Unique Pathology in Simian Immunodeficiency Virus-Infected Rapid Progressor Macaques Is Consistent with a Pathogenesis Distinct from That of Classical AIDS

Article (PDF Available)inJournal of Virology 81(11):5594-606 · July 2007with29 Reads
DOI: 10.1128/JVI.00202-07 · Source: PubMed
Simian immunodeficiency virus (SIV) infection of macaques and human immunodeficiency virus type 1 (HIV-1) infection of humans result in variable but generally fatal disease outcomes. Most SIV-infected macaques progress to AIDS over a period of 1 to 3 years, in the face of robust SIV-specific immune responses (conventional progressors [CP]). A small number of SIV-inoculated macaques mount transient immune responses and progress rapidly to AIDS (rapid progressors [RP]). We speculated that the underlying pathogenic mechanisms may differ between RP and CP macaques. We compared the pathological lesions, virus loads, and distribution of virus and target cells in SIVsmE660- or SIVsmE543-infected RP and CP rhesus macaques at terminal disease. RP macaques developed a wasting syndrome characterized by severe SIV enteropathy in the absence of opportunistic infections. In contrast, opportunistic infections were commonly observed in CP macaques. RP and CP macaques showed distinct patterns of CD4(+) T-cell depletion, with a selective loss of memory cells in RP macaques and a generalized (naive and memory) CD4 depletion in CP macaques. In situ hybridization demonstrated higher levels of virus expression in lymphoid tissues (P < 0.001) of RP macaques and a broader distribution to include many nonlymphoid tissues. Finally, SIV was preferentially expressed in macrophages in RP macaques whereas the primary target cells in CP macaques were T lymphocytes at end stage disease. These data suggest distinct pathogenic mechanisms leading to the deaths of these two groups of animals, with CP macaques being more representative of HIV-induced AIDS in humans.
JOURNAL OF VIROLOGY, June 2007, p. 5594–5606 Vol. 81, No. 11
0022-538X/07/$08.000 doi:10.1128/JVI.00202-07
Unique Pathology in Simian Immunodeficiency Virus-Infected Rapid
Progressor Macaques Is Consistent with a Pathogenesis Distinct
from That of Classical AIDS
Charles R. Brown,
Meggan Czapiga,
Juraj Kabat,
Que Dang,
Ilnour Ourmanov,
Yoshiaki Nishimura,
Malcolm A. Martin,
and Vanessa M. Hirsch
Laboratory of Molecular Microbiology
and Research Technology Branch,
NIAID, NIH, 4 Center Drive,
Bethesda, Maryland 20892
Received 29 January 2007/Accepted 14 March 2007
Simian immunodeficiency virus (SIV) infection of macaques and human immunodeficiency virus type 1
(HIV-1) infection of humans result in variable but generally fatal disease outcomes. Most SIV-infected
macaques progress to AIDS over a period of 1 to 3 years, in the face of robust SIV-specific immune responses
(conventional progressors [CP]). A small number of SIV-inoculated macaques mount transient immune
responses and progress rapidly to AIDS (rapid progressors [RP]). We speculated that the underlying patho-
genic mechanisms may differ between RP and CP macaques. We compared the pathological lesions, virus loads,
and distribution of virus and target cells in SIVsmE660- or SIVsmE543-infected RP and CP rhesus macaques
at terminal disease. RP macaques developed a wasting syndrome characterized by severe SIV enteropathy in
the absence of opportunistic infections. In contrast, opportunistic infections were commonly observed in CP
macaques. RP and CP macaques showed distinct patterns of CD4
T-cell depletion, with a selective loss of
memory cells in RP macaques and a generalized (naive and memory) CD4 depletion in CP macaques. In situ
hybridization demonstrated higher levels of virus expression in lymphoid tissues (P < 0.001) of RP macaques
and a broader distribution to include many nonlymphoid tissues. Finally, SIV was preferentially expressed in
macrophages in RP macaques whereas the primary target cells in CP macaques were T lymphocytes at end
stage disease. These data suggest distinct pathogenic mechanisms leading to the deaths of these two groups of
animals, with CP macaques being more representative of HIV-induced AIDS in humans.
Human immunodeficiency virus type 1 (HIV-1), the caus-
ative agent of AIDS in humans, results in a progressive deple-
tion of CD4
T lymphocytes that culminates in fatal immuno-
deficiency. Although it is clear that HIV is responsible for
massive early destruction of memory CD4
T lymphocytes (7),
the subsequent disease course is prolonged and variable, sug-
gesting additional mechanisms in the chronic stages of the
disease. The long asymptomatic period of infection is associ-
ated with increased immune activation particularly of the re-
maining memory CD4
T cells, increased rates of spontaneous
apoptosis, abnormalities in CD4
T-cell function (13, 14, 20,
21, 24), and destruction of the lymphoid architecture (69). The
degree of T-cell activation has value in predicting HIV infec-
tion survival time (13, 24). This has led many to suggest that
the mechanisms underlying AIDS are more complex than a
simple model of slow virus-induced CD4
T-cell death (6, 18,
28, 29).
Simian immunodeficiency virus (SIV) originating in sooty
mangabey monkeys is perhaps the most relevant model of
human AIDS currently available. This lineage includes the
virus most commonly used in the field, SIVmac239, which
arose following the introduction of SIVsm into rhesus ma-
caques housed in U.S. primate centers (12, 71). However,
equally relevant models have been developed by using SIVsm
strains from captive sooty mangabeys, including the closely
related SIVsmE660 and molecularly cloned SIVsmE543-3
strains derived in our laboratory (1, 31, 34, 35, 42, 63). SIVsm/
mac and HIV are remarkably similar in terms of biology and
pathogenesis in vivo. Both target CD4
cells, including T cells
and macrophages, and use the chemokine coreceptor CCR5,
resulting in direct destruction of memory CD4
T cells at
mucosal sites (7, 48, 50, 60, 66). SIV infection of macaques
covers a continuum in terms of rate of disease progression,
similar to HIV infection of humans (5, 19, 31, 33, 34, 38, 49, 62,
73, 79). The level at which the virus stabilizes following primary
infection is predictive of the rate of disease progression in both
SIV and HIV infections (33, 53, 76). Both are associated with
intense immune activation during the chronic phase of disease
(13, 24, 48, 70, 72), and the pathological manifestations of
AIDS are similar (5, 47, 51, 71). End stage disease is charac-
terized by terminal blood and tissue CD4
T-cell depletion,
infections with a variety of opportunistic pathogens (5, 47, 51,
71), and the development of virus-induced encephalitis (3, 17,
26, 57, 61, 62, 68, 81, 82).
Despite remarkable similarities between simian AIDS and
human AIDS, there are a number of differences that must be
considered. First, the disease course of SIV infection of ma-
caques is compressed relative to that of HIV infection, with a
median survival time of 1 to 3 years versus 10 years in un-
treated human AIDS. Second, SIVsm/mac virus isolates use
CCR5 as their primary coreceptor; the emergence of CXCR4-
using isolates observed in many human AIDS patients is only
rarely observed in SIV infection (45). However, since isolates
* Corresponding author. Mailing address: LMM, NIAID, NIH,
Building 4, Room B1-41, 4 Center Drive, Bethesda, MD 20892. Phone:
(301) 496-0559. Fax: (301) 480-3129. E-mail: vhirsch@niaid.nih.gov.
Published ahead of print on 21 March 2007.
recovered early in infection with HIV preferentially use CCR5
as a coreceptor, the pathogenesis of SIV and that of HIV
appear more similar to one another than to that of CXCR4-
using SIV-HIV isolates (39, 60). Third, while rare individuals
infected with HIV-1 rapidly develop disease (22, 23, 54, 80),
the frequency of an accelerated disease course is much higher
in SIV-infected macaques. About 10 to 25% of rhesus ma-
caques inoculated with pathogenic strains of SIVmac/sm fail to
mount measurable immune responses and develop disease in
less than 6 months (5, 16, 34, 71, 79). Finally, the pathology of
simian AIDS includes descriptions of SIV-induced disease in
which multinucleated giant cells (MNGC) in the lymphoid
tissues, brain, and lungs are a prominent feature (5, 47, 51).
With the exception of the brains of HIV-infected patients with
encephalitis, MNGC are rarely observed in tissues of humans
with AIDS. In our studies, the presence of MNGC was associated
with extremely high viremia and transient immune responses,
both signs of rapid progression (31, 37, 46). Retrospective studies
have also shown that SIV encephalitis (SIVE) is highly correlated
with rapid disease progression (3, 61, 62, 77). We speculated that
rapid progression of SIV infection of macaques might constitute
a unique pathological syndrome, distinct from the classical de-
scriptions of SIV-induced AIDS. The goal of the present study
was to compare pathology, virus distribution, and target cells by in
situ hybridization (ISH) and confocal microscopy in terminal tis-
sues of conventional progressors (CP) and rapid progressors (RP)
infected with SIVsmE660 or SIVsmE543-3.
Viruses and animal study design. The rhesus macaques evaluated in this study
were inoculated intravenously with either uncloned SIVsmE660 (33, 34, 37) or
the molecular clone SIVsmE543-3 (31, 33). These isolates are closely related
genetically (92% identity in envelope) to one another and were derived by
macaque passage from the SIVsmF236 isolate from the Tulane National Primate
Research Center. SIVsmF236 was derived by one macaque passage of virus from
a naturally infected sooty mangabey monkey (5).
Study animals were selected from a group of 72 SIVsm-infected Indian rhesus
macaques (25, 49). This group of animals included nine RP (see Table 1) that
were identified by a lack of SIV-specific antibody responses by 4 weeks postin-
oculation and progression to disease in 6 months or less. CP macaques for
comparison were chosen on the basis of seroconversion and survival for 1 year or
longer; any animals that died prematurely because of unrelated causes, such as
anesthetic accidents, were eliminated. The CP macaques selected for the present
study were not significantly different from the remainder of the CP macaques in
the cohort in terms of CD4
T-cell numbers at the time of euthanasia (mean
CD4 count of 85 versus 188/l; P 0.11) or survival time (median survival time
of 58 versus 76 weeks; P 0.27).
The same clinical criteria were used for euthanasia in both groups. All animals
were euthanized if they lost 20% of their body weight or developed intractable
diarrhea that was unresponsive to supportive or antibiotic treatment, respiratory
signs with radiographic evidence of pneumonia, persistent anorexia and lethargy,
or neurologic signs. Animals were exsanguinated under deep anesthesia, per-
fused with 1 liter of saline, followed by 1 liter of 10% buffered formalin, prior to
collection of tissues. Representative samples were taken for formalin fixation. All
sections were stained with hematoxylin and eosin for routine histopathology. All
animals were housed in accordance with American Association for Accreditation
of Laboratory Animal Care standards. The investigators adhered to the Guide
for the Care and Use of Laboratory Animals prepared by the Committee on Care
and Use of Laboratory Animals of the Institute of Laboratory Resources, Na-
tional Resource Council, and the NIAID Animal Care and Use Committee-
approved protocols.
Lymphocyte immunophenotyping. EDTA-anticoagulated blood samples were
collected sequentially for analysis of plasma viral RNA loads and lymphocyte
subsets by flow cytometry. EDTA-treated blood samples were stained for flow
cytometric analysis as described previously (58, 60), by using combinations of the
following fluorochrome-conjugated monoclonal antibodies: CD3 (fluorescein
isothiocyanate [FITC] or phycoerythrin [PE]), CD4 (PE, peridinin chlorophyll
protein-Cy5.5 [PerCP-Cy5.5], or allophycocyanin [APC]), CD8 (PerCP or APC),
CD28 (FITC or PE), CD95 (APC), and Ki-67 (FITC or PE). All antibodies were
obtained from BD Biosciences (San Diego, CA), and samples were analyzed by
four-color flow cytometry (FACScalibur; BD Biosciences Immunocytometry Sys-
tems). Analysis of data was performed with CellQuest Pro (BD Biosciences) and
FlowJo (TreeStar, Inc., San Carlos, CA). In this study, naive CD4
T cells were
identified by their CD95
phenotype, whereas memory CD4
T cells
were CD95
or CD95
in the CD4
small lymphocyte
gate (60, 67).
Plasma SIV RNA quantitation. A plasma SIV RNA viral load real-time quan-
tification assay based on the Applied Biosystems Prism Sequence Detection
TABLE 1. Summary of clinical and pathological outcomes of study animals
Category and
time (wk)
Major pathological findings
H147 E660 14 SIVE and pneumonia with MNGC, severe enteritis (protozoal), MNGC in GI tract and prostate
H168 E660 22 SIVE and pneumonia with MNGC, severe enteritis, MNGC in lymph nodes
H538 E660 9 SIVE and pneumonia with MNGC, severe enteritis, MNGC in ovary and fallopian tube
H567 E660 10 SIVE and mild pneumonia with MNGC, severe enteritis, MNGC in lymph nodes and GI tract
H426 E660 20 SIVE and pneumonia with MNGC, severe enteritis, MNGC in GI tract and eye
18655 E660 16 SIVE and pneumonia with MNGC, severe enterocolitis, MNGC in thymus, lymph nodes, spleen,
GI tract, kidney, and testis
H445 E543-3 15 SIVE and pneumonia with MNGC, severe enteritis
CK2F E543-3 31 Mild diffuse SIVE, pneumonia with MNGC, severe enteritis
CK2K E543-3 15 SIVE and pneumonia, severe lymphoid depletion with MNGC, moderate-to-severe enteritis
H119 E660 51 Disseminated M. avium enteritis and lymphadenitis, lymphoid hyperplasia and involution
lymphoma (mesenteric lymph node), focal SIVE
H120 E660 54 M. avium enteritis, lymphadenitis, lymphoid hyperplasia and involution
H133 E660 58 P. carinii pneumonia, enteritis, lymphoid depletion
H187 E660 54 Pyogranulomatous lymphadenitis, cytomegalovirus orchitis, lymphoid depletion
H454 E660 72 Multicentric lymphoma (heart, adrenals, urinary bladder, ovaries, eye)
H063 E543-3 164 M. avium enteritis and lymphadenitis, lymphoid depletion
H460 E543-3 100 Cryptosporidial enteritis, cholecystitis, cholangiohepatitis, lymphoid depletion
XGE E543-3 77 Suppurative pneumonia with pulmonary thrombosis, lymphoid depletion, enteritis with amyloidosis
H679 E543-3 60 Abscessation of kidney, secondary peritonitis (Staphylococcus aureus), lymphoid depletion
System was used as previously described (74). Viral RNA was isolated from
plasma with a QIAamp viral RNA kit (QIAGEN Inc., Santa Clarita, CA) and
treated with amplification grade DNase I (Life Technologies, Gaithersburg, MD)
as recommended by the manufacturer. Replicate aliquots of the test RNA were
subjected to RT-PCR by a two-step, two-enzyme protocol with SIV-Gag con-
sensus primers S-GAG03 and S-GAG04 and SIV-Gag consensus TaqMan probe
P-SUS-05. The poly(A)-tailed full-length RNA control template was purified
on oligo(T)-agarose and quantified by A
measurements based on the
calculated extinction coefficient for the transcript sequence. A serial fivefold
dilution series of the standard RNA template was assayed in duplicate to
generate a standard curve for each assay. RT-PCR for each plasma sample
was performed in triplicate. Assay results were normalized to the volume of
plasma extracted and expressed as numbers of SIV RNA copy equivalents per
milliliter of plasma, as described for HIV-1 (64, 65). Interassay variation was
less than 25% (confidence value).
SIV-specific ISH. Formalin-fixed, paraffin-embedded tissues were assayed for
SIV viral RNA expression by ISH as previously described (32). Briefly, the
sections were hybridized overnight at 50°C with either a sense or an antisense
SIVmac239 digoxigenin-UTP-labeled riboprobe. The hybridized sections were
blocked with 3% normal sheep and horse serum in 0.1 M Tris, pH 7.4, and then
incubated with sheep anti-digoxigenin–alkaline phosphatase (Roche Molecular
Biochemicals) and nitroblue tetrazolium–5-bromo-4-chloro-3-indolyl--
topyranoside (BCIP; Vector Labs). ISH-stained tissues from mesenteric lymph
nodes and the gastrointestinal (GI) tract (gut-associated lymphoid tissue
[GALT] and lamina propria) were visualized and photographed with a Zeiss
Axiophot microscope. Five representative fields of view of each stained tissue
section were photographed and analyzed. The number of SIV
cells was calcu
lated by using a segmentation function in the IPLab software (Scanlytics Inc.,
Rockville, MD). SIV
cells in several ISH-stained control sections were counted
FIG. 1. Hematoxylin-and-eosin-stained sections of the ileum of an
RP (top) and comparison of the lymph nodes (LN) of a representative
RP macaque and a CP macaque. (A) Section of ileum of RP macaque
H445 showing the blunting and fusion of intestinal villi that was char-
acteristic of RP intestinal tissues. (B) Mesenteric lymph node of RP
macaque H445 showing depletion of the paracortex and a lack of
secondary germinal centers. (C) Inguinal lymph node of CP macaque
H133 showing large germinal centers with irregular mantles and
hyalinization indicative of involution.
FIG. 2. Plasma viremia in RP and CP macaques is shown over the
time course of infection. (A) Sequential plasma viral loads in RP (red)
and CP (blue) macaques. (B) Comparison of peak, set point, and
terminal plasma viral RNA levels in RP and CP macaques with statis-
tically significant differences between the two groups at each of these
time points (Mann-Whitney U test).
5596 BROWN ET AL. J. V
manually, and the size and threshold values for the automatic segmentation
function were determined.
Confocal microscopy to identify SIV-expressing cells. Formalin-fixed, paraffin-
embedded tissues were stained for SIV viral RNA by ISH by a modified method
previously described (31). Briefly, the sections were stained with the same SIV
probe and procedure as mentioned above, with the following changes. The
samples were treated with methanol-hydrogen peroxide, and the hybridized
tissue sections were incubated with sheep anti-digoxigenin–horseradish peroxi-
dase (SAD-HRP; Roche Molecular Biochemicals). SAD-HRP was detected by a
fluorescent tyramide signal amplification technique (TSA Plus FITC, NEL741;
Perkin-Elmer). After completion of the ISH assay, the sections were incubated
in mouse anti-human macrophage antibody (HAM56; DAKO M0632) and
stained with goat anti-mouse immunoglobulin M-Alexa 633 (Invitrogen) and
then incubated with rabbit anti-human CD3 (T-cell marker; DAKO A0452),
followed by goat Alexa 594 anti-rabbit immunoglobulin G antibody. The triple-
stained sections were mounted in Vectashield Hardset mounting medium (Vec-
tor Laboratories, Burlingame, CA) and photographed with a Leica confocal
scanning microscope. Ten representative 40 fields of mesenteric lymph node
and ileum tissues from five RP macaques and four CP macaques were photo-
graphed, and the SIV
T cells and macrophages were counted.
Statistical analyses. Viral loads in plasma, numbers of CD4
T cells in blood,
and numbers of SIV-expressing cells in tissues were compared by unpaired
two-tailed t tests with GraphPad Prism (San Diego, CA), and Kaplan-Meier
survival plots were compared by using log rank tests.
A retrospective analysis was undertaken to examine end
stage virus distribution and infected target cells in rhesus ma-
caques inoculated with SIVsm (25, 33, 49) that progressed
rapidly (RP macaques) or underwent conventional progression
(CP macaques) to disease. As shown in Table 1, RP macaques
developed an illness that necessitated euthanasia significantly
earlier than CP macaques, with a median survival of 15 versus
72 weeks (log rank test; P 0.001).
The apparent disease phenotypes in RP and CP macaques
also appeared to be quite distinct. RP macaques succumbed to
a wasting syndrome with chronic diarrhea. Pathological find-
ings in these macaques included severe enteritis, characterized
by villus blunting, and fusion, as shown in a representative
section of ileum in Fig. 1A, similar to the SIV enteropathy
previously described in SIV infection (30, 75, 78) and are in
keeping with the major impact of SIV on the GI tract (75).
Opportunistic infections (OIs) were not observed in either the
GI tract or other organs, suggesting that clinical disease in
these animals was the direct result of SIV infection rather than
secondary to immune suppression. In addition to enteritis,
SIV-induced pneumonia and SIVE with MNGC were con-
sistently observed in RP macaques and MNGC were fre-
quently observed in a variety of other tissues (Table 2). RP
macaques did not develop reactive lymphoid hyperplasia as
assessed by sequential peripheral lymph node measure-
ments (data not shown), and consistent with this observa-
FIG. 3. CD4
T cells in the peripheral blood of RP and CP macaques over the time course of infection. (A) Total CD4
T cells are compared
in RP (red) and CP (blue) macaques. (B) Comparison of total CD4
T cells in peripheral blood at the time of euthanasia shows significantly lower
total CD4
T-cell counts in CP versus RP macaques (Mann-Whitney U test). Comparison of memory (C) and naive (D) CD4
T cells in the blood
of two RP and two CP macaques inoculated with SIVsmE543-3 shows the selective depletion of memory CD4
T cells that occurs in RP macaques,
as determined by CD28 and CD95 expression. In contrast, CP macaques demonstrate early depletion of memory CD4
T cells with partial
replenishment but subsequent progressive loss of both memory and naive CD4
T-cell subsets during long-term progression.
tion, lymph nodes were relatively quiescent, with a lack of
germinal centers (Fig. 1B).
In contrast, CP macaques presented with a disease syndrome
more similar to human AIDS. As shown in Table 1, OIs with
agents such as Mycobacterium avium (n 3), Pneumocystis
carinii (n 1), cryptosporidia (n 1), cytomegalovirus (n
1), or overwhelming bacterial infections were commonly seen
in CP macaques (n 2). Lymphoma, presumably due to Ep-
stein-Barr virus infection, was also observed (n 1). SIVE was
only rarely observed in CP macaques (H119) and was consid-
erably less severe than in RP macaques. MNGC were only
rarely observed in tissues of CP macaques (H460, H119). CP
macaques had evidence of prior reactive lymphoid hyperplasia
with prominent germinal centers often in the process of invo-
lution and fibrosis (Fig. 1C).
Prior to the evaluation of end stage tissues, sequential
plasma viral loads and circulating CD4
T-cell levels of the two
groups were compared. As shown in Fig. 2, RP macaques
exhibited higher levels of plasma viral RNA at peak (P 0.02),
set point (P 0.0001), and death (P 0.0001) than CP ma-
caques. By the time of euthanasia, plasma viral RNA levels of
RP macaques were 2 to 3 logs higher than those of CP ma-
caques. The two SIV-infected cohorts also differed with re-
spect to circulating CD4
lymphocyte counts at the time of
euthanasia. As shown in Fig. 3A, only modest CD4
depletions were observed in either group during the first 30
weeks of infection (37 versus 16% decline in RP and CP,
respectively). At 30 weeks, the extent of depletion did not
differ significantly between the two groups (Mann-Whitney U
test, P 0.05). Many of the RP macaques were euthanized
FIG. 4. Representative fields of view of ISH for SIV viral RNA in the mesenteric lymph nodes of RP and CP macaques. (A, B, and C) RP lymph
nodes showing numerous SIV-expressing cells (dark blue) within the paracortex, follicle, intrafollicular zone, and cortex of the lymph nodes of
SIV-infected RP macaques H538 (A; magnification, 10), H445 (B; magnification, 10), and H168 (C; magnification, 40) showing large
numbers of SIV-expressing cells. Lower numbers of SIV-expressing cells were observed in the mesenteric lymph nodes of representative CP
macaques H063 (D), H119 (E), and H460 (F). Magnification, 40.
with only moderate blood CD4
T-cell depletion (Fig. 3B)
(36). In contrast, CP macaques consistently showed severe
depletion of total blood CD4
T cells at the time of euthanasia
(Fig. 3A and B); terminal blood CD4
T cells were signifi
cantly lower in CP macaques than in RP macaques (Fig. 3B;
Mann-Whitney U test, P 0.0001).
Because of the retrospective nature of this study, CD4 sub-
sets could not be evaluated in the majority of the animals.
However, in a parallel study we examined CD4
T-cell subsets
during disease progression in a cohort of 12 macaques (59).
Memory and naive CD4
T-cell subsets of two representative
RP (CK2F and CK2K) and CP (XGE and H679) macaques are
detailed in Fig. 3C and D. These animals were typical for the
RP and CP disease course and pathology, respectively (Table
1). All four animals experienced a dramatic decline in CD4
memory cells during the primary stage of infection (Fig. 3C), as
previously observed by others (50, 66). However, partial re-
plenishment of this T-cell subset only occurred in CP ma-
caques, followed by a more precipitous decline terminally. The
depletion of CD4
memory cells in RP macaques was rapid
and progressive, reaching extremely low levels by the time of
euthanasia. Naive CD4
T cells were preserved in RP ma
caques (Fig. 3D), as previously reported (59, 60, 66). In con-
trast, progressive loss of naive CD4
T cells was also observed
in the CP macaques by the time of euthanasia (Fig. 3C, right).
The total depletion of circulating CD4
T cells in the remain
der of the cohort (Fig. 3A) is consistent with the depletion of
both memory and naive subsets in these animals.
High virus expression in the lymph nodes and GI tracts of
RP macaques. ISH to detect SIV expression in lymphoid
FIG. 5. Representative fields of view of ISH for SIV viral RNA in the ilea of RP (left) and CP (right) macaques. (A and B) Representative
sections of the ilea of RP macaques H147 and H538 showing large numbers of SIV-expressing cells in the GALT, crypt regions, and lamina propria
(magnification, 10). Panel C shows a higher magnification of the ileum of macaque 18655 (magnification, 40). (D, E, and F) Representative
sections of the ilea of CP macaques H187 and H063 showing a scattering of SIV-expressing cells in the GALT (magnifications: D and E, 10;
F, 40).
tissues and the GI tract revealed much higher expression
levels in RP macaques at the time of euthanasia, as evident
in representative lymph node (Fig. 4) and ileum (Fig. 5)
sections. SIV-expressing cells in tissues of the RP macaques
appeared to be primarily macrophages on the basis of their
morphology, i.e., large, irregularly shaped cells containing
abundant, foamy cytoplasm (Fig. 4C and 5C). In contrast,
CP macaque tissues contained SIV-expressing cells that
were small and round with a high nucleus-to-cytoplasm ra-
tio, consistent with their identification as T cells (Fig. 4F and
5F). The difference in morphology between SIV
cells in RP
and CP lymphoid tissues is clearly demonstrated by compar-
ison of Fig. 4C and F.
The number of SIV-expressing cells in lymphoid tissues and
the GI tract were assessed by scoring these tissues on a scale of
0 to 4. A score of 0 indicated that no SIV
cells were observed,
whereas a score of 4 indicated 10 SIV
cells per field of view.
As shown in Table 2, the number of virus-expressing cells was
consistently higher in the lymphoid tissues and GI tracts of RP
versus CP macaques. The majority of CP tissues scored at level
1 or lower (89%), whereas the majority of the scores for RP
tissues were 3 or greater (73%). IPLab software was used to
count the SIV-expressing cells in representative (40) fields of
the ileum and mesenteric lymph node sections of each of the
RP and CP macaques. As shown in Fig. 6, the mean number of
SIV-expressing cells in lymph nodes and the GALT and lamina
propria of the ileum in samples collected at necropsy was
significantly higher in RP macaques compared with CP ma-
caques (Mann-Whitney U test).
Identification of target cells by confocal microscopy. Based
on cell morphology in ISH-stained sections, the predominant
cell type expressing SIV appeared to differ in RP and CP
macaques. Triple-label confocal microscopy with SIV-specific
ISH (green) and immunohistochemistry for CD3
T cells (red)
and HAM56
macrophages (white) was used to identify SIV-
expressing cells in lymph nodes and the GI tract. Representa-
tive confocal fields from the mesenteric lymph nodes of RP
and CP macaques are shown in Fig. 7A to D. The majority of
SIV-expressing cells in the RP lymph nodes were identified
as macrophages on the basis of coexpression of HAM56
(Fig. 7A and B). Rare SIV-expressing CD3
T cells were
also identified (asterisks). In contrast, the majority of SIV-
expressing cells in CP tissues were T cells on the basis of
CD3 coexpression (Fig. 7C and D). Macrophages (HAM56)
were also present but only rarely expressed SIV (not
shown). To determine the proportion of SIV-expressing T
cells and macrophages in tissues of RP and CP macaques, 10
representative fields of the lymph nodes or ileum of five RP
and four CP macaques were photographed and the SIV
cells of each type were counted. As shown in Fig. 8A, similar
numbers of SIV-infected T cells were observed in both RP
and CP tissues. However, a large population of SIV-express-
ing macrophages was also observed in RP tissues such that
SIV-infected macrophages greatly outnumbered T cells. The
ratio of SIV
T cells to macrophages in CP macaques (Fig.
8B) was significantly higher than in RP macaque lymph
nodes and GI tracts (0.97 versus 0.11; P 0.0001), consis-
tent with a difference in end stage target cell preference
between the two disease courses.
Wider distribution of SIV expression in RP macaques by
ISH. ISH for SIV viral RNA was used to examine the distri-
bution of SIV-expressing cells in a variety of other, nonlym-
phoid, tissues such as lung, kidney, liver, reproductive tract,
eye, and brain tissues. Representative fields of the latter
TABLE 2. Distribution and estimates of viral loads in tissues of SIV-infected rhesus macaques
147 168 538 567 445 426 18655 119 120 133 187 063 454 460
Thymus 243132 2 1000111
ALN 224442 3 1112111
ILN 334432 4 1112111
TBLN 342243 3 1111100
MLN 444442 4 1112221
Spleen 324441 4 1112111
Ileum 334443 4 1011112
Colon 333442 4 1111110
Lung 444343 4 0000111
Liver 114231 2 0000000
Kidney 322332 3 0000100
Heart 232222 2 0000000
Brain 234433 4 1
Spinal cord 2321ND2 2 0000000
Eye 223332 1 0000000
Uterus NA NA 4 3 NA NA NA NA NA NA NA 1 0 0
Ovary NA NA 4 ND NA NA NA NA NA NA NA 0 0 0
Prostate 3 3 NA NA 3 0 2 0000NANANA
Testis 3 3 NA NA 2 1 3 0001NANANA
ALN, axillary lymph node; ILN, inguinal lymph node; TBLN, tracheobronchial lymph node; MLN, mesenteric lymph node.
0, no SIV
cells observed in entire section; 1, rare SIV
cell per high-power field; 2, 1 to 5 SIV
cells per high-power field; 3, 5 to 10 SIV
cells per high-power
field; 4, 10 SIV
cells per high-power field; ND, not determined; NA, not applicable.
One focal area or a single positive cell was observed.
tissues are shown in Fig. 9. SIV-expressing giant cells were
observed in the lung, choroid plexus, brain parenchyma,
retina (Fig. 9A to D, respectively), glomeruli of the kidney
(Fig. 9E), portal triads of the liver (Fig. 9F), and female and
male urogenital tracts, respectively (Fig. 9G and H). This
high level of expression of SIV in MNGC in nonlymphoid
tissues was unique to the RP macaques. As detailed in the
bottom half of Table 2, the distribution of virus expression
in nonlymphoid tissues was much broader in the RP ma-
caques than in CP macaques. SIV expression was generally
only observed in the lymphoid tissues and GI tracts of CP
macaques, whereas virus expression was observed in tissue
macrophages residing in virtually every tissue of RP ma-
caques that was examined, including privileged compart-
ments such as the eye, brain, and testes.
This study demonstrates that RP macaques form a distinct
group among SIVsmE660- and E543-3-inoculated macaques
that is unique in terms of pathology and, by inference, patho-
genesis. The end stage tissues we examined from RP macaques
in this study exhibited primary SIV-induced pathological le-
sions in lymphoid tissues, as well many other, nonlymphoid,
tissues. Despite evidence of immune suppression, OIs were
rarely observed in RP macaques and thus their severe disease
is presumably due to a direct effect of SIV replication in tis-
sues, particularly in the lymphoid tissues, lungs, intestinal tract,
and brain. SIVE was strongly associated with rapid progres-
sion, and there was a strong preference for SIV replication in
macrophages. In contrast, the clinical disease in CP macaques
could often be attributed to infections with opportunistic
pathogens, a pathological picture reminiscent of HIV-induced
AIDS in humans.
The clinical phenomenon of rapid disease progression in
SIV-infected macaques is clearly observed with all pathogenic
SIVsm and SIVmac strains (42). However, the pathology in RP
macaques has not been comprehensively compared with CP
macaques in other SIV models. Thus, it is not certain whether
RP macaques inoculated with other SIV strains would segre-
gate so clearly from slower progressors, as observed for
SIVsmE543 and SIVsmE660 in the present study. The patho-
logical features that we associated with rapid progression, such
as MNGC formation (4), macrophage tropism (2, 17, 55), SIV
enteropathy (30, 43, 78), and SIVE (3, 61, 77, 81), are not
unique to one specific strain of SIV. For example, previous
studies of SIVmac239 and SIVsm strains have demonstrated
that SIVE is highly associated with lack of SIV-specific anti-
body responses and a rapid disease course (3, 61, 77). Addi-
tionally, immunologic features such as transient SIV-specific
immune responses and early depletion of memory CD4
cells are similar between SIVmac239 and SIVsmE543-3-in-
fected RP macaques (59, 60, 66). Although OIs were rare in
the RP macaques in the present study, they have been ob-
served in other RP macaques infected with other strains of SIV
(61, 66). Previous studies of three of these RP macaques
(H445, H538, H567) demonstrated that they are profoundly
immunosuppressed, as evident by their lack of SIV-specific
immune responses and inability to mount recall and new an-
tigen responses (36). Therefore, the lack of detectable OIs in
the present study may be a matter of timing, i.e., SIVsm-
infected RP macaques develop clinical disease as a result of the
severity of primary SIV lesions in the intestine, lungs, and
central nervous system before the manifestations of other OIs
become pathologically evident. Certainly, the SIV-induced le-
sions in the intestine and lungs are sufficient to explain the
clinical deterioration of these animals. Nevertheless, the sever-
ity of the primary SIV lesions appeared to be more pro-
nounced in SIVsmE660- and SIVsmE543-inoculated RP ma-
caques compared to reports of SIVmac239-inoculated RP
What is responsible for the difference in end stage pathology
in RP macaques compared to CP macaques? We hypothesized
that primary SIV lesions observed in RP macaques were the
result of an extremely high viral load, resulting in nearly com-
plete elimination of memory CD4
T cells and redirection of
FIG. 6. Comparison of the numbers of SIV-expressing cells per
high-power field (HPF) in the mesenteric lymph nodes and ilea of RP
and CP macaques. Five random fields of view (magnification, 40) of
ISH-stained tissues from seven RP and seven CP macaques were
photographed from each tissue, and the mean number of SIV
per high-power field for each tissue was calculated. Scatter plots of
mean numbers of SIV
cells in the mesenteric lymph nodes (A),
GALT (B), and lamina propria (C) of the ileum show that RP ma-
caque tissues contained significantly more SIV
cells per high-power
field compared to CP macaques (Mann-Whitney U test).
FIG. 7. Confocal microscopic analysis of mesenteric lymph nodes of representative RP and CP macaques. Triple-label confocal microscopy of
lymph nodes from an RP macaque (A and B) and a CP macaque (C and D) to identify macrophages (HAM56, white), SIV RNA expression (ISH,
green), and T cells (CD3, red). The majority of SIV-expressing cells in the tissues from the RP macaque coexpressed Ham56, a marker for
macrophages (magnification, 63). Rare CD3
T cells coexpressing SIV are indicated by asterisks. In contrast (C and D), the majority of
SIV-expressing cells in tissues of the CP macaques (H187 and H063) coexpressed CD3 (magnification, 63).
FIG. 8. Graphic representation of the proportions of SIV-expressing macrophages and T cells in the lymph nodes and ilea of RP and CP
macaques. (A) The mean numbers of SIV
cells per high-power field (HPF) of each type in various tissues of RP and CP macaques are shown
by a bar graph where the filled bars represent T cells and the open bars represent macrophages. (B) Ratios of infected T cells to total infected
cells in RP and CP tissues, demonstrating a significant difference between these two groups.
5602 BROWN ET AL. J. V
the virus to infect tissue macrophages. Early destruction of
mucosal CD4
T cells occurs in both RP and CP macaques
(50, 59, 66, 75). However, compromised production and inad-
equate mucosal transport result in near total depletion of
memory cells very early in the infection of SIV-infected
RP macaques (59, 66). This loss is probably responsible for the
failure of RP macaques to maintain SIV-specific immune re-
sponses, and as a consequence, RP macaques also do not
develop the lymphadenopathy generally associated with hyper-
immune activation in HIV and SIV infections. Consistent with
lack of immune activation, RP macaques show only transient
increases in T-cell proliferation with declining levels of Ki-67-
expressing CD4
T cells during disease progression (59). Loss
of these cells as targets for the virus also presumably forces the
virus to replicate in macrophages. This is similar to the sce-
nario observed in X4-tropic SIV-HIV infections of macaques
(39, 60), where total early loss of all CD4
T cells results in
increased macrophage tropism (40, 41).
This forced adaptation to macrophages is probably also re-
sponsible for the specific evolution of SIV variants that we
FIG. 9. ISH for SIV RNA in a wide range of tissues of RP macaques. (A) A section of lung tissue from RP macaque H168 shows abundant
SIV-expressing giant cells (magnification, 10). (B) SIV-expressing giant cells in the choroid plexus of macaque H567 (magnification, 20).
(C) ISH of the brain of RP macaque H567 showing perivascular infiltration of SIV-expressing macrophages and giant cells (magnification, 10).
(D) SIV-expressing macrophages in the retina of RP macaque H538 (magnification, 20). (E) SIV-expressing cells within glomeruli of RP
macaque H538. (F) SIV-expressing cells in periportal infiltrates in the liver of RP macaque H538. (G) SIV
giant cells in the interstitial areas of
the fallopian tube of female macaque H538. (H) SIV-expressing cells adjacent to seminiferous tubules of male macaque H168.
have observed in previous studies of SIVsmE543-3-inoculated
RP macaques (15, 46). Sequence analysis of viruses in three
SIVsmE543-3-infected RP macaques (8) revealed a unique
convergent pattern of substitutions in env, including the loss of
a highly conserved potential glycosylation site in the V
region (N158D/S or S160N/G), substitutions in the cysteine
loop that is analogous to HIV-1 V
(P337T/S/H/L and
R348W), and substitutions in the highly conserved GDPE mo-
tif (G386R and D388N/V). These substitutions are associated
with the development of CD4-independent use of CCR5 (15),
which has previously been associated with an increased ability
of viruses such as SIVmac316 to infect macrophages (56), as
well as with increased sensitivity to neutralization (52). Both of
these factors may drive the evolution of SIV in RP macaques
and may be responsible for the difference in end stage pathol-
ogy between RP and CP macaques.
The pathogenesis of CP macaques appears to be more sim-
ilar to AIDS in humans than in RP macaques. CP macaques
exhibit an early phase of memory CD4 T-cell loss at mucosal
sites (7, 50); an asymptomatic phase characterized by persis-
tent viremia and chronic immune activation (10, 13, 14, 70)
with lymphadenopathy, lymphoid expansion, and fibrosis (69);
eventual immunologic collapse; and development of OIs.
Transient partial replenishment of memory CD4
T cells was
observed in many CP macaques (59, 66). An increase in mem-
ory CD4
T-cell turnover, as assessed by Ki-67 expression, was
observed in previous studies of CP macaques, and CD4 pro-
liferation was sustained throughout the course of infection
(59). As previously observed in other SIV studies (11, 71), CP
macaques developed a lymphadenopathy also consistent with
intense immune activation (8, 9). Similar to human AIDS, the
onset of disease in CP macaques was associated with an overall
decline in circulating CD4
T cells, including both memory
and naive subsets (59). Despite the steady decline and the
severe terminal CD4
T-cell depletion in CP macaques, a
generalized switch or preference to infect macrophages was
not observed. In addition to the early destruction of memory
T cells, CP macaques also experience a slower loss of
these cells during the chronic stages of infection that is accom-
panied by a significant loss of naive CD4
T cells (59).
It is not clear why naive CD4
T cells would be affected in
CP macaques, since SIVsmE543-3 and SIVsmE660 use CCR5
for entry (15, 27). Since CCR5 is primarily expressed on mem-
ory CD4
T cells, depletion of this subset is an expected
outcome. Evolution of CXCR4-using SIV variants such as seen
in approximately 50% of late-stage human AIDS patients, is
only rarely observed in SIV infection (44, 66). Although iso-
lates from these animals were not examined for coreceptor use,
a consistent switch to X4 tropism in each of these animals is
unlikely. Indeed, loss of naive cells also occurs in HIV infection
in the absence of the emergence of CXCR4-using variants.
This makes the pattern of CD4 T-cell loss in CP macaques
more similar to AIDS in humans. We speculate that the addi-
tional loss of naive CD4
T cells in CP macaques is not a direct
result of virus-induced cell death. The decline in naive CD4 T
cells by the terminal stages of disease is probably due to failure
of the homeostatic and regenerative process from the contin-
ual, unrelenting loss of CCR5
memory CD4
T cells by virus
killing and immune activation-induced cell death. Factors
speculated to be involved include (i) exhaustion of the regen-
erative capacity of T-cell precursors to produce naive cells, (ii)
bystander killing by immune activation-induced cell death, and
(iii) limited repopulation and survival of T cells in lymphoid
tissues damaged by collagen deposition (69).
The present study clearly demonstrated that rapid progres-
sion of SIV infection in macaques constitutes a pathological
phenomenon that is distinct from classic AIDS. Nonetheless,
the study of rapid progression to AIDS is important for a
couple of reasons. First, RP macaques allow us to examine the
direct effects of virus-induced cytopathology, uncomplicated by
the secondary effects of immune activation-induced cell death
seen in CP macaques. Second, it is important to understand the
similarities and differences between SIV and HIV infections in
using them as models for AIDS. Whereas the pathological and
clinical descriptions of HIV-1 in humans are fairly uniform,
SIV infection in macaques spans a wider spectrum of disease
syndromes and pathological manifestations.
We thank Russell Byrum for conducting animal studies; Robert
Goeken, Sonya Whitted, and Simoy Goldstein for technical assistance;
and Owen Schwartz of the Confocal Laboratory of the Research Tech-
nologies Branch for assistance with confocal microscopy.
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    • "Remarkably, we showed directly in situ that CD4-depletion induces a significant shift in the nature of productively infected cells, with 80% of SIV vRNA + cells in LN and mucosal tissues being CD3 + T-cells in undepleted controls but CD68/CD163 + macrophages in CD4- depleted RMs. It has recently been described that increased activation and rapid turnover of monocytes, in particular those with a CD14 + CD16 + phenotype, associates with macrophage destruction in tissues and predicts the tempo of progression to AIDS in SIV-infected RMs91011. In response to activation, CD163 is cleaved from the cellular surface of monocytes/ macrophages and is shed as sCD163 [23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Author Summary CD4+ T-cells are both mediators of antiviral immune response and critical targets for HIV replication. We have previously shown that experimental depletion of CD4+ T-cells prior to SIV infection in rhesus macaques results in higher viremia and the emergence of CD4-independent SIV-envelopes. The findings reported in this new study of CD4 depletion address key unanswered questions about the phenotype, location, and lifespan of the sources of the increased viral replication in the absence of CD4+ T-cells. Altogether, our new data indicate that depletion of CD4+ T-cells prior to SIV infection results in activation of monocyte and massive infection of tissue-resident macrophages, which appear to be the predominant population of productively infected cells. Furthermore, our analysis of the slope of viremia decline after initiation of antiretroviral therapy suggests that the lifespan of these virus targets is markedly shorter than those previously estimated for macrophages. In summary, in the context of CD4+ T-cell depletion macrophages can be highly infectable, exhibit rapid turnover, and short in vivo lifespan. These finding raises a suggestive hypothesis that eradication of HIV from this reservoir could be enhanced by therapeutics able to modulate monocyte/macrophage turnover.
    Full-text · Article · Oct 2014
    • "There was also evidence of lymphoid fibrosis, opportunistic infections, and lymphoma. On the other hand, these animals were more similar to SIV-infected rapid progressor macaques [37]. CD4 T-cell decline was rapid, there were no anti-HIV antibody responses, and viral production was mainly from macrophages at the later stages of infection. "
    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 exhibits a narrow host range, hindering the development of a robust animal model of pathogenesis. Past studies have demonstrated that the restricted host range of HIV-1 may be largely due to the inability of the virus to antagonize and evade effector molecules of the interferon response in other species. They have also guided the engineering of HIV-1 clones that can replicate in CD4 T-cells of Asian macaque species. However, while replication of these viruses in macaque hosts is persistent, it has been limited and without progression to AIDS. In a new study, Hatziioannou et al., demonstrate for the first time that adapted macaque-tropic HIV-1 can persistently replicate at high levels in pigtailed macaques (Macaca nemestrina), but only if CD8 T-cells are depleted at the time of inoculation. The infection causes rapid disease and recapitulates several aspects of AIDS in humans. Additionally, the virus undergoes genetic changes to further escape innate immunity in association with disease progression. Here, the importance of these findings is discussed, as they relate to pathogenesis and model development.
    Full-text · Article · Sep 2014
    • "Also, in SHIV-infected macaques, after depletion of CD4+ T cells, macrophages were identified as the virus producing cells (Igarashi et al., 2001 ). In macaques inoculated with rapidprogressing SIVsmm, the majority of SIV-positive cells in the lymph nodes and gastrointestinal tract were macrophages (Brown et al., 2007). As already reported, macrophages have the potential to produce viruses at the end stage of infection. "
    [Show abstract] [Hide abstract] ABSTRACT: To understand the pathogenicity of acquired immune deficiency syndrome (AIDS), it is important to clarify where, when and how the virus replicates in the body of infected individuals. To identify the major virus replication site at the end stage of SHIV infection, we investigated the systemic tissues of SHIV-infected monkeys that developed AIDS-like disease. We quantified proviral DNA, and compared the mutation patterns of the viruses in various systemic tissues and in peripheral blood through phylogenetic analysis of the full genome sequence. We found that the amounts of proviral DNA detected in internal tissues were higher than those in peripheral blood mononuclear cells. In the sequence and phylogenetic tree analyses, the mutation patterns of the viruses in each tissue were generally different. However, the mutation pattern of the viruses in the jejunum and mesenteric lymph node were most similar to that of plasma viral RNA among the tissues examined in all three monkeys. In two of the three monkeys, which were euthanized earlier, viruses in the jejunum and mesenteric lymph node occupied the root position of the phylogenetic tree. Furthermore, in these tissues, more than 50% of SHIV-expressing cells were identified as macrophages based on co-expression of CD68. These results suggest that macrophages of the small intestine and/or mesenteric lymph node are the major virus production site at the end stage of SHIV infection of macaques.
    Full-text · Article · Jul 2013
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