associated with dramatic increases in cytokine/chemokine
595
production (66, 68, 69). Finally, we did not employ large stock volumes that would have
596
induced changes in the soluble markers and the volume of plasma used for in vivo
597
infections of PTMs were similar between P1 and P2.
598
We next tested the hypothesis that strain selection associated with the virus
599
passage was responsible for the different outcomes of infection. To this goal, we
600
performed
601
transmitted/founder viruses in the two groups. We also characterized the viral diversity
602
of the two stocks. This analysis showed that the total diversity of the original stock was
603
retained, but was shifted after passage in PTM BH66, and as such, our analysis
604
suggests that adaptation of SIVsab to the new PTM host was associated with and
605
probably necessary for increased pathogenicity upon SIV cross-species transmission.
606
Note, however, that sequence analyses were limited to the env gene, which did not
607
permit us to identify the genetic signature(s) of increased pathogenicity, a process
608
which would require dedicated studies to identify all mutations throughout the genome,
609
generate clones of these various combinations, and test each combination in vitro and
610
in vivo to show what mutations were important for increased pathogenesis. Such
611
experiments were beyond the scope of the current study. In the past, viral determinants
612
of increased pathogenicity, mainly occurring as point mutations, were reported but not
single
genome
amplification
24
and
sequence
analysis
of
the
613
always confirmed as being clearly associated with induced pathogenicity (70).
614
Therefore, the viral mechanisms of increased pathogenicity of SIVs in new hosts are still
615
unclear.
616
Our study demonstrates that the ability of the virus to stimulate the innate
617
immune system is probably the main determinant of increased pathogenicity and virus
618
adaptation to a new host. Identification of the specific motifs/genes associated with
619
increased pathogenicity may contribute to the design of new therapeutic approaches to
620
control HIV.
621
We conclude that a stringent selection of virus variants inducing high levels of
622
immune activation/inflammation and increasing the availability of target cells in the initial
623
stages of infection are conditions for successful viral adaptation to a new host. One may
624
argue that such conditions are achieved only in experimental conditions and that serial
625
passage is not characteristic of viral adaptation. Yet, similar conditions may be achieved
626
when cross-species SIV transmissions occur in association with concurrent infections
627
which contribute to a coincidental inflammatory response. Furthermore, transmission to
628
humans may involve serial passage through injections with unsterilized material (15).
629
Our study shows that irrespective of the circumstances of successful SIV cross-species
630
transmission, virus adaptation is a prerequisite. These results explain the scarcity of
631
documented cross-species transmissions of SIVs to humans, in the context of extensive
632
exposure of humans to a plethora of SIVs in Central Africa (16).
633
In addition to the characterization of SIV adaptation to a new host, we report here
634
the development of a new animal model for progressive SIV infection. SIVsabBH66
635
infection in PTMs is very similar to SIVmac infection in both RMs and PTMs (70-72), the
636
reference NHP models for AIDS vaccine and pathogenesis studies. Furthermore, as per
637
previous studies, SIVsab-infected PTM is the model of choice for the study of SIV25
638
associated comorbidities (i.e., cardiovascular disease), due to the similarities of SIVsab
639
infection in PTMs to HIV-1 infection in humans.
640
Finally, this new model completes a new animal system for performing
641
comparative studies between pathogenic, nonpathogenic and controlled SIV infections.
642
In the only other available system, comparisons between the nonpathogenic SIVsmm
643
infection of sooty mangabeys and the pathogenic SIVsmm/mac infection of RMs (73,
644
74), is considered a major advantage, as it permits meaningful comparative studies to
645
identify the host factors associated with progression to AIDS (75). Our new model also
646
enables similar comparisons between the pathogenic infection of PTMs and natural host
647
(AGM) infection by employing a single SIVsab strain {Pandrea, 2012 #4776}, with
648
several major advantages over the conventional RM/sooty mangabey/SIVmac/smm
649
system: AGMs are not endangered and can thus accommodate invasive studies, which
650
are prohibited in sooty mangabeys. Furthermore, since SIVsab infection of RMs is
651
functionally cured (19), this system expands to comparative studies between
652
pathogenic, nonpathogenic and elite-controlled infections (10, 35), to gain insight into
653
the mechanisms of immunodeficiency associated with lentiviral infection. As such, this
654
new system is unique and can be used to assess essential aspects of AIDS
655
pathogenesis; among these are identifying the discrete immunopathogenic mechanisms
656
of SIV infection and comparing the genetic evolution of SIV in different host species
657
(76), both of which are needed to develop effective strategies for prevention of AIDS
658
disease progression.
659 660
Acknowledgements
661
We thank Preston Marx, Ronald Veazey and Andrew Lackner for helpful
662
discussion. We also thank the veterinary and animal care staff of TNPRC and University
26
663
of Pittsburgh for their service and expertise. This work was supported by funds from
664
grants RO1 AI064066 and R01 AI065325 and P20 RR020159 (CA) from the National
665
Institute of Allergy and Infectious Diseases (NIAID), RO1 RR025781 (CA and IP) from
666
the National Center for Research Resources (NCRR), RO1 HL117715 (IP) from the
667
National Heart, Lung and Blood Institute and in part with federal funds from the National
668
Cancer Institute under contract HHSN261200800001E. The funders had no role in
669
study design, data collection and analysis, decision to publish, or preparation of the
670
manuscript.
671
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Silvestri G, Sodora DL, Koup RA, Paiardini M, O'Neil SP, McClure HM,
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Staprans SI, Feinberg MB. 2003. Nonpathogenic SIV infection of sooty
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mangabeys is characterized by limited bystander immunopathology despite
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chronic high-level viremia. Immunity 18:441-452.
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Schmitz JE, Silvestri G. 2009. Toward an AIDS vaccine: lessons from natural
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simian immunodeficiency virus infections of African nonhuman primate hosts. Nat
960
Med 15:861-865.
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Silvestri G. 2005. Naturally SIV-infected sooty mangabeys: are we closer to
76.
Fischer W, Apetrei C, Santiago ML, Li Y, Gautam R, Pandrea I, Shaw GM,
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Hahn BH, Letvin NL, Nabel GJ, Korber BT. 2012. Distinct evolutionary
965
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966
immunodeficiency virus lineages. J Virol 86:13217-13231.
967 968
39
969
Figure legends
970
Figure 1. More rapid and consistent progression to AIDS in pigtailed
971
macaques infected with a serially passaged virus than in those infected with a
972
directly cross-species transmitted virus. Survival, as measured by days until AIDS-
973
defining illnesses and death in 5 PTMs infected with an SIVsab directly derived from an
974
African green monkey (black), compared to that in 7 PTMs infected with the virus
975
collected from the rapid progressor from the first group (red). The p value was
976
determined using the Gehan-Breslow-Wilcoxon test.
977
Figure 2. Dynamics of viral replication in SIVagm.sab-infected PTMs. Viral
978
replication was assessed by quantifying SIVsab RNA loads in plasma. Shown are the
979
genometric means (a) and individual viral loads (b). SIVsab RNA loads in PBMCs (c).
980
Animals infected with the SIVsab92018 strain directly derived from an acutely-infected
981
AGM are shown as black lines and symbols. Animals infected with SIVsabBH66,
982
collected from an SIVsab92018-infected PTM (BH66) rapid progressor (passaged virus)
983
are shown as red lines and symbols. In (b), rapid progressors from both P1 and P2
984
groups are illustrated using green symbols. Viral replication in different tissues was
985
assessed in cells separated from necropsy samples collected from 4 animals (d). BH66
986
(P1) and ET30 (P2) progressed to AIDS within 1 year postinfection. EC10 (P1)
987
progressed to AIDS after 2.5 years of follow-up. Remaining PTM BK42 (P1) died from
988
complications of surgery. Untested tissues are illustrated with an asterisk (*).
989
Abbreviations: PW-peritoneal wash; BAL-bronchoalveolar lavage; BM-bone marrow;
990
Ing-inguinal; AX-axillary; Mes-mesenteric; LN-lymph node; Duo-Duodenum; Jej-
991
jejunum; Ile-ileum; IEL-intraepitelial lymphocytes; LPL-lamina propria lymphocytes.
992
Plasma viral loads are expressed as SIVsab RNA copies/ml of plasma; detection limit of
993
the assay is 102 copies/ml. SIVsab RNA levels in tissues are expressed per 105 cells
994
with a detection limit of 10 copies. 40
995
Figure 3. CD4+ T cell dynamics in blood and tissues of SIVsab-infected PTMs.
996
Significant acute depletion followed by a limited and transient restoration of circulating
997
CD4+ T cells (a). CD4+ T cell depletion in the lymph nodes (b). Catastrophic acute CD4+
998
T cell depletion in the intestine of SIVsab-infected PTMs from both groups, followed by
999
only modest restoration in animals infected with the cross-species transmitted virus and
1000
virtually
no
restoration
in
PTMs
infected
with
the
passaged
virus
(c).
1001
Immunohistochemical assessment of CD4+ T cell depletion in the lymph nodes (d) and
1002
intestine (e). Massive depletion of CD4+ T cells can be observed at both sites at the
1003
chronic time points (42 and 60 dpi) compared to baseline levels (0 dpi) (d, e). The CD4
1004
staining pattern was different between LNs collected at the baseline (d), in which CD4+
1005
cells were small, round cells, and those collected after the peak of viral replication, in
1006
which the stained cells were larger, sometimes multinucleated cells (most probably
1007
macrophages). Animals infected with the SIVsab92018 strain directly derived from an
1008
acutely-infected AGM are shown as black lines and symbols. Animals infected with
1009
SIVagm.sabBH66, collected from an SIVagm.sab92018-infected rapid progressor PTM
1010
(BH66) (passaged virus) are shown as red lines and symbols. The graphs show the
1011
average levels of immune cells ± standard error of means (sem).
1012
Figure 4. Assessment of microbial translocation in pigtailed macaques
1013
infected with SIVsab. The baseline levels of microbial translocation (LPS, endocab
1014
and sCD14) were similar between the PTMs used for the two virus passages and
1015
between controllers, normal progressors and rapid progressors (a). Shown are PTMs
1016
infected with the unpassaged (P1) virus (illustrated as black and white circles) and with
1017
the passaged (P2) virus (illustrated as red circles). RP-rapid progressors; NP-nornal
1018
progressors; C-controllers. Changes in the levels of LPS between PTMs infected with
1019
the unpassaged (P1) virus and those infected with the passaged (P2) virus (b).
1020
Changes in the levels of sCD14 between PTMs infected with the unpassaged (P1) virus
41
1021
and those infected with the passaged (P2) virus (c). Shown are the average fold
1022
increases from the baseline ± standard error of the means (sem).
1023
Figure 5. Kinetic expression of immune activation and T cell proliferation in
1024
SIVsab-infected PTMs. Dynamics of CD4+ and CD8+ T cell immune activation (as
1025
defined by changes in the expression of –DR markers) in blood (a and b). Dynamics of
1026
CD4+ and CD8+ T cell proliferation (as defined by changes in the expression of Ki-67) in
1027
blood (c and d). Animals infected with the SIVsab92018 strain directly derived from an
1028
acutely-infected AGM are shown as black lines and symbols. Animals infected with
1029
SIVsabBH66 collected from an SIVsab92018-infected rapid progressor PTM (BH66)
1030
(passaged virus) are shown as red lines and symbols. Shown are the average fold
1031
increases from the baseline ± standard error of the means (sem).
1032
Figure 6. “Cytokine storm” during acute infection of PTMs infected with
1033
serially passaged SIVsabBHH66. Significantly higher increases in proinflammatory
1034
cytokines in PTMs infected with the serially-passaged SIVagm.sabBH66 (red lines and
1035
dots) versus PTMs infected with the AGM-derived SIVagm.sab92018 strain (black lines
1036
and dots): IL-1β (a); IL-2 (b); IL-4 (c); IL-6 (d); IL-12 (e); and TNFα (f). Shown are the
1037
average fold increases from the baseline ± standard error of the means (sem).
1038
Figure 7. In vitro assessment of the intrinsic virulence of the plasma SIVsab
1039
stocks used to infect PTMs. (a) In vitro replication of the SIVsab92018 (P1) and
1040
SIVsabBH66 (P2) plasma stocks on CD4+ enriched PBMCs from four uninfected PTMs,
1041
as assessed by the measurement of P27 production in the supernatants. Shown are the
1042
average fold increases from the baseline ± standard error of the means (sem). (b)
1043
Comparative assessment of induction of cytokine/chemokine production in vitro in
1044
PBMCs collected prior to infection from the PTMs challenged with SIVsab92018 (P1)
1045
and SIVsabBH66 (P2). The levels of cytokines/chemokines in supernatants harvested
1046
at 6 and 24 hours after incubation with the two viral stocks are shown as grey (P1) or 42
1047
black columns (P2). Controls (cells incubated in the same conditions in the absence of
1048
virus exposure) are shown as white columns. Differences between multiple experiments
1049
are illustrated as standard error of the means (sem). Statistical differences (p
