Download Pig-tailed Macaques Infected with Human Immunodeficiency Virus

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Pig-tailed Macaques Infected with Human Immunodeficiency Virus (HIV) Type
2G B 1 2 2 or Simian/HIV8 9 . 6 p Express Virus in Semen during Primary Infection:
New Model for Genital Tract Shedding and Transmission
Jennifer K. Pullium,1,4 Debra R. Adams,1
Eddie Jackson,2 Caryn N. Kim,1 Dawn K. Smith,3
Robert Janssen,3 Kenneth Gould,5 Thomas M. Folks,1
Sal Butera,1 and Ron A. Otten1
1
HIV/AIDS and Retrovirology Branch, Division of AIDS, STD,
and TB Laboratory Research, and 2Scientific Resources Program,
National Center for Infectious Diseases, and 3Division of HIV/AIDS
Prevention—Surveillance and Epidemiology, National Center for HIV,
STD, and TB Prevention, Centers for Disease Control and Prevention,
and 4Division of Animal Resources and Department of Pathology
and Laboratory Medicine and 5Yerkes Regional Primate Research
Center, Emory University, Atlanta, Georgia
Characterizing human immunodeficiency virus (HIV) expression in semen during primary
infection remains essential to understanding the risk of sexual transmission. This investigation
represents the first systematic evaluation of male genital tract shedding to use a nonhuman
primate model, including the impact of exposure route and viral virulence. Male macaques
were inoculated with either a chronic disease–causing virus (HIV-2GB122; n p 4 intravenous;
n p 4 intrarectal) or an acutely pathogenic simian/HIV strain (SHIV89.6P; n p 2 intravenous).
All macaques were systemically infected, and seminal plasma virion-associated RNA (vRNA)
levels were ∼10-fold lower than those in blood. In HIV-2GB122 infection, seminal virus was
delayed by 1–2 weeks compared with that in blood. Intrarectal inoculation resulted in a shorter
duration of seminal vRNA expression and intermittent seminal cell provirus. No delays, higher
peaks (∼50-fold), or longer durations in seminal virus expression were noted for SHIV89.6P
infection. This novel model definitively establishes that virus dissemination results in early
peak seminal levels and provides a basis for evaluating interventions targeting male genital
tract expression.
Most new human immunodeficiency virus (HIV) infections are
acquired via sexual transmission involving seminal fluids [1, 2].
Analysis of human specimens has demonstrated the presence of
both cell-associated and cell-free virus in semen [3, 4]. Sophisticated fractionation experiments have provided solid evidence
Received 18 October 2000; revised 18 December 2000; electronically published 8 March 2001.
Presented in part: 18th Annual Symposium on Nonhuman Primate Models for AIDS, Madison, Wisconsin, October 2000 (abstract 105).
All animal procedures were approved by the Centers for Disease Control
and Prevention Animal Care and Use Committee. The animals were housed
according to the Guide for the Care and Use of Laboratory Animals. Use of
trade names is for identification only and does not constitute endorsement
by the Public Health Service or by the US Department of Health and Human
Services.
Financial support (in part): Appointment to the Research Participation
Program for the Centers for Disease Control and Prevention, National Center for Infectious Diseases, Division of AIDS, STD, and TB Laboratory
Research (DASTLR), administered by the Oak Ridge Institute for Science
and Education through an agreement between the Department of Energy
and DASTLR.
Reprints or correspondence: Dr. Ron A. Otten, HIV/AIDS and Retrovirology Branch, MailStop G-19, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Disease, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333 (rxo1
@cdc.gov).
The Journal of Infectious Diseases 2001; 183:1023–30
This article is in the public domain, and no copyright is claimed.
0022-1899/2001/18307-0005
that T lymphocytes and macrophages in semen can harbor provirus. There was, however, no convincing virus association with
spermatozoa [5]. At present, the relative contributions of cellassociated versus cell-free virus in semen for successful transmission remain undefined.
In general, HIV-1 levels in human semen have been found
to be lower and much more intermittent than are those in
peripheral blood [3, 6, 7]. Identification of differing viral populations expressed in semen versus blood has indicated that the
male genital tract may serve as a distinct compartment for HIV1 replication [6, 8–13]. Controversy exists amid disparate findings regarding the impact of blood virus levels, peripheral CD41
cell counts, and disease stage on seminal virus shedding [7, 11,
14]. The existing human data collectively indicate that HIV-1
shedding in the male genital tract can occur at any time during
the course of infection [reviewed in 2].
Recent human studies [15–17] have assessed the impact of
antiretroviral therapy on seminal HIV-1 burden during chronic
infection, in an effort to alter overall transmissibility. Evaluating virus replication during early primary infection, especially
in the genital tract compartment, however, remains essential to
understanding sexual transmission potentials that may be perpetuating the pandemic. Very limited human data suggest that
virus expression occurs in semen during primary infection
[18–21], which may correlate with the greatest period of trans-
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Pullium et al.
mission risk [21]. Comprehensive characterization of the earliest
virologic events after exposure requires appropriate animal
models, because problems with routine recognition of acute
retroviral syndromes preclude such studies in humans.
The only previous attempt to investigate male genital tract
shedding in nonhuman primates [22] was hampered by insensitive culturing methods, as were early human investigations.
The main hurdle in using other macaque systems (rhesus) to
evaluate seminal shedding has historically involved specimen
coagulation problems immediately following collection, as
highlighted elsewhere [17]. In the present study, we have overcome these logistical difficulties, and we report the first systematic evaluation of male genital tract virus shedding in a nonhuman primate model for acute-phase HIV infection.
Important aspects, not feasible to investigate during human
HIV infection, were explored in this study, such as the timing,
level, and duration of seminal virus expression early after exposure; the impact of exposure route on virus-shedding characteristics; and the effect of virus virulence on expression from
the male genital tract.
Methods
Animals and housing. Ten male pig-tailed macaques (Macaca
nemestrina), 4–9 years of age and weighing 6.0–12.0 kg (Charles
Rivers Laboratories), were used in this study. These animals were
housed individually under biosafety level 3 containment conditions.
The cohort tested negative for malaria, tuberculosis, simian immunodeficiency virus (SIV), simian retrovirus type D, HIV-1, and
HIV-2 at baseline.
Virus stocks and inoculations. The utility of HIV-2GB122, a primary isolate derived from an AIDS patient residing in Bissau,
Guinea Bissau (West Africa), for productively infecting nonhuman
primates has been documented elsewhere [23–25]. A cell-free virus
inoculum was generated by limited propagation of the original
human isolate onto a mixture of phytohemagglutinin-p (PHAp)–activated peripheral blood mononuclear cells (PBMC) from 4
source macaques. The titer of the cell-free virus challenge stock
was determined by standard coculturing techniques [26, 27] and
consisted of 100 TCID per milliliter. Intravenous (iv) exposures
consisted of 1 mL of virus stock injected into the saphenous vein.
Intrarectal (ir) virus exposures involved atraumatic inoculation of
cell-free virus (5 mL per exposure) into the rectum via a sterile
gastric feeding tube. The ir inoculations were separated by 30 min,
during which the anesthetized macaques remained recumbent to
promote localized absorption. Specific strain characteristics of simian/HIV, SHIV89.6p, have been detailed elsewhere [28, 29]. The virus
stock used for this investigation was obtained from a recent virus
expansion on rhesus macaque PBMC (F. Villinger, personal communication) that was passaged onto PHA-p–blasted pig-tailed macaque PBMC with a starting MOI of 0.01. SHIV89.6p (derived from
pig-tailed macaque PBMC) was propagated for 28 days and yielded
a final stock with sufficient titer (100 TCID/mL) to be used for in
vivo inoculations; iv exposures consisted of 1 mL of virus stock
injected into the saphenous vein.
Specimen collection and processing. Longitudinal blood spec-
JID 2001;183 (1 April)
imens were collected and were processed to isolate PBMC and
plasma, cryopreserve PBMC, and derive cellular lysates, as described elsewhere [24]. Whole-blood and serum specimens were also
routinely collected for blood chemistry analysis and complete blood
cell counts. Semen specimens were obtained by mild rectal probe
electrostimulation (RPE), which produces rhythmic contractions of
pelvic muscles, resulting in emission of semen, usually yielding
lower sperm concentrations compared with a true ejaculate [30].
A rectal probe (2.0–2.5 cm in diameter) with 2 flush-mounted electrodes was inserted into the rectum of anesthetized macaques. Mild
electrostimulation required for emission of semen ranged between
4 and 15 V. Current was delivered intermittently, in a sequence of
∼3 s of stimulus and 3 s of rest for a maximum of 15 min. The
current was progressively increased until an emission resulted. After
collection, semen was incubated at 377C for 30 min, to allow the
semen to liquify as much as possible. Semen was then diluted 1:1
with RPMI containing 1000 mg/mL streptomycin and 1000 U/mL
penicillin and was centrifuged at 600 g for 15 min [31]. The seminal
plasma supernatant was recovered for cell-free virus detection and
quantitation. Seminal cell pellets were lysed and were evaluated for
the presence of cell-associated provirus. Evidence derived from human specimens indicated that cell-associated virus localizes exclusively to mononuclear cells present in semen [5]. Thus, no attempts
were made to minimize the amount of spermatozoa in seminal cell
specimens harvested for analysis.
Cell-associated provirus detection. Nested DNA–polymerase
chain reaction (PCR) techniques were used to monitor the establishment of HIV-2GB122 or SHIV89.6p provirus in PBMC and seminal
mononuclear cells. The sensitivity (>10 copies/106 cells) and high
specificity of HIV-2 proviral DNA-PCR assays with a 105 PBMC
input have been described elsewhere [24]. Seminal cell extracts were
derived for nucleic acid purification by using a standard Nuclisens
protocol (Organon Teknika). Because routine seminal cell counts
were not feasible for each collection, owing to the potential for
substantial sample loss, primary amplification of b-actin was used
to ensure that cellular input amounts were standardized before
subsequent virus detection. Duplicate input samples, corresponding
to ∼20% of the total specimen obtained, were used for virus-specific
nested PCR analysis, with a maximum input of 4 3 10 5 mononuclear cells. All nested PCR reactions were performed as described
elsewhere for PBMC specimens [24]. The presence of amplifiable
DNA in both PBMC and seminal cells was confirmed by b-actin–specific PCR by use of the primers BAF1 (50-GTGCTATCCCTGTACGCCTCT-30) and BAR1 (50-GGCCGTGGTGGTGAAGCTGTA-30). The primers were derived from the human b-actin
mRNA sequence (GenBank accession number X00351). Cycler
conditions were identical to those used for HIV-2 protease PCR
[24]. SHIV89.6p provirus detection was accomplished in a similar
manner, except that HIV-1 env-gp41 was the targeted region for
nested DNA-PCR amplification using highly conserved and sensitive primer sets [32].
Cell-free virion-associated RNA (vRNA) quantitation. A reverse transcriptase–mediated PCR (RT-PCR) prototype assay system, similar in design to that originally detailed by Kwok and
colleagues [33], was used for quantitative measurement of vRNA
in blood and seminal plasma. Specific details regarding genomic
positions of the utilized long terminal repeat (LTR) primers and
assay conditions have recently been reported for monitoring HIV-
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HIV Shedding in Macaque Semen
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Table 1. Cellular provirus detection (peripheral blood/semen) for pig-tailed macaques
exposed to human immunodeficiency virus type 2 (HIV-2GB122) or simian/HIV (SHIV89.6p).
HIV-2GB122 (iv route)
SHIV89.6p
(iv route)
HIV-2GB122 (ir route)
Week
2GA
3ET
72–79
72–55
2GP
3AW
72–243
52–59
3DK
2FW
1
2
3
4
6
8
12
1/2
1/1
1/1
1/2
1/1
1/1
1/2
1/2
1/2
1/2
1/1
1/2
1/2
1/2
1/2
1/1
1/1
1/1
1/1
1/2
1/2
1/2
1/1
1/1
1/1
1/2
1/1
1/2
1/2
1/1
1/1
1/2
1/2
1/2
1/2
1/2
1/1
1/2
1/2
1/2
1/2
1/2
1/2
1/2
1/1
1/2
1/2
1/2
1/2
1/2
1/2
1/1
1/1
1/2
1/2
1/2
1/1
2/NT
2/2
1/1
2/1
1/1
1/1
1/1
1/1
1/2
1/NT
1/2
1/1
1/1
NOTE. A plus sign (1) represents successful nested DNA–polymerase chain reaction (PCR)
amplification and detection of conserved pol-protease gene sequences for HIV-2GB122–exposed macaques
or conserved env-gp41 sequences in the case of SHIV89.6p-exposed macaques. A minus sign (2) indicates
that no virus-specific signal was detected, whereas the external cellular (b-actin) DNA-PCR control
reaction was positive. ir, Intrarectal; iv, intravenous; NT, not tested.
2 vRNA in the context of dual infections with HIV-1 [34]. Very
minor base-pair mismatching among this LTR primer set for SIV
strains has enabled this vRNA quantitative assay to monitor these
viruses effectively, as well as SHIV89.6p, as documented recently
elsewhere [35]. Deviation from assay methodology reported earlier
related to the desire to standardize extraction procedures for blood
and genital tract specimens, to avoid any possible PCR inhibition
[3]. Toward this end, vRNA from 150 mL of blood or seminal
plasma, along with a spiked RNA quantitation standard (25
copies), was extracted by using Nuclisens methodologies (Organon
Teknika). Single-tube RT-PCRs (rTth DNA polymerase, Roche
Molecular Systems) were performed, using a 50 mL specimen equivalent per reaction. Assay sensitivity was routinely 100 copies/mL,
and characterized stocks of SIVmac251 and HIV-2NIH-Z, with associated virus particle counts, were included as assay standards.
Measurement of virus-specific serologic responses and lymphocyte
phenotyping analysis. Sensitive detection of virus-specific serologic
responses was achieved by using a synthetic peptide EIA (Genetic
Systems HIV-1/HIV-2). Reactive samples were confirmed by HIV2–specific Western analysis (Cambridge Biotech or Genelabs Diagnostics). Virus-specific serologic titers were determined by performing
2-fold dilutions and analyzing reactivity by EIA. Standard cell phenotyping reagents (BD-Pharminogen), demonstrating strong cross-reactivity with macaque cell markers, were used to monitor pig-tailed
macaque CD41 and CD81 lymphocyte populations during the course
of infection, as performed elsewhere [23, 24].
Results
To study the dynamics of virus load in both blood and semen
during acute HIV infection, 10 male macaques were stratified
into groups receiving a single iv dose corresponding to 100
TCID of HIV-2GB122 (n p 4); 2 ir doses (1000 TCID total) of
HIV-GB122 (n p 4); or a single iv dose (100 TCID) of SHIV89.6p
(n p 2). Multiple inoculation routes were utilized to determine
the impact of exposure route on genital tract shedding. Acutely
pathogenic SHIV89.6p was compared with the more chronic
HIVGB122, to determine any role of viral virulence on genital
tract shedding. All inoculations outlined in this investigation
resulted in systemic infection.
Virologic characterization after HIV-2GB122 infection. Macaques infected with HIV-2GB122 were DNA-PCR positive for
provirus in PBMC by week 1 after inoculation and at all time
points through 12 weeks (table 1). Furthermore, all 8 macaques
had >1 seminal cell provirus–positive result during the course
of study; however, detection of virus signals in genital tract
secretions was much more intermittent in comparison with that
in peripheral blood, especially in the ir group (table 1). The
last positive signal detected in the ir group was at week 4 after
inoculation, whereas multiple seminal cell provirus–positive signals were observed for the majority of those in the iv group,
with week 8 representing the latest detection time point. During
12 weeks of monitoring, 13 (46%) of 28 seminal cell specimens
collected from the iv group were provirus positive, versus 5
(18%) of 28 specimens collected from the ir group (P p
.0437, Fisher’s exact test).
The onset of detectable blood plasma virus loads for all 8 HIV2GB122–infected macaques (figure 1A) occurred at week 1. Significant differences (P p .0262 , unpaired t test) were observed between the iv and ir groups at this very early time point (mean
values of 104.04 vs. 103.43 copies/mL, respectively), which may reflect slower virus dissemination due to ir inoculation. Peak vRNA
levels were reached by weeks 1–2 after inoculation in the iv group
(mean, 104.64 copies/mL; range, 104.05–104.97 copies/mL) and by
weeks 2–3 after inoculation in the ir group (mean, 105.02 copies/
mL; range, 104.73–105.21 copies/mL). No significant differences in
absolute levels achieved were noted (P p .0842 , unpaired t test),
and vRNA expression for the majority of macaques in both the
iv and ir groups remained above the quantitation limit at week
6 after inoculation (figure 1A).
Seminal plasma vRNA levels (figure 1B), compared with levels
in blood plasma, were delayed by >1 week in all but 1 macaque
(iv group); were quantitatively lower (mean peak, 103.49 copies/
mL; range, 102.48–103.63 copies/mL for the iv group, and mean
peak, 102.96 copies/mL; range, 102.17–103.33 copies/mL for the ir
group); and were much more transient in nature with regard to
detectability, especially in the ir group. It is interesting that the
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Pullium et al.
JID 2001;183 (1 April)
Figure 1. Longitudinal virion-associated RNA (vRNA) levels in blood plasma (A) and seminal plasma (B) through 12 weeks after intravenous
(n p 4; solid symbols) or intrarectal (n p 4; open symbols and dashed lines) exposure to human immunodeficiency virus type 2 (HIVGB122).
Longitudinal vRNA levels in blood plasma (C) and seminal plasma (D) through 12 weeks after intravenous (n p 2) exposure to simian/HIV
(SHIV89.6p). Results are reported as log10 copies/mL. The sensitivity limits are 100 copies/mL (as indicated on each graph), with a 50 mL sample
equivalent input into the assay system.
delay in onset of detectable seminal plasma vRNA did not translate into a significant delay in the timing to achieve peak levels,
compared with that observed for blood plasma (P p .0824, unpaired t test). The trend toward higher peak levels in the iv versus
the ir group (0.53 log10 difference among mean values) was not
significant (P p .0821, unpaired t test). By week 6 after inoculation, seminal plasma vRNA levels for these macaques fell below
the limit of quantitation, with no rebound through week 12.
Virologic characterization after SHIV89.6p infection. By comparison, SHIV89.6p infection also resulted in early (week 1) detectable provirus in PBMC (table 1), although continuous detection was observed for only 1 macaque (2FW). It is noteworthy
that provirus-positive seminal cells were detected earlier, at week
1 after inoculation, in both SHIV89.6p-infected macaques, compared with those in the HIV-2GB122 group (table 1). The number
of positive genital tract specimens (9 of 12) was also greater over
the 12-week study course, although it did not reach significance
(P p .1654, Fisher’s exact test).
Initial detection and peak vRNA levels (range, 106.2–106.5 copies/mL) in blood plasma for both SHIV89.6p-infected macaques
occurred at week 1 after inoculation and remained above the
detection limit at week 12 (figure 1C). The mean peak vRNA
value observed for these macaques (106.37 copies/mL) was 1.73
log10 higher than that for the HIV-2GB122–infected iv group (104.64
copies/mL). Seminal plasma vRNA levels (figure 1D) in these
macaques also had higher peak levels (range, 104.5–105.5 copies/
mL) at weeks 1–2 after inoculation but remained lower than those
noted for corresponding blood plasma.
Seminal plasma expression declined by weeks 3–4 after inoculation, reaching a magnitude similar to that observed for
HIV-2GB122–infected macaques. At weeks 6 and 8 after inoculation, vRNA levels for 1 macaque (2FW) fell below quantitation limits; however, both SHIV89.6p-infected macaques were
found to shed genital tract virus again at week 12 (figure 1D).
A single SHIV89.6p-infected macaque (3DK) became ill at
week 15 after inoculation, with evidence of a ruptured spleen,
and was promptly euthanized. Histopathologic analysis revealed splenic lymphoid hypoplasia similar to that found in
previous studies [36]. The testes showed atrophy of the seminiferous tubules and hypospermatogenesis, which is consistent
with the findings of postmortem studies in humans [37]. This
is the first report of such findings in SHIV89.6p-infected macaques. All major organs, including testes, from 2 age-matched
HIV-2GB122–infected macaques that were euthanized and un-
JID 2001;183 (1 April)
HIV Shedding in Macaque Semen
derwent necropsy at week 12 after inoculation were histologically normal. Further study involving additional macaques and
a greater length of follow-up would be required to document
other evidence of localized or systemic pathogenic consequences
of infection.
Virus-specific antibody (Ab) responses and lymphocyte fluctuations during primary infection. As anticipated, all 8 HIV2GB122–infected macaques developed EIA-reactive, Western blot
(WB)–confirmed Ab responses by week 3 or 4 after inoculation
(titer range, 10–25,600). The strength, timing, and duration of
the systemic Ab responses observed were consistent with those
we have observed previously in this macaque model for iv or
vaginal route exposures [23, 25]. Virus-specific, WB-confirmed
Ab responses were also observed in both SHIV89.6p-infected macaques, starting at 6 or 8 weeks after inoculation. These responses, however, were much lower (titer range, 10–200) than
were those of the HIV-2GB122–infected macaques.
No overall differences were observed in the CD41 (figure 2A)
or CD81 (figure 2B) cell populations among the HIV2GB122–infected macaque groups. Notable CD41 cell losses, ac-
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companied by increased CD81 cells, were observed for most macaques early within primary infection (2–4 weeks after
inoculation), with distinct rebound patterns characteristic of
HIV-2GB122 replication in pig-tailed macaques [23]. SHIV89.6p infection resulted in a rapid total CD41 cell depletion by week 2
after inoculation that was maintained for the remainder of the
study course (figure 2C). This severe depletion is a hallmark of
infection for acutely pathogenic SHIV strains. Corresponding
increases in CD81 cells (figure 2D) were also observed.
Discussion
We sought to model male genital tract shedding in nonhuman
primates (pig-tailed macaques) during primary retroviral infection. Novel aspects reported here include (1) longitudinal collection and virologic evaluation of macaque semen specimens
after exposure; (2) direct assessment of cell-free and cell-associated virus within macaque seminal fluid versus peripheral
blood; (3) the impact of distinct exposure routes (iv and ir) on
seminal virus shedding, using the chronic HIV-2GB122 strain; and
Figure 2. CD41 and CD81 cell population profiles for human immunodeficiency virus type 2 (HIV-2GB122)–inoculated macaques (A and B)
and for simian/HIV (SHIV89.6p)–inoculated macaques (C and D). Independent cellular baseline values for each animal were calculated from blood
specimens collected before virus inoculation and were set at 100% for week 0. Results are presented as percentages of established baseline values,
to detect fluctuations over the course of the 12 weeks after virus exposure.
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Pullium et al.
(4) seminal and peripheral virus expression after infection with
a chronic versus a highly pathogenic virus. Furthermore, this
report documents the first attempts at infecting pig-tailed macaques with the highly virulent virus, SHIV89.6p.
A majority of earlier human genital tract investigations were
limited to chronically infected subjects [6, 38], whereas recent
efforts have been more successful in identifying limited numbers
of individuals early after HIV-1 acquisition [18, 19, 39]. Despite
the successes of these human studies, an animal modeling system remains crucial to understanding the very earliest events
occurring after infection. In the only previous report, which
was limited because of insensitive culturing methods, virusinfected mononuclear cells were detected in just 2 of 10 semen
specimens collected from a single chronically SIV-infected rhesus macaque [22]. In contrast, we used molecular methods for
retroviral detection, which have been shown to have enhanced
sensitivity in human studies [3, 38, 40, 41].
Elegant human studies have shown that virus levels observed
in human semen were lower than those in peripheral blood and
were more intermittent, probably because of local environmental (e.g., possibly mucosal immune-based) pressures and compartmentalization or selected growth of virus quasi species [3,
6, 7, 42]. These trends were also evident in this animal model
study. In particular, for HIV-2GB122 infection, the majority (63%)
of seminal cell provirus-positive specimens occurred with detectable seminal plasma vRNA, whereas only 16% of seminal
cell provirus-negative specimens were associated with virus
shedding in seminal plasma. Specifically, the ability to detect
seminal cell virus correlated with the level of seminal plasma
vRNA (P ! .0001; r p .5037, Spearman’s rank correlation). A
larger sample size would be required to determine whether a
similar correlation exists for SHIV89.6p infection. Intermittent
detectable genital tract shedding and the presence of detectable
seminal cell virus in the absence of detectable seminal plasma
vRNA signals were also noted during both HIV-2GB122 and
SHIV89.6p infection.
Measurement of vRNA levels has been accepted as a surrogate marker of active virus production, even though it is not
an actual infectious virus measurement, and it has been shown
to correlate with an ability to isolate virus in peripheral blood
and semen compartments [3, 31]. By applying this technology,
we observed peak levels of seminal plasma vRNA in all macaques within the first few weeks of primary infection, similar
to those observed in blood plasma, although at consistently
lower levels. The existence and transient nature of an early peak
in seminal virus shedding were supportive of a hyperinfectious
state during primary infection, a finding that has never been
shown conclusively in humans. It is interesting to note that the
observed peak levels were short-lived, lasting only a few weeks,
and generally corresponded with changes in peripheral blood
levels. These findings, extrapolated to human infections, would
suggest that a brief period of increased sexual infectiousness
might occur during primary infection. In addition, these data,
JID 2001;183 (1 April)
coupled with seminal cell provirus detection, confirm limited
existing human data [18–20, 43] showing that the male genital
tract serves as an early site of dissemination and productive
infection after retroviral exposure.
Furthermore, our investigation provides the first indication of
possible differences in the longevity of seminal virus shedding as
a function of exposure route. Overall, HIV-2GB122–infected macaques that were ir exposed had abbreviated shedding, as indicated by both seminal plasma vRNA and seminal cell provirus
signals, compared with those that were iv infected, even in light
of the higher ir inoculum. This disparity of viral shedding in the
seminal compartment was not explained by differences in the
peripheral blood virus load between these groups. Slower dissemination of virus to the genital tract than to blood was also
observed for the majority (7 of 8) of HIV-2GB122–infected macaques. These observations serve as a foundation to address the
highly relevant issue of early therapeutic intervention to counter
initial virus dissemination to the genital tract.
In contrast, SHIV89.6p infection of pig-tailed macaques resulted
in high levels of virus replication in both peripheral and genital
tract compartments, with no delays in the appearance of cellfree or cell-associated virus in seminal fluid. Rapid CD41 cell
depletion and extremely high blood plasma vRNA levels have
been the hallmarks of this acutely pathogenic virus in the rhesus
system [29, 44], characteristics that have been extended to pigtailed macaques by our report. Comparing pig-tailed macaque
infection with SHIV89.6p versus HIV-2GB122 has provided direct
evidence that increased viral virulence may be associated with
increased shedding in semen for a longer period during primary
infection. Also, the lack of seminal plasma virus at weeks 6 and
8 after inoculation that was noted for one of the 2 SHIV89.6pinfected macaques highlights the transient nature of genital tract
virus expression, even in light of the pronounced virulence of the
infecting strain used in this study. It is also interesting that the
increased viral virulence observed in both macaques resulted not
only in a more prolonged shedding in semen, but also in a longer
seronegative window. By extrapolation, such observations are of
great concern in humans, in whom infection with a highly virulent
HIV strain may be missed by standard serologic detection methods, most likely increasing transmission potentials during this
period of high infectiousness.
A potential weakness of this investigation, as with most other
nonhuman primate modeling studies, was the ability to detect
only marked differences among measured parameters because
of the small number of animals per experimental group. Also,
collection of semen by RPE may not be entirely representative
of a true ejaculate, although detection and characterization of
both cell-free and cell-associated virus shedding during primary
infection were successful. Furthermore, extrapolation of results
from any animal model to the human condition remains a constant challenge. The virus-host interactions that result from
HIV-2GB122 or SHIV89.6p infections seemingly represent minority
JID 2001;183 (1 April)
HIV Shedding in Macaque Semen
extremes in virus virulence and resulting pathogenesis, compared with most human infections.
Assessing early genital tract events remains vital to understanding factors associated with sexual transmission of HIV. The
ability to address these critical issues in the pig-tailed macaque
system, with males or females [25], by using chronic or acutely
pathogenic viruses enables important transmission questions to
be more easily investigated. Transmission-related parameters,
such as virus threshold levels and the relative contributions of
seminal plasma versus seminal cell virus required for successful
in vivo transmission, can now be explored by using semen specimens collected from infected pig-tailed macaques.
In summary, this report provides vital insight into early
events associated with HIV expression in the male genital tract
and establishes a reproducible nonhuman primate system in
which to study factors that govern transmission risk among
infected individuals.
Acknowledgments
We are grateful to Shirley Kwok and Cindy Christopherson (Roche
Molecular Systems, Alameda, CA) for support with HIV-2 vRNA
quantitation issues and reagents; to Dr. Gale Galland and Archer Miller
(Scientific Resources Program, CDC), for serving as the attending veterinarian for this study protocol (G.G.) and monitoring and maintenance of our macaque cohort (A.M.), respectively; to Drs. Harold Jaffe
(Division of AIDS, STD, and TB Laboratory Research, CDC) and
Lynn Paxton (Division of HIV/AIDS Prevention—Surveillance and
Epidemiology, National Center for HIV, STD, and TB Prevention,
CDC), for valuable input and discussions of pertinent issues related to
sexual transmission of HIV; to Dr. Dirk Dillehay (Division of Animal
Resources and Department of Pathology and Laboratory Medicine,
Emory University, Atlanta), for pathology services; and to Bill Switzer
(HIV/AIDS and Retrovirology Branch, Division of AIDS, STD, and
TB Laboratory Research, CDC), for assistance related to amplification
of nonhuman primate b-actin sequences.
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