JOURNAL OF VIROLOGY, May 2010, p. 4769–4781
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 9
Generation of the Pathogenic R5-Tropic Simian/Human
Immunodeficiency Virus SHIVAD8by Serial
Passaging in Rhesus Macaques?†
Yoshiaki Nishimura,1Masashi Shingai,1Ronald Willey,1Reza Sadjadpour,1Wendy R. Lee,1
Charles R. Brown,1Jason M. Brenchley,1Alicia Buckler-White,1Rahel Petros,2
Michael Eckhaus,3Victoria Hoffman,3Tatsuhiko Igarashi,1‡
and Malcolm A. Martin1*
Laboratory of Molecular Microbiology1and Comparative Medicine Branch,2National Institute of Allergy and Infectious Diseases,
and Diagnostic and Research Services Branch, Division of Veterinary Resources, Office of the Director,3
National Institutes of Health, Bethesda, Maryland 20892
Received 28 October 2009/Accepted 31 January 2010
A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial
passaging in rhesus macaques. All 13 animals inoculated with SHIVAD8passaged lineages experienced marked
depletions of CD4?T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual
losses of both memory and naïve CD4?T lymphocytes, generated antiviral CD4?and CD8?T cell responses,
and sustained chronic immune activation while maintaining variable levels of plasma viremia (102to 105RNA
copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic
infections caused by Pneumocystis carinii, Mycobacterium avium, and Campylobacter coli that required eutha-
nasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4?
T lymphocytes (92 to 154 cells/?l) following 1 to 2 years of infection. When tested for coreceptor usage, the
viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIVAD8-
inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of
>1 ? 107RNA copies/ml and rapid irreversible loss of memory CD4?T cells that required euthanasia between
weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4?T lymphocytes, and
induction of AIDS make the SHIVAD8lineage of viruses a potentially valuable reagent for vaccine studies.
Simian immunodeficiency virus (SIV)/macaque models of
AIDS have been extensively used as surrogates for human
immunodeficiency virus type 1 (HIV-1) in studies of virus-
induced immunopathogenesis and vaccine development. As is
observed for the HIVs recovered from a majority of individuals
during the asymptomatic phase of their infections, pathogenic
SIVs utilize the CCR5 coreceptor to enter their CD4?T lym-
phocyte targets in vivo (36). This leads to the elimination of
memory CD4?T cells circulating in the blood and residing at
effector sites (gastrointestinal [GI] tract, mucosal surfaces, and
lung), particularly during acute HIV and SIV infections (5, 29,
32, 49). In contrast to naturally occurring SIVs and HIVs,
SIV/HIV chimeric viruses (simian/human immunodeficiency
viruses [SHIVs]) were constructed in the laboratory by insert-
ing a large segment of the HIV genome, including the env
gene, into the genetic backbone of the molecularly cloned
SIVmac239(44). SHIVs were developed because they expressed
the HIV envelope glycoprotein and could be used in vaccine
experiments to evaluate neutralizing antibodies (NAbs) elic-
ited by HIV-1 gp120 immunogens. The commonly used patho-
genic SHIVs generated high levels (107to 108RNA copies/ml)
of plasma viremia and induced an extremely rapid, systemic,
and nearly complete depletion of the entire CD4?T cell pop-
ulation, resulting in death from immunodeficiency beginning at
3 months postinoculation (23, 26, 41). Unlike SIVs, however,
these pathogenic SHIVs exclusively targeted CXCR4-express-
ing CD4?T cells during infections of rhesus monkeys (36).
Despite their extraordinary virulence, most vaccine regimens
(naked DNA, peptides, proteins, inactivated virions, recombi-
nant modified vaccinia virus Ankara (MVA), and DNA prime/
recombinant viral-vector boosting) were effective in controlling
intravenous (i.v.) and mucosal X4-tropic SHIV challenges (1,
3, 33, 42, 46). When it became apparent that the same vac-
cination strategies that were effective in suppressing patho-
genic SHIVs failed to control SIV infections, concerns were
raised about whether X4 SHIVs were appropriate surro-
gates for HIV in vaccine experiments (13).
The unusual biological properties of the X4 SHIVs plus the
discrepant outcomes of SIV and X4 SHIV vaccine experiments
have become a driving force for developing CCR5-utilizing
(R5) SHIVs. Although several clade B and clade C R5-tropic
SHIVs have been constructed (7, 15, 21, 30, 38), the SHIVSF162
lineage viruses are the best-characterized and most widely used
R5 SHIVs (20). They have been employed in microbicide (10),
neutralizing monoclonal antibody (MAb) passive-transfer (16,
17), and vaccination (2) studies.
* Corresponding author. Mailing address: Bldg. 4, Room 315A, 4
Center Drive MSC 0460, National Institutes of Health, Bethesda, MD
20892-0460. Phone: (301) 496-4012. Fax: (301) 402-0226. E-mail: malm
‡ Present address: Laboratory of Primate Models, Institute for Virus
Research, Kyoto University, 53 Shogoinkawaramachi, Sakyo-ku, Kyoto
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 10 February 2010.
In the aftermath of the failed STEP HIV vaccine trial, there
was general consensus that additional SIVs and SHIVs should
be developed, particularly for use as heterologous challenge
viruses in vaccine studies (12). With this goal in mind, we
report the generation of a new pathogenic R5-tropic SHIV
bearing the env gene from the HIV-1Adaisolate (14). HIV-1Ada
was selected because it is a prototypical macrophage-tropic
strain (8), uses CCR5 for cell entry (53), and has the potential
for eliciting NAbs against HIV-1 gp120, and we had previously
constructed a full-length infectious molecular clone (pHIV-
1AD8) (48). Based on previous experience in obtaining patho-
genic X4-tropic SHIVs, serial passaging in macaques, treated
with an anti-CD8 MAb at the time of virus inoculation, was
used to expedite the adaptation of R5-SHIV sequences in a
nonhuman primate host. Of the 13 animals inoculated with in
vivo-passaged SHIVAD8#2(see below) and its immediate de-
rivatives, 10 exhibited a normal-progressor (NP) phenotype,
sustaining gradual depletions of both memory and naïve CD4?
T cells from the circulation and memory CD4?T cells at an
effector site (lung) while maintaining variable viral-RNA loads
(102to 105RNA copies/ml) for up to 3 years postinfection
(p.i.). Five of these monkeys developed immunodeficiency with
associated opportunistic infections requiring euthanasia. Three
other NPs currently have total CD4?T cell counts of 92 to 154
cells/?l plasma after 1 to 2 years of infection. The remaining 3
of the 13 SHIVAD8-inoculated macaques experienced a rapid-
progressor (RP) clinical course and were euthanized between
weeks 19 and 23 p.i. because of intractable diarrhea and
marked weight loss. The sustained viremia, associated deple-
tion of CD4?T lymphocytes, and induction of AIDS make the
SHIVAD8lineage of viruses a potentially valuable reagent for
MATERIALS AND METHODS
Construction of SHIVAD8. SHIVAD8contains the env gene from the R5-tropic
HIV-1Ada(14)-derived molecular clone pHIVAD8(48). A 3.04-kb segment from
pHIVAD8, including a portion of the vpr gene and the entire tat, rev, vpu, and env
genes, was PCR amplified using the forward primer TGAAACTTATGGGGA
TACTTGGGC, which begins at nucleotide 141 of the AD8 vpr gene, allowing the
incorporation of a unique EcoRI site, located 21 nucleotides downstream from
the primer, into the PCR product. The reverse PCR primer (TCCACCCATAA
GCTTATAGCAAAGTCCTTTCCAAGCCC) generated a HindIII site adjacent
to and encompassing the last 2 nucleotides of the env reading frame, as well as
a substitution of a Thr for a Leu 3 codons upstream from the env termination
codon. PCRs were performed using 10 pmol each of the forward and reverse
primers, Platinum PCR SuperMix High Fidelity (Invitrogen), and 1 ?l of
pHIVAD8in a final volume of 50 ?l. The reaction mixtures were heated to 94°C
for 2 min, followed by 30 cycles of 94°C for 20 s, 59°C for 30 s, and 70°C for 3 min
and a 7-min extension at 70°C. The PCR product was gel extracted using a
Qiaquick gel extraction kit (Qiagen) and digested with EcoRI and HindIII, and
the resulting 3.04-kb restriction fragment was cloned directly into the previously
described and similarly digested pSHIVDH12(45) to generate pSHIVAD8. DNA
sequencing of the entire 3.04-kb insert in pSHIVAD8was conducted to verify that
no spurious changes had been introduced during the PCR amplification and
Preparation of SHIVAD8virus stocks. HeLa cells were transfected with 25 ?g
of pSHIVAD8, and the virus present in the supernatant at 48 h was pelleted in an
ultracentrifuge and resuspended in RPMI 1640 medium as previously described
(52). Stocks of the cloned SHIVAD8were prepared by infecting PM1 cells (31)
or concanavalin A (ConA)-activated rhesus monkey peripheral blood mononu-
clear cells (PBMC) with the HeLa-derived SHIVAD8, as previously described
(23, 24), and pooling the supernatant media at the times of peak reverse tran-
scriptase (RT) production from both infections.
SHIVAD8stock 2 (SHIVAD8#2) was prepared from PBMC and bone marrow
(BM), spleen, and lymph node (LN) samples collected from macaque CK1G on
day 6 p.i. Cell suspensions from axillary, inguinal, iliac, and mesenteric LNs,
PBMC, and BM were cocultivated with PBMC from uninfected animals; the
culture supernatants were monitored daily for RT activity, pooled, and desig-
nated SHIVAD8#2. The infectious titer of SHIVAD8#2was 1.5 ? 103tissue
culture infective doses (TCID50)/ml, as determined in rhesus macaque PBMC.
SHIVAD8lymph node virus (SHIVAD8LN) was prepared from supernatant
medium collected from cocultures of lymph node suspensions plus PBMC re-
covered from animal CJ8B at week 59 p.i. (Table 1) and PBMC from uninfected
rhesus monkeys. The infectious titer of SHIVAD8LNwas 6.4 ? 103TCID50/ml, as
determined in rhesus macaque PBMC.
SHIVAD8PBMC virus (SHIVAD8PBMC) was prepared from supernatant me-
dium collected from cocultures of PBMC recovered and pooled from animals
CK15 and CJ58 at week 4 p.i. (Table 1) and PBMC from uninfected rhesus
monkeys. The infectious titer of SHIVAD8PBMCwas 1.1 ? 104TCID50/ml, as
determined in rhesus macaque PBMC.
SHIVAD8#2.d30was prepared by infecting ConA-stimulated pig-tailed ma-
caque (PT) PBMC with SHIVAD8#2. Fresh ConA-stimulated PT PBMC were
added to the infected cultures on days 10 and 20, and the supernatant medium
collected on day 30, designated SHIVAD8#2.d30, had an infectious titer of 8.5 ?
104TCID50/ml, as determined in rhesus macaque PBMC.
Virus replication assay in rhesus monkey PBMC. The preparation and infec-
tion of rhesus monkey PBMC have been described previously (25). Briefly,
PBMC stimulated with concanavalin A and cultured in the presence of recom-
binant human interleukin-2 (IL-2) were spinoculated (1,200 ? g for 1 h) (37) with
virus normalized for RT activity. Virus replication was assessed by RT assay of
the culture supernatant as described above.
Animal experiments. Rhesus macaques (Macaca mulatta) were maintained in
accordance with the guidelines of the Committee on Care and Use of Laboratory
Animals (9) and were housed in a biosafety level 2 facility; biosafety level 3
practices were followed. Phlebotomies, i.v. virus inoculations, euthanasia, and
tissue sample collections were performed as previously described (11). Bron-
choalveolar lavage (BAL) fluid lymphocytes were prepared from uninfected and
infected animals using a pediatric bronchoscope (Olympus BF3C40; Olympus
America, Inc., Melville, NY), as previously described (22).
Serial in vivo passaging of SHIVAD8was initiated by transferring whole blood
(10 ml) and BM (2 ml) to a recipient animal previously treated with the anti-
CD8?T cell-depleting MAb cM-T807 (10 mg/kg of body weight) on days ?1 and
?3 p.i. In subsequent passages, spleen, LN (axillary, inguinal, iliac, and mesen-
TABLE 1. Infection of rhesus macaques with SHIVAD8#2and
CK15 ...............Blood transfusion from CJ8B (wk 60)
CJ58.................Blood transfusion from CJ8B (wk 60)
CE8J................Lymph node virusa(SHIVAD8#2LN, 3.2 ? 105
TCID50) from CJ8B (wk 59)
CJ35.................Lymph node virus (SHIVAD8#2LN, 3.2 ? 105TCID50)
from CJ8B (wk 59)
CJ3V................PBMC virusb(SHIVAD8#2PBMC, 5.9 ? 104TCID50)
from CK15 ? CJ58 (wk 4)
CK5G..............PBMC virus (SHIVAD8#2PBMC, 5.9 ? 104TCID50)
from CK15 ? CJ58 (wk 4)
DB99...............Blood transfusion from CJ8B (wk 117) ? CK15
(wk 57) ? CJ58 (wk 57)
DA1Z..............Blood transfusion from CJ8B (wk 117) ? CK15
(wk 57) ? CJ58 (wk 57)
A4E008 ...........Blood transfusion from DA1Z (wk 1) ? DB99 (wk 1)
DA4W.............Blood transfusion from DA1Z (wk 1) ? DB99 (wk 1)
CL5A...............SHIVAD8#2passaged in vitro for 30 days
(SHIVAD8#2.d30, 4.3 ? 105TCID50)
CL98................SHIVAD8#2 passaged in vitro for 30 days
(SHIVAD8#2.d30, 4.3 ? 105TCID50)
aLymph node virus: SHIVAD8derivative prepared from the supernatant me-
dium collected from cocultures of lymph node suspensions plus PBMC, recov-
ered from animal CJ8B at week 59 p.i., and PBMC from uninfected rhesus
bPBMC virus: SHIVAD8derivative prepared from the supernatant medium
collected from cocultures of PBMC, recovered from the indicated infected ani-
mals at week 4 p.i., and PBMC from uninfected rhesus monkeys.
4770NISHIMURA ET AL.J. VIROL.
teric), PBMC, and BM cell suspensions were prepared from infected donors at
the time of necropsy and transferred (1 ? 108to 3 ? 108mononuclear cells and
1 ? 108to 10 ? 108BM cells) to a new recipient by the i.v., intraperitoneal (i.p.),
and BM routes.
Quantitation of proviral-DNA and plasma viral-RNA levels. The number of
viral-DNA copies in PBMC was measured by quantitative DNA PCR (45).
Viral-RNA levels in plasma were determined by real-time reverse transcription-
PCR (ABI Prism 7700 sequence detection system; Applied Biosystems, Foster
City, CA) as previously reported, using reverse-transcribed viral RNA in plasma
samples from SIVmac239-inoculated rhesus macaques (11).
Lymphocyte immunophenotyping and data analysis. EDTA-treated blood
samples and BAL fluid lymphocytes were stained for flow cytometric analysis as
described previously (34, 36), using combinations of the following fluorochrome-
conjugated MAbs: CD3 (fluorescein isothiocyanate [FITC] or phycoerythrin
[PE]), CD4 (PE, peridinin chlorophyll protein-Cy5.5 [PerCP-Cy5.5], or allophy-
cocyanin [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 (FACS-
Calibur; BD Biosciences Immunocytometry Systems). Data analysis was per-
formed using CellQuest Pro (BD Biosciences) and FlowJo (TreeStar, Inc., San
Carlos, CA). For Ki-67 staining, cells were fixed with fluorescence-activated cell
sorter (FACS) lysing solution (Becton Dickinson), treated with FACS perme-
abilization buffer 2 (Becton Dickinson), and stained with Ki-67 MAb or a control
isotype IgG1. In this study, naïve CD4?T cells were identified by their CD95low
CD28highphenotype, whereas memory CD4?T cells were CD95highCD28highor
CD95highCD28lowin the CD4?small lymphocyte gate (36, 39).
Intracellular-cytokine assays. Stimulation was performed on frozen lympho-
cytes as described previously (40). Freshly thawed lymphocytes were resuspended
(106/ml) in RPMI medium supplemented with antibiotics and glutamine. Anti-
CD28 conjugated to Alexa 594-PE was used for costimulation. Staphylococcus
enterotoxin B (1 ?g/ml; Sigma-Aldrich, St. Louis, MO) was used to stimulate T
cells mitogenically through the T cell receptor as a positive control. A negative
control (cells treated only with costimulatory anti-CD28) was included in every
experiment. Peptides used to stimulate SIV-specific T cells were 15 amino acids
(aa) in length, overlapping by 11 amino acids, and encompassed SIVmac239Gag
(New England Peptide, Gardner, MA). The concentration of each peptide was 2
?g/ml for stimulations, which were performed in the presence of brefeldin A
(BFA) (1 ?g/ml; Sigma-Aldrich, St. Louis, MO) for 16 h at 37°C. All cells were
surface stained with the dead-cell exclusion dye Aqua Blue (Invitrogen Corp.,
Carlsbad CA), followed by staining with anti-CD3 Alexa 700 (BD Biosciences),
anti-CD4 Cy5.5-PE (eBioscience Inc., San Diego, CA), anti-CD8 Pacific Blue
(BD Biosciences), and anti-CD95 Cy5-PE (BD Biosciences). The cells were then
fixed, permeabilized, and stained with anti-gamma interferon (IFN-?) Cy7-PE
(BD Biosciences), anti-IL-2 APC (BD Biosciences), tumor necrosis factor (TNF)
FITC (BD Biosciences), and Mip1-? PE (BD Biosciences). SIV-specific CD8 T
cell responses are reported as the frequency of memory CD8 T cells, gated by
characteristic light scatter properties; then as Aqua Blue?, CD3?, CD8?, CD4?,
or CD95?; and by production of either TNF or Mip-1?. All data are reported
after background subtraction.
Virus neutralization assays. Autologous plasma samples (1:20 dilution) from
SHIVAD8-infected macaques were incubated with (i) the same uncloned SHIV-
AD8derivative used for inoculation or (ii) the SHIVAD8isolated from PBMC at
week 4 p.i. (for monkeys CJ58 and CK15) in quadruplicate in 96-well flat-bottom
culture plates in a total volume of 50 ?l for 1 h at 37°C. Prechallenge plasma
samples from each animal served as controls. Freshly trypsinized TZM-bl cells
(50) (1.5 ? 104in 150 ?l Dulbecco’s modified Eagle’s medium [DMEM] con-
taining 20 ?g/ml DEAE dextran) were added to each well, and the cultures were
maintained in a 37°C incubator for 28 h. The amount of virus-induced luciferase
activity, measured as relative light units (RLU), present in cell lysates was
determined as previously described (51), and the average neutralization activity
for each plasma sample was determined. The average number of RLU for the
prechallenge plasma controls ranged from 1 ? 105to 2 ? 105. Any sample
resulting in a 50% reduction of luciferase activity compared to that obtained with
the uninfected control sample was considered positive for NAbs. To determine
neutralizing-antibody titers, 40 ?l of diluted virus, sufficient to generate the
desired numbers of RLU, was mixed with 10 ?l of appropriately diluted plasma
samples in a 96-well plate and incubated for 1 h at 37°C. TZM-bl cells were
added, cultures were maintained for an additional 28 h, and intracellular lucif-
erase activity was measured as described above.
Coreceptor utilization assays. Freshly trypsinized TZM-bl cells (1 ? 104per
well) in 135 ?l DMEM containing 10% fetal calf serum (FCS) and DEAE
dextran (15 ?g/ml) were seeded in flat-bottom 96-well plates. Twenty-five mi-
croliters of coreceptor antagonists (AD101 against CCR5, AMD3100 against
CXCR4, or both, at final concentrations ranging from 0.1 nM to 1,000 nM) was
added to each well. Following incubation for 1 h at 37°C, 10 TCID50of replica-
tion-competent virus, determined in TZM-bl cells as previously described, in 40
?l was added to each well. After 24 h of incubation at 37°C, luciferase activity was
determined. The percent infectivity reported was derived from the mean of
To generate 293T cell-derived SHIVAD8pseudotyped viruses, two separate
plasmids were constructed. The first (pNLenv1) contained a frameshifted muta-
tion in the leader peptide region of gp120 (43). Plasmids expressing the
SHIVAD8(RIG?) and SHIVAD8(RIG–) env genes [pCMV-AD8(RIG?) and
AD8(RIG?)] were generated by reverse transcription-PCR of plasma viral
RNA, collected from macaque DB99 at the time of euthanasia, and subcloning
into NotI and (newly created) XbaI sites of the pCMVbeta expression plasmid
(Clonetech, Palo Alto, CA). Both plasmids [pNLenv1 and pCMV-AD8(RIG) in
a 5:1 ratio] were cotransfected into 293T cells using Lipofectamine 2000 (In-
vitrogen, Carlsbad, CA). The titers of pseudotyped-virus preparations were de-
termined, and they were assayed for coreceptor usage 48 h following infection of
TZM-bl cells, as described for replication-competent virus.
Construction of a CCR5-tropic SHIV. We previously re-
ported the construction of a full-length infectious HIV-1 mo-
lecular clone (pHIV-1AD8) derived from the prototypical mac-
rophage-tropic CCR5-utilizing HIV-1Adaisolate (14, 48). A
SHIV expressing the env gene from pHIVAD8was obtained by
inserting the 3.04-kb EcoRI-to-HindIII DNA fragment (in-
cluding a portion of vpr and the entire tat, rev, vpu, and env
genes) into the genetic background of pSHIVDH12(45), as
described in Materials and Methods. The resulting molecular
clone, pSHIVAD8, directed the production of progeny virions
following the transfection of HeLa cells. Virus stocks were
prepared by infecting PM1 cells or ConA-stimulated rhesus
PBMC with virions pelleted from HeLa cell transfection cul-
It is not generally appreciated how daunting it is to generate
an R5-tropic SHIV able to maintain detectable levels of set-
point viremia, exclusively target memory CD4?T cells, and
induce immunodeficiency in inoculated rhesus monkeys. Sim-
ply replacing orthologous SIV sequences with a DNA segment
including a CCR5-utilizing HIV-1 env gene does not usually
result in a SHIV exhibiting robust replication kinetics in vivo
and a disease-inducing phenotype. This was, in fact, the case
for SHIVAD8: levels of plasma viremia following virus inocu-
lation (1 ml of undiluted virus by the i.v., i.p., and BM routes)
were promptly and durable suppressed, and the numbers of
memory CD4?T lymphocytes did not change appreciably, as
shown for a representative infected animal (CJ7H) in Fig. 1a.
To be certain that we were on the right track with respect to
the targeting and elimination of memory, not naïve, CD4?T
cells in vivo, a second macaque (CJ9F) was treated with the
CD8?T lymphocyte-depleting MAb cM-T807 24 h prior to
SHIVAD8inoculation, as well as on days 3 and 6 post-virus
infection, to promote a vigorous in vivo infection. Unlike un-
treated macaque CJ7H, the levels of plasma viral RNA in
monkey CJ9F rapidly rose to 3.8 ? 107copies/ml by day 10 p.i.
and were associated with a rapid and irreversible decline of
circulating memory CD4?T cells (Fig. 1b). In contrast, the
numbers of naïve CD4?T lymphocytes in animal CJ9F were
maintained in the 600- to 800-cell/?l range during this period.
This result, therefore, confirmed that SHIVAD8could sustain
high virus loads and preferentially target the memory CD4?T
VOL. 84, 2010 GENERATION OF SHIVAD8BY SERIAL PASSAGING IN MACAQUES 4771
cell subset in vivo, but only in an animal with a compromised
In vivo passaging of SHIVAD8. The prompt control of plasma
viremia and the nonpathogenic phenotype of SHIVAD8ob-
served in untreated macaques were reminiscent of the infec-
tivity patterns observed with first-generation X4-tropic SHIVs
(28, 44). We therefore initiated serial animal-to-animal pas-
saging of SHIVAD8with macaque CJ9F as the “founder” in-
fected monkey (Fig. 2a). This approach had previously been
used to generate X4 SHIVs exhibiting more robust replicative
and pathogenic properties (26, 41). Unfortunately, in vivo se-
rial passaging of virus to optimize infectivity is an empirical and
stochastic process. One never knows when or if an R5 SHIV
has acquired an augmented replicative phenotype. The ulti-
mate proof that such a change has occurred requires the inoc-
ulation of additional animals and waiting several months to
assess the resultant viral replication kinetics and CD4?T cell
The strategy employed was to maximize the emergence of
disease-inducing SHIV variants, putatively present in an in-
creasingly genetically diverse virus population, by serially
transferring large numbers of infected cells by i.v., i.p., and BM
routes into recipient animals previously treated with an anti-
CD8 depleting MAb. As indicated in Fig. 2a, whole blood and
bone marrow cells were transferred from macaque CJ9F to
macaque H681 by these three routes. In subsequent passages,
cell suspensions were prepared from spleen, LN (axillary, in-
guinal, iliac, and mesenteric), PBMC, and BM cells collected at
the time of necropsy, as described in Materials and Methods.
With one exception (macaque CJ7F), the depleting anti-CD8
MAb was administered to a recipient animal on days ?1 and
?3 p.i. to facilitate unrestricted replication in vivo. Animal
CJ7F did not receive anti-CD8 MAb at the time of virus trans-
fer to investigate the possibility that SHIVAD8had acquired
improved replication properties in vivo following the initial two
in vivo passages. Because this was not the case (its plasma
viral-RNA loads had declined to 360 RNA copies/ml at week
10 p.i.), macaque CJ7F was treated with anti-CD8 MAb at
week 13 p.i. and sustained an immediate burst of virus produc-
tion that reached 1.4 ? 106RNA copies/ml of plasma at week
FIG. 1. Infectivity of the original SHIVAD8in rhesus monkeys. Macaques CJ7H (a) and CJ9F (b) were inoculated with 1 ml of undiluted
SHIVAD8by the i.v., i.p., and BM routes. Macaque CJ9F was treated with the depleting anti-CD8 MAb cM-T807 as indicated.
FIG. 2. Serial animal-to-animal passage of SHIVAD8. (a) Passage history of SHIVAD8and origin of SHIVAD8#2. (b) Rhesus monkey PBMC
were infected with SHIVAD8or the passaged SHIVAD8#2virus stock, normalized for RT activity. CPM, counts per minute.
4772 NISHIMURA ET AL.J. VIROL.
14 p.i. CJ7F was euthanized at week 15 p.i., and cell suspen-
sions were prepared as described above and transferred by the
i.v., i.p., and BM routes into macaque CK1A, previously
treated with anti-CD8 MAb (Fig. 2a). Following the fifth in
vivo passage, macaque CK1G was euthanized on day 6 p.i., and
cell suspensions, prepared at the time of necropsy, were cocul-
tivated with ConA-stimulated PBMC from uninfected rhesus
monkeys as described in Materials and Methods; the culture
supernatants were monitored for the presence of reverse tran-
scriptase activity, pooled, and designated SHIVAD8#2.
Inoculation of rhesus macaques with SHIVAD8#2and its
immediate derivatives resulted in sustained plasma viremia
and loss of CD4?T lymphocytes. To ascertain whether serial
passaging of SHIVAD8in vivo had resulted in the acquisition of
improved replicative properties, ConA-stimulated rhesus mon-
key PBMC were infected with SHIVAD8#2or the starting
SHIVAD8virus preparation, both normalized for RT activity.
As shown in Fig. 2b, SHIVAD8#2replicated to much higher
levels in cultured macaque PBMC than the original SHIVAD8.
To determine whether this improved infectivity of SHIVAD8#2
for rhesus PBMC was correlated with augmented replication in
an animal not treated with the depleting anti-CD8 MAb, ma-
caque CJ8B was inoculated i.v. with 1.5 ? 104TCID50of
SHIVAD8#2. As shown in Fig. 3a, this monkey experienced a
marked but transient depletion of memory CD4?T cells in
BAL specimens during the acute infection and maintained
detectable levels of plasma viremia. Because animal CJ8B
subsequently experienced a decline in the total circulating
CD4?T lymphocyte population from 565 to 175 cells/?l at
week 56 p.i. (Fig. 3b), whole blood or virus propagated ex vivo
from CJ8B lymph node suspensions (lymph node virus
[SHIVAD8LN]) was inoculated into four additional macaques
(CK15, CJ58, CE8J, and CJ35) (Fig. 4). Four other animals
(DB99, DA1Z, A4E008, and DA4W) received blood transfu-
sions, and two (CJ3V and CK5G) were inoculated with PBMC
coculture virus (SHIVAD8PBMC) derived from monkeys CK15
and CJ58 (Fig. 4). In addition, because it was unknown at the
time of its preparation whether SHIVAD8#2had acquired aug-
mented in vivo infectivity properties, SHIVAD8#2was propa-
gated for an additional 30 days ex vivo in macaque PBMC as
described in Materials and Methods. Because the resulting
derivative, designated SHIVAD8#2.d30, exhibited robust infec-
tivity in both pigtailed and rhesus macaque PBMC (data not
shown), it was inoculated intravenously into two rhesus mon-
keys (CL5A and CL98) (Fig. 4). The inocula used to infect
rhesus monkeys with SHIVAD8#2and its immediate derivatives
are listed in Table 1. None of these monkeys received the
depleting anti-CD8 MAb.
Ten of the 13 animals infected with SHIVAD8#2or its im-
mediate derivatives experienced an NP clinical course charac-
terized by set-point virus loads that varied widely (from less
than 103to more than 105RNA copies/ml) and a gradual
depletion of circulating CD4?T lymphocytes (Fig. 5a and b).
Transient, and in some cases quite significant, losses of mem-
ory CD4?T cells in BAL samples was a common finding
during the acute infection (Fig. 5c). The loss of circulating
CD4?T lymphocytes in the 10 SHIVAD8#2-infected NPs af-
fected both memory and naïve subsets (Fig. 6). With one ex-
ception (monkey CJ35), these animals sustained depletions of
circulating memory CD4?T cells to the 200-cell/?l level by
week 100. NPs also experienced increased memory CD4?T
lymphocyte turnover, as monitored by Ki-67 expression, par-
ticularly during the first 10 weeks and the final stages of the
infection (see Fig. S1 in the supplemental material). The loss
of naïve CD4?T lymphocytes in NP monkeys was even more
profound. By week 80 p.i., this subset had declined to below
100 cells/?l in all of the animals (Fig. 6b). At the time of their
euthanasia, five NPs (CJ8B, CE8J, CJ3V, CK15, and CL98)
had only 1, 3, 6, 12, and 68 circulating naïve CD4?T cells/?l,
respectively. We previously reported that SIVsmE543-infected
FIG. 3. SHIVAD8#2induces sustained plasma viremia and loss of CD4 T cells in an inoculated rhesus macaque. Plasma viremia and the
percentage of BAL fluid CD4?T cells (a) or the absolute numbers of circulating CD4?T cells (b) in rhesus macaque CJ8B inoculated
intravenously with SHIVAD8#2are shown. RT-PCR, reverse transcription-PCR.
FIG. 4. SHIVAD8#2and its immediate derivatives cause immuno-
deficiency in rhesus macaques. The dashed arrows indicate virus trans-
fer by blood transfusion. The thick arrows indicate LN or PBMC
specimens used to generate virus stocks by coculturing with PBMC
from uninfected donors. †, euthanized animals.
VOL. 84, 2010GENERATION OF SHIVAD8BY SERIAL PASSAGING IN MACAQUES4773
NPs had also experienced a marked loss of naïve CD4?T cells
as early as 20 weeks p.i. (35). It was therefore not unexpected
that NP SHIVAD8-infected monkeys might also sustain a de-
pletion of their naïve CD4?T cell subset.
Three of the 13 macaques inoculated with SHIVAD8#2and
its immediate derivatives became RPs, requiring euthanasia
between weeks 19 and 23 p.i. because of anorexia, intractable
diarrhea, and marked weight loss (Fig. 7). Virus set points in
the RPs exceeded 107RNA copies/ml, memory CD4?T cells
in BAL specimens rapidly and irreversibly declined, and at the
time of death, all of the animals had sustained marked losses of
circulating CD4?T cells.
Immune responses to SHIVAD8.In the context of its use as a
challenge virus in vaccine experiments, it was important to
show that SHIVAD8elicited both cellular and humoral im-
mune responses during infections of rhesus monkeys. There-
fore, anti-SHIVAD8Gag-specific CD8?T lymphocyte re-
sponses were measured by flow cytometry for 6 of the 10 NPs
by intracellular staining of cells expressing TNF-? and/or
IFN-? following stimulation with a 15-mer peptide pool
spanning SIVmac239Gag. The levels of virus-specific CD8?T
cells in this group of rhesus monkeys ranged from 0.33 to
1.68% during the second year of their infection (see the
table in the supplemental material). A similar analysis of
Gag-specific responses in memory CD4?T cells at these
times in the same animals indicated that 0.90 to 2.90%
expressed TNF-? and/or IFN-? (see the table in the supple-
NAbs were detected in several of the NPs during the course
of their infections (Fig. 8). The seven macaques evaluated had
been inoculated with SHIVAD8#2or two immediate derivatives
(SHIVAD8#2LNand SHIVAD8#2PBMC). Plasma neutralizing
activity directed against the same virus used for animal chal-
lenge was evaluated in monkeys CJ8B, CE8J, CJ35, CJ3V, and
FIG. 5. Total CD4?T lymphocytes are gradually lost in normal progressors following infection with SHIVAD8#2and its immediate derivatives.
The levels of plasma viremia (a), absolute numbers of peripheral CD4?T cells (b), and percentages of BAL fluid CD4?T cells (c) are shown. The
five normal progressors that developed AIDS and were euthanized are indicated (†).
4774 NISHIMURA ET AL.J. VIROL.
CK5G. The neutralization sensitivity of autologous virus
(SHIVAD8#2PBMC) was monitored using plasma collected from
PBMC of macaques CK15 and CJ58 (Fig. 4). The time of
appearance of neutralization activity varied widely (week 20 to
week 78 p.i.) and was generally correlated with levels of set-
point viremia. In the three macaques producing the highest
levels of anti-SHIVAD8NAbs, the actual 50% inhibitory con-
centration (IC50) neutralization titers determined by limiting
plasma dilution were 1:159 (CJ8B at week 89), 1:102 (CJ58 at
week 30), and 1:143 (CE8J at week 52).
Coreceptor usage by SHIVAD8lineage viruses. The env gene
of SHIVAD8was derived from the prototypical macrophage-
tropic HIV-1Ada, previously shown to use CCR5 for cell entry
(53). When tested in a TZM-bl entry assay with inhibitors that
specifically target CXCR4 or CCR5, the original SHIVAD8,
SHIVAD8#2(data not shown), and SHIVAD8#2LNexclusively
utilized CCR5 (see Fig. S2 in the supplemental material). The
marked depletion of circulating naïve CD4?T cells in all
SHIVAD8NPs (Fig. 6b) raised the possibility that a coreceptor
switch had occurred, enabling these viruses to enter and elim-
inate naïve CD4?T cells, which express high levels of surface
CXCR4, but not CCR5. Accordingly, virus was recovered from
three NPs (CK15, CE8J, and CL98) immediately prior to eu-
thanasia. When tested for coreceptor usage, the viruses iso-
lated from all three NPs remained R5 tropic (see Fig. S2 in the
supplemental material), indicating that the loss of naïve CD4?
T cells was not due to direct virus-induced cell killing.
As noted earlier, three monkeys infected with SHIVAD8#2
derivatives exhibited an RP phenotype. By week 10 p.i., these
macaques (DB99, A4E008, and CL5A) had experienced mas-
sive loss of memory CD4?T cells in samples collected by BAL
(Fig. 7c) but had little change in the number of circulating
naive CD4?T lymphocytes (data not shown). However, by
week 19 p.i., the levels of total CD4?T cells in the blood
had declined significantly in all three RPs (Fig. 7b), raising
again the possibility that coreceptor usage might have
changed. To assess a possible coreceptor switch, virus was
collected from RP monkeys DB99 and A4E008 at the time
of euthanasia and evaluated in the TZM-bl assay with spe-
cific CXCR4 and CCR5 inhibitors. As shown in Fig. 9a,
blocking the entry of SHIVAD8-DB99required both inhibi-
tors, whereas SHIVAD8-A4E008was inhibited only by the
CCR5 inhibitor. This result indicates that SHIVAD8-DB99had
acquired the capacity to use CXCR4 during its infection of
macaque DB99 and that SHIVAD8-A4E008had remained R5
Reverse transcription-PCR cloning and sequencing of env
genes amplified from the plasma of macaque DB99 at the time
of its euthanasia revealed that 28 of 29 recovered clones con-
tained a 3-aa insertion (RIG) located 2 residues upstream of
the GPGR sequence in the crown of the gp120 V3 region (Fig.
9b). A similar analysis of the env gene from virus circulating in
monkey A4E008 revealed a different 3-aa insertion (HIG) at
the same location in its V3 loop. The V3 loop sequences
amplified from the plasma of both animals at week 2 p.i. did
not contain any insertion. The gp120 region amplified from the
third RP (macaque CL5A) at the time of euthanasia contained
no insertion (Fig. 9b).
One of the 28 viral-DNA clones amplified from macaque
DB99 plasma at the time of euthanasia containing the RIG
insertion in V3 and the single clone simultaneously obtained
from this animal lacking the V3 insertion were used to prepare
pseudotyped virus for testing in the entry assay, as described in
Materials and Methods. As shown in Fig. 9c, the V3 RIG
insertion conferred usage of both CCR5 and CXCR4 corecep-
tors on SHIVpsAD8(RIG?)compared to the exclusive utilization
of CCR5 by SHIVpsAD8(RIG–), which lacks the gp120 V3 in-
SHIVAD8-infected macaques developed immunodeficiency.
The clinical statuses and disease outcomes of all 13 animals
inoculated with SHIVAD8#2and its immediate derivatives dur-
ing a 2- to 3-year observation period are presented in Table 2.
As noted above, 10 of these 13 macaques were NPs and expe-
rienced gradual and irreversible depletions of both memory
and naïve CD4?T lymphocyte subsets (Fig. 6). Five of these
animals were euthanized with symptoms of AIDS, and 3 addi-
tional NPs currently have CD4?T cell counts ranging from 92
to 154 cells/?l plasma (Table 2). Histopathological studies
performed on specimens collected at the time of necropsy
revealed the presence of Pneumocystis carinii, Mycobacterium
avium, and Campylobacter coli infections in individual
macaques (see Fig. S3 in the supplemental material). In addi-
tion, 3 of the 13 R5-SHIV-infected monkeys experienced an
RP syndrome characterized by sustained plasma viremia of
FIG. 6. Marked depletion of naïve and memory CD4?T lymphocytes characterizes long-term SHIVAD8infection in NP rhesus monkeys.
Absolute numbers of memory CD4?T cells (a) and naive CD4?T cells (b) in 10 normal-progressor macaques during 200 weeks of SHIVAD8
infection are shown. †, euthanized animals.
VOL. 84, 2010 GENERATION OF SHIVAD8BY SERIAL PASSAGING IN MACAQUES4775
?1 ? 107RNA copies/ml; rapid and irreversible loss of mem-
ory CD4?T cells in the blood and at an effector site (BAL);
and intractable diarrhea, anorexia, and weight loss requiring
euthanasia between weeks 19 and 23 p.i.
The results presented clearly show that the generation of a
pathogenic R5-SHIV was not a trivial undertaking. Animal-to-
animal passaging eventually gave rise to SHIVAD8#2, possess-
ing greatly augmented infectivity for rhesus PBMC compared
to the starting SHIVAD8construct. Although it was not appre-
ciated at the time, SHIVAD8#2had also acquired improved in
vivo properties, as evidenced by its and its immediate deriva-
tives’ capacity to cause fatal immunodeficiency in 8 of 13 in-
oculated rhesus monkeys (Fig. 4 and Table 2). The most con-
sistent and distinguishing property of the passaged SHIVAD8
family of viruses during infections of rhesus macaques was the
slow and unremitting loss of both memory and naïve CD4?T
cells (Fig. 6), a pattern of depletion observed in all 10 NPs.
Surprisingly, and in contrast to both SIVmac and SIVsmE
lineages, the pace of CD4?T lymphocyte decline was not
correlated with plasma virus loads. Although the geometric
mean plasma viral-RNA level at week 50 in the SHIVAD8-
infected monkey cohort was 1.7 ? 103RNA copies/ml, the
set-point virus loads varied widely in the 10 infected animals
FIG. 7. Patterns of virus replication and CD4?T cell dynamics in SHIVAD8rapid progressors. The levels of plasma viremia (a), absolute
numbers of peripheral CD4?T cells (b), and percentages of BAL fluid CD4?T cells (c) are shown. †, euthanized animals.
4776NISHIMURA ET AL. J. VIROL.
FIG. 8. Neutralizing-antibody activities detected in normal-progressor macaques following infection with SHIVAD8#2or its immediate deriv-
atives. Plasma samples (1:20 dilution) from the indicated SHIVAD8-infected macaques were incubated in quadruplicate for 1 h at 37°C with the
virus isolates shown in parentheses and then used as an inoculum to infect TZM-bl cells. The luciferase activity present in cell lysates at 28 h p.i.
was measured, and the average percent neutralization activity in plasma at each time point was determined. Prechallenge plasma samples served
as negative controls and baselines for zero neutralizing-antibody activity.
VOL. 84, 2010 GENERATION OF SHIVAD8BY SERIAL PASSAGING IN MACAQUES4777
FIG. 9. Coreceptor utilization of SHIVAD8derivatives isolated from rapid progressors. (a) TZM-bl cells were infected in quadruplicate
with viruses (SHIVAD8-DB99and SHIVAD8-A4E008) recovered from rapid progressors DB99 and A4E008, respectively, in the presence of the
indicated amounts of the small-molecule coreceptor inhibitors AD101 (CCR5), AMD 3100 (CXCR4), or both. SIVmac239and SHIVDH12RCL-7
were also analyzed as representative R5-tropic and dual-tropic viruses, respectively. The luciferase activities present in cell lysates 24 h p.i.
were measured, and percent infectivities were determined in the absence or presence of coreceptor inhibitors. (b) gp120 sequences from the
N-terminal V3 regions of SHIVAD8variants, recovered from three RP animals, were aligned with the starting SHIVAD8V3 loop. The V3
regions of the R5-tropic SHIVSF162P3and its SHIVSF162-BR24Nderivative, which also emerged in an RP, are included in the alignment. (c)
Coreceptor utilization of virus, pseudotyped with Envs present in RP DB99 at the time of necropsy, containing or lacking the 3-aa RIG V3
4778NISHIMURA ET AL. J. VIROL.
(1.6 ? 102to 1.5 ? 105RNA copies/ml). This variability was
also observed in pairs of animals inoculated with identical
SHIVAD8#2derivatives (viz. CK15 and CJ58, and DB99 and
DA1Z). An extreme example of the nonlinkage between viral-
RNA levels and CD4?T cell loss with SHIVAD8occurred with
animal CK5G, which had 43 and 42 circulating naïve and
memory CD4?T cells/?l, respectively, at week 86 p.i. and a
plasma viral load of only 5.4 ? 102RNA copies/ml. During the
chronic phase of SHIVAD8infections, the loss of naïve CD4?
T cells was more rapid and more marked than the depletion of
the memory subset, as was previously observed in SIVsmE543-
infected animals (35) (Fig. 6). By week 80, for example, NPs
had sustained an 87 to 93% loss of naïve CD4?T cells from
their preinoculation levels, whereas the depletion of memory
cells was significant, but not as pronounced. The dissociation of
plasma virus loads and CD4?T cell loss is reminiscent of the
previously reported infection of pig-tailed macaques with
SIVl’hoest and SIVsun (4). In that study, 8 of 12 infected
animals developed immunodeficiency over a 5-year period
while maintaining set-point viremia between 102and 103RNA
We do not presently understand why naïve CD4?T lympho-
cytes are lost in SHIVAD8NPs. Based on coreceptor expres-
sion, this T cell subset expresses CXCR4, not CCR5, on its
surface and should therefore be refractory to infection by R5-
tropic SHIVs and virus-induced cell killing. An assessment of
the coreceptor utilization status of late-stage viruses recovered
from SHIVAD8NPs, in fact, revealed that a coreceptor switch
had not occurred in these animals (see Fig. S2 in the supple-
mental material). Although a dissociation between viral-RNA
levels and memory/naïve CD4?T cell loss was observed, the
NPs did experience increased memory CD4?T lymphocyte
turnover (see Fig. S1 in the supplemental material), even in
animals with very low plasma virus loads. Activation-induced
proliferation and killing of memory CD4?T cells during the
lengthy chronic SHIVAD8infection might therefore be respon-
sible for driving the differentiation of naïve CD4?lymphocytes
into memory cells and impose an unsustainable drain on this
CD4?T cell subset. It is also possible that SHIVAD8infection
of rhesus macaques negatively affects naïve CD4?T lympho-
cyte homeostasis in the thymus, thereby impeding the differ-
entiation or emigration of this T cell subset. It has also recently
been reported that the loss of naive CD4?T cells during
SIVsmE543 infections was associated with the presence of
autoreactive antibodies to CD4?T lymphocytes, platelets,
double-stranded DNA, and phospholipid (27). Increased num-
bers of circulating IgG-coated CD4?T cells were observed in
that study, and the levels of autoreactive antibodies were cor-
related with the extent of naïve CD4?T cell depletion.
Approximately 20% of rhesus monkeys infected with
SIVmac/SIVsm lineage viruses become RPs, experiencing per-
sistently high virus set points, rapid and complete losses of
memory CD4?T cells, undetectable or transient antiviral an-
tibody responses, and early onset (3 to 6 months p.i.) of symp-
tomatic disease (6). Despite losing virtually all of their memory
CD4?T lymphocytes, SIV RPs, at the time of death, usually
maintain preinoculation levels of naïve CD4?T cells (35). This
was not the case for SHIVAD8RPs. Although all three expe-
rienced early and massive depletions of memory CD4?T cells,
two of the infected macaques had lost virtually all of their naïve
CD4?T cells at the time of euthanasia. In one of these animals
(DB99), the virus recovered at the time of euthanasia, as well
as a virus pseudotyped with an Env possessing the RIG inser-
tion in the V3 loop, had acquired the capacity to infect cells
expressing CXCR4 (Fig. 9a and c). Interestingly, coreceptor
switching has been previously reported to occur during RP
infections of macaques inoculated with a different R5-tropic
SHIV, SHIVSF162P3(18, 19, 47). In one of the SHIVSF162P3
coreceptor-switching events, the insertion of two positively
charged amino acids (HR) immediately upstream of the V3
loop GPGR crown (Fig. 9b) was shown to confer X4 tropism
(18). In the case of SHIVAD8-DB99, a 3-aa (RIG) insertion, also
located in the N-terminal V3 stem and which increased the net
charge of the V3 loop from ?3 to ?5, was responsible for the
acquisition of CXCR4 usage. The insertion of HIG at the same
location of the SHIVAD8-A4E008V3 region did not affect the
net charge and did not confer tropism for CXCR4-expressing
Independent and unrecognized cross-species transmissions
and spread of SIVsm at different U.S. primate facilities during
the 1970s contributed to the emergence of SIVmac and
SIVsmE660 lineages with distinctive replicative and patho-
genic phenotypes. The serial passaging of SHIVAD8in rhesus
monkeys described here also resulted in an AIDS-inducing
primate lentivirus with its own characteristic properties. First,
in contrast to commonly used pathogenic SIVs, SHIVAD8#2
and its immediate derivatives generated sustained but, as pre-
viously noted, highly variable set-point virus loads in NPs.
Similarly variable viral loads were also observed in eight rhesus
monkeys inoculated with four independent SHIVAD8stocks
prepared from macaques CK15, CE8J, CL98, and CJ58 at the
time of their euthanasia (data not shown). Profound depletions
of both memory and naïve CD4?T cells, which accompany
relatively low virus set points (geometric mean level, 1.7 ? 103
RNA copies/ml) in NPs, is a second property that distinguishes
the R5-tropic SHIVAD8from pathogenic SIVs. Finally, unlike
SIVs, SHIVAD8RPs experience an initial loss of memory
CD4?T lymphocytes and a later rapid deletion of naïve CD4?
T cells prior to death, which in one animal occurred following
a CCR5-to-CXCR4 coreceptor switch. Based on the results
shown in Fig. 4 and Table 2, we plan to use and distribute
TABLE 2. Clinical and pathological findings in rhesus monkeys
infected with SHIVAD8#2and its immediate derivatives
Animal Clinical data/pathological findings
CJ8B ...................Euthanized (wk 199); uncontrolled diarrhea; wt loss
CK15...................Euthanized (wk 112); P. carinii pneumonia
CJ58....................Total CD4?T cells, 154/mm3(wk 111)
CE8J ...................Euthanized (wk 117); uncontrolled diarrhea, C. coli
CJ35....................Total CD4?T cells, 270/mm3(wk 129)
CJ3V...................Euthanized (wk 135); uncontrolled diarrhea;
CK5G..................Total CD4?T cells: 101/mm3(wk 101)
DB99...................Euthanized (wk 23); rapid progressor
DA1Z..................Total CD4?T cells, 545/mm3(wk 65)
A4E008...............Euthanized (wk 20); rapid progressor
DA4W ................Total CD4?T cells, 92/mm3(wk 64)
CL5A..................Euthanized (wk 19); rapid progressor
CL98 ...................Euthanized (wk 100); disseminated M. avium
VOL. 84, 2010 GENERATION OF SHIVAD8BY SERIAL PASSAGING IN MACAQUES 4779
SHIVAD8#2LN, SHIVAD8#2PBMC, or the SHIVs recovered
from NPs at the time of euthanasia (SHIVAD8–CL98,
SHIVAD8–CK15, or SHIVAD8–CE8J) as challenge viruses in vac-
cine experiments. Animals inoculated with cell-free prepara-
tions of the last group of viruses have experienced variable but
sustained plasma viremia associated with a gradual but signif-
icant CD4?T cell loss during 30 weeks of infection. Some of
these macaques have developed a rapid-progressor clinical
We are indebted to Keith Reimann and the NIH Nonhuman Pri-
mate Reagent Resource for providing cM-T807; to the NIH AIDS
Research and Reference Reagent Program for providing AMD3100;
and to Julie Strizki, Schering-Plough, for providing AD101. We thank
John Mascola for TZM-bl cells and instructions for performing virus
neutralization assays, Robin Kruthers and Ranjini Iyengar for deter-
mining viral RNA levels, and Vanessa Hirsch for critical comments
during the preparation of this paper. We appreciate the contributions
of Boris Skopits in diligently assisting in the care and maintenance of
This work was supported by the Intramural Research Program of the
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health.
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