JOURNAL OF VIROLOGY, Aug. 2009, p. 7517–7523 Vol. 83, No. 150022-538X/09/$08.00�0 doi:10.1128/JVI.00552-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Is the Gut the Major Source of Virus in Early SimianImmunodeficiency Virus Infection?�

Matthew D. H. Lay,1† Janka Petravic,1 Shari N. Gordon,2 Jessica Engram,2Guido Silvestri,2,3 and Miles P. Davenport1*

Complex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Kensington NSW 2052,Australia1; Departments of Pathology and Laboratory Medicine and Microbiology, University of Pennsylvania

School of Medicine, Philadelphia, Pennsylvania2; and Yerkes National Primate Research Center,Emory University, Atlanta, Georgia3

Received 18 March 2009/Accepted 11 May 2009

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV)infection are characterized by rapid and profound depletion of CD4� T cells from the guts of infectedindividuals. The large number of CD4� T cells in the gut (a large fraction of which are activated and expressthe HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of thisCD4� T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosamay be the major source of virus driving the peak viral load. Here, we used data on CD4� T-cell proportionsin the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sootymangabeys (which do not progress) to show that in both species, the depletion of CD4� T cells from thismucosal site and its maximum loss rate are often observed several days before the peak in viral load, with fewCD4� T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate ofCD4� T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus.Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4� T cellsin the intestinal mucosa are a highly susceptible population for infection but not a major source of plasmavirus in acute SIV infection.

The acute phase of human immunodeficiency virus (HIV)infection is characterized by moderate CD4� T-cell depletionin blood, followed by a transient partial restoration of CD4�

T-cell numbers and eventually by a slow long-term CD4� T-cell decline in the chronic phase that lasts for several years.Studies of CD4� T-cell depletion in mucosal sites, often con-ducted with simian immunodeficiency virus (SIV)-infected ma-caques, have demonstrated that mucosal CD4� T-cell deple-tion is more rapid and profound (3, 10, 13, 19, 21). The severedepletion of cells in the gut in early infection is thought to bedriven in part by the phenotype of the cells present, which arepredominantly CCR5� and in general more activated thantheir circulating counterparts. As such, these mucosal CD4� Tcells are highly susceptible to productive infection with thedominant CCR5-tropic strains of HIV and SIV present in earlyinfection (20). The rapid depletion of CD4� T cells at mucosalsites is accompanied by relatively high numbers of infectedcells (10, 13) and is temporally associated with the peak viralload in plasma, suggesting that the infection of mucosal CD4�

T cells may be responsible for the majority of virus replicationoccurring during acute infection (10, 15, 21, 22).

The size of the CD4� T-cell pool in the gut is a matter of

some controversy, with estimates ranging from �5 to 50% ofthe total body pool of these cells (reviewed in reference 5).Regardless of the precise numbers, the gut (and particularlythe mucosal lamina propria) contains a significant proportionof the body CD4� CCR5� memory T cells, which are depletedvery early in infection. However, whether CD4� T cells in thegut are merely a target of early infection or whether they are amajor driver of early viral growth and peak viral loads in acuteinfection is unclear. Here we use a combination of experimen-tal data and modeling to demonstrate that the gut is unlikely tobe a major source of virus production in acute SIV infection.

MATERIALS AND METHODS

Experimental data. The data and experimental protocols for analysis of viraland CD4� T-cell levels refer to 15 SIVmac239-infected rhesus macaques and fiveSIVsmm-infected sooty mangabeys and have been published previously (7) orare in the course of publication (4a). Briefly, the SIVmac239 studies involved 15MaMu-A*01-positive Indian rhesus macaques. Two groups of five animals werevaccinated with modified vaccinia virus Ankara vectors bearing SIV Gag and Tatantigens, and one group of five was left uninfected. All 15 animals were chal-lenged with 10,000 50% tissue culture infective doses of SIVmac239. The five sootymangabeys were infected using 1 ml of plasma passaged from another experi-mentally infected animal. Viral levels in plasma were measured using reversetranscription-PCR as described previously (6), and the proportions of CD4� Tcells in the various tissues examined (peripheral blood [PB], lymph nodes [LNs],bronchoalveolar lavage [BAL] specimens, and rectal biopsy [RB] specimens)were measured by flow cytometry as previously described (7).

Analysis of peak viral and CD4� T-cell depletion. The percentage of residualCD4� T cells at each studied time point post-SIV infection was calculated as aproportion of the uninfected value (taken 4 weeks prior to infection). The rateof CD4� T-cell loss was calculated from the linear slope between adjacent datapoints and can be considered an average over the interval between the datapoints. The midpoint time of the maximum loss rate interval was then used tocompare to the time of peak viral load. The day of peak viral load was taken to

* Corresponding author. Mailing address: Complex Systems in Bi-ology Group, Centre for Vascular Research, University of New SouthWales, Kensington NSW 2052, Australia. Phone: 61 2 9385 2762. Fax:61 2 9385 1389. E-mail: m.davenport@unsw.edu.au.

† Present address: Commonwealth Scientific and Industrial Re-search Organisation, Materials Science and Engineering, Clayton, VIC3168, Australia.

� Published ahead of print on 20 May 2009.

7517

be the time of the highest viral load or, in cases where two subsequent viral loadsdiffered by �2-fold, the average of the times of the two highest measurementswas used.

The difference between the time of peak viral load and the time of the fastestCD4� T-cell loss for all animals was assessed for significance with a Wilcoxonsigned rank test using Prism (ver. 5.01; GraphPad Software). This test was alsoused to compare CD4� T-cell levels from RB specimens with those from BALspecimens.

Modeling of CD4� T-cell kinetics. The interaction between the viral load andCD4� T-cell kinetics was modeled using a simplified version of a previouslypublished differential equation model for HIV and SIV (14) adapted to considerdifferent compartments of susceptible cells:

dTi

dt� �kiVTi (1)

dIi

dt� kiVTi � �iIi (2)

dVdt

� �i�1

3

piIi � cV (3)

where Ti and Ii are the numbers of target and infected cells in the ith compart-ment, respectively; ki is the infectivity; �i is the death rate of infected CD4� Tcells; and pi is the production rate of virus from infected cells in that compart-ment. The clearance rate of free virus (V) is c. This simplified model assumes thatthe production rate and the natural death rate of uninfected cells are negligibleover the course of acute infection. We modified this basic model of infection toinclude three separate “compartments” of infected cells. These compartmentscould differ in various ways, including in size, in death rate (�i), in infectivity (ki),and in viral production (pi).

For the purpose of this model, the cell numbers needed to be in the sameproportion as the total numbers in the respective compartments. We chose therepresentative numbers so that the sizes of the compartments were roughly10-fold different. Thus, T was initially 900, 90, and 9 cells in compartments 1 to3, respectively.

The equations were solved using the ode45 function in Matlab (ver. 7.4;Mathworks). The observed total CD4� T-cell numbers contained both unin-fected and infected CD4� T cells (since infected cells still express CD4). Fromthe model, combining equation 1 and equation 2, we find that the observed lossrate of total CD4� T cells,

d�Ti � Ii�

dt� ��iIi (4)

is proportional to the number of infected cells in the compartment. Thus, themaximum observed CD4� loss rate will coincide with the peak of infected cellsand the peak virus production in the compartment.

RESULTS

Dynamics of CD4� T-cell depletion in RB specimens duringacute SIVmac239 infection. In order to study the dynamics of theviral load and CD4� T-cell depletion in the gut, we obtainedsequential plasma and tissue samples from the rectums ofrhesus macaques infected with SIVmac239. Of note, the exam-ined samples contained for the most part CD4� T cells resid-ing in the intestinal lamina propria, an immune effector mu-cosal site, and did not provide a representative sampling of theimmune inductive mucosal sites (i.e., Peyer patches and iso-lated lymphoid follicles). Consistent with previous studies, weobserved a rapid depletion of CD4� T cells in the rectal mu-cosa during acute infection (Fig. 1a). We then examined thedynamics of CD4� T-cell depletion compared to the viral load.Surprisingly, we observed that in a significant proportion ofanimals, CD4� T cells were severely depleted from the gutseveral days before the peak viral load (Fig. 1b). As expectedin a group of outbred animals, the exact timing of CD4� T-celldepletion and peak viral loads varied between individual SIV-

infected macaques. Although 8 of the 15 animals showed aclearly defined peak in the viral load at day 10, others animalsshowed a slightly broader peak and could show a peak as lateas day 14. Therefore, we aligned the peak viral load in eachanimal, and observed the level of CD4� T-cell depletion in therectum at different sampling times leading up to the peak viralload (Fig. 1b). In 8 of the 15 animals, CD4� T cells weredepleted to 0.3 to 31% of preinfection levels 2 to 5 days priorto the peak viral load.

The clearance rate of free virus in HIV and SIV is extremelyhigh (16), and thus, the viral load levels provide a good esti-mate of the viral production rate around the time of measure-ment, and also the number of productively infected cells. If gutCD4� T cells were a major source of virus, one would concludethat the relatively small fraction of gut CD4� T cells remainingat the time of the peak viral load was sufficient to cause thishigh level of virus replication. However, the daily loss rate ofCD4� T cells in the gut prior to the peak viral load in some

FIG. 1. Plasma viral load and CD4� T-cell kinetics in rhesus ma-caques. (a) The plasma viral load (black) and percent CD4� T cellswere measured in PB, LNs, rectum, and BAL specimens for an indi-vidual animal. The peak viral load (indicated by the dashed verticalline) varied between individual animals. The kinetics of CD4� T-celldepletion in the rectum (b) BAL specimens (c) LNs (d), and PB (e) areshown relative to the time of the peak viral load in each animal. Thisreveals that depletion in the rectum often significantly precedes thepeak in the viral load. Animals that deplete early in the rectum (indi-cated in red) do not necessarily deplete early in other sites. Note thatBAL specimen CD4� kinetics for animal RNr8 was not included, asbaseline levels were not available.

7518 LAY ET AL. J. VIROL.

cases exceeded the total number of cells remaining at the peak.Thus, more cells died each day (presumably as a result ofinfection) than were even available for infection by the time ofthe peak viral load. Moreover, since the viral loads at the timeof rapid loss of CD4� T cells in the gut were often 10-foldlower than the peak viral loads, this would require that theminority population of CD4� T cells remaining in the gut atthe peak viral load were all infected and produced many morevirions on a per cell basis than infected CD4� T cells earlier ininfection. Since the level of CD4� T cells in the rectum did notchange between the viral peak and day 28 after infection (Fig.2), it seems extremely unlikely that all of these cells could havebeen infected at the peak.

Dynamics of CD4� T-cell depletion in other compartmentsduring acute SIVmac239 infection. Data on the kinetics ofCD4� T-cell depletion during acute SIVmac239 infection werealso available from BAL, LN biopsy, and PB samples obtainedfrom the same macaques. CD4� T-cell depletion in PB and LNdid not reach the same high levels seen in RB specimens (likelydue to their enrichment in naïve CD4� T cells, which are moreresistant to infection), and thus it was not possible to directlycompare depletion levels across all compartments. CD4� T-cell depletion in RB specimens over the course of acute infec-tion was similar to that seen in BAL specimens compared atday 28 (median, 5.4% versus 9.5%, respectively) (Fig. 2). How-ever, when depletion rates at the time of the peak viral loadwere compared, the rectum showed significantly higher deple-tion than the BAL specimens (median, 11.9% versus 62.7%,respectively; P � 0.0001; Wilcoxon matched pairs), indicatingthat early depletion was limited to the gut, despite similarlong-term depletion levels in both RB specimens and BALspecimens.

In order to compare the timing of CD4� T-cell loss betweenanatomic compartments in which the overall levels of deple-tion differed greatly, we first determined the time at which themaximal depletion rate was observed. The loss rate of CD4� Tcells was calculated from the available CD4� T-cell data atdifferent times, allowing us to estimate the loss rate over theinterval between any data points. The rate is assumed to beconstant over this interval; however, it is possible that there iseither an early or late depletion within this interval and thatthe interval merely serves as the range of possible times when

this could occur. Consistent with the observed severe CD4�

T-cell depletion prior to the peak viral load, we observed thatthe peak rates of CD4� T-cell loss in the rectum preceded thepeak viral load in 9 out of the 15 animals (Fig. 3). By compar-ing the midpoint of the time interval in which the peak CD4�

T-cell loss rate occurred to the time of peak virus (Fig. 4), wefound that the peak CD4� T-cell depletion rate in the gutoccurred significantly before the peak viral load (P � 0.001;

FIG. 2. CD4� T-cell depletion in RB and BAL specimens. Therectum (RB) shows significantly higher levels of CD4� T-cell depletionthan the BAL specimens at the time of peak viral load (a) (P �0.0001). However, by 28 days postinfection, the depletions are similarin both compartments (b) (P � 1.0).

FIG. 3. The timing of peak CD4� T-cell depletion rates in RB (a),BAL (b), PB (c), and LN (d) specimens relative to the time of peakviral load in rhesus macaques. The animals are aligned according tothe time of the peak viral load, and the relative times of the peak CD4�

T-cell depletion rate are indicated for samples from different tissues.The time period in which the peak loss of CD4� T cells (i.e., maximumpercent per day) occurred is indicated as a horizontal bar.

FIG. 4. CD4� T-cell depletion occurs significantly earlier in therectum. The time of peak CD4� T-cell depletion relative to the peakviral load is indicated in different tissues for rhesus macaques (a) andsooty mangabeys (b). The time of peak depletion is taken as themidpoint of the interval in which the maximum depletion was ob-served. Each point represents an individual animal. The gray barsindicate the median differences. Peak depletion in RB samples fromrhesus macaques occurred significantly before the peak viral load (P �0.001; Wilcoxon). However, in other compartments, the time of peakdepletion was not significantly different from the timing of the peakviral load.

VOL. 83, 2009 IS THE GUT THE MAJOR VIRUS SOURCE IN EARLY SIV INFECTION? 7519

Wilcoxon). In contrast, the time of the maximum rate of loss inPB, LN, and BAL specimens was not significantly differentfrom the time of the peak plasma viral load (P 0.50 for LN,BAL specimens, and PB).

Dynamics of CD4� T-cell depletion during acute SIV infec-tion of sooty mangabeys. The observed disconnect between thetiming of gut CD4� T-cell depletion and the peak viral loads inSIVmac239 infection of rhesus macaques may reflect physiolog-ical factors specific to the host or viral factors specific to thisstrain of SIV. In order to investigate whether early depletionwas seen in other models of HIV infection, we chose to studyintestinal CD4� T-cell depletion in a “natural-host” model ofSIV infection in sooty mangabeys (18). These animals do notsuffer from severe CD4� T-cell depletion in the PB and do notprogress to AIDS but still experience profound CD4� T-celldepletion in mucosal sites in acute SIV infection (7). Thismodel thus provides a different host and disease outcome tocompare with SIVmac239. Figure 5 illustrates the timing of themaximum CD4� T-cell loss rate and the viral-load peak in fiveexperimentally infected sooty mangabeys. Consistent with our

observations in rhesus macaques, the majority of these animalsagain showed that CD4� T-cell depletion in the gut precededthe viral peak, while the timing coincided with the viral peak inother compartments. When we compared the timing of thehighest CD4� T-cell loss rate for these animals with the timingof the peak viral load in plasma, we saw a strong trend (P 0.055) toward earlier depletion in the gut, before the peak virallevel (Fig. 4b and 6), whereas differences in other compart-ments were not significant (BAL specimens, P 0.409; LN,P 0.099; PB, P 0.581).

Modeling susceptibility versus production of virus in acuteinfection. In order to understand the dynamics of CD4� T-celldepletion in different physiological compartments in acuteSIV, we adapted the standard mathematical model of HIVinfection (9, 14) to consider infection in different “compart-ments” that differed in “size” and “susceptibility to infection”(Fig. 7). When all compartments had equal susceptibilities toinfection, all were infected and depleted synchronously (andcoincident with the peak viral load) (Fig. 7a). However, differ-ent dynamics were observed when the compartments differedin their susceptibilities to infection. When the largest compart-ment was the most susceptible, infection in the compartmenteffectively drove the peak in the viral load, and thus the rapidCD4� T-cell depletion occurred at the same time in the othercompartments. Thus, all compartments were depleted synchro-nously and coincidentally with the peak viral load (Fig. 7b).However, when the largest compartment was the least suscep-tible to infection, it was the last to be depleted. Regardless ofthe susceptibility of the largest compartment, the peak in in-fection here was always coincident with the peak viral load(since it provided the major source of virus during infection).The smaller compartments were infected at earlier times de-pending on their susceptibility to infection but had little impacton the peak viral load. In cases where the minor compartmentswere more susceptible to infection than the largest compart-ment, they were profoundly depleted prior to the peak viral

FIG. 5. Plasma viral load and CD4� T-cell kinetics in sooty manga-beys. (a) The plasma viral load (black) and the percent CD4� T cellswere measured in PB, LNs, rectum, and BAL specimens for an indi-vidual animal. The peak viral loads (indicated by the dashed verticalline) varied between individual animals. The kinetics of CD4� T-celldepletion in the rectum (b), BAL specimens (c), LN (d), and PB (e)are shown relative to the time of the peak viral load in each animal.The animals were aligned according to the timing of the peak viral loadin order to observe the timing of depletion in different anatomicalcompartments.

FIG. 6. Difference between the time of the peak CD4� T-cell de-pletion rate and the peak viral load in RB (a), BAL (b), PB (c), and LN(d) specimens from sooty mangabeys. The times of peak CD4� T-celldepletion (indicated as horizontal bars) and peak viral load (indicatedas a vertical line) are shown in different compartments for SIV-in-fected sooty mangabeys. The peak rate of CD4� T-cell depletion(taken as the midpoint of the time interval) in the rectum occurredbefore the peak viral load in all animals (although this is not significantdue to the small number of animals studied). The depletion in othercompartments occurred closer to the peak viral load.

7520 LAY ET AL. J. VIROL.

load. Thus, the timing of the depletion of minor compartmentscould be dissociated from that of major compartments, de-pending on the susceptibility and dynamics of infection.

Another factor that could drive the earlier depletion in thegut would be if infected cells in the gut had a higher death ratethan infected cells elsewhere. However, as shown in Fig. 7d,even a twofold difference in infected-cell death rates betweencompartments had relatively little impact on the timing ofdepletion between compartments if they had the same suscep-tibility to infection.

The rapid depletion of cells in the gut is best explained by anincreased susceptibility to infection. However, the conclusionthat the gut is not a major source of virus does not require thatthere must be overall fewer CD4� T cells in the gut, as it is alsopossible that infected cells in the gut simply produced lessvirus. Figure 7e illustrates the predicted outcome of infectionwith three compartments of the same size that differed insusceptibility to infection. In this case, all of the compartmentscontributed to the peak viral load, and all were depleted afterthe peak. However, if the compartments differed in both sus-ceptibility to infection and viral production per infected cell(Fig. 7f), then we observed essentially the same behavior as

seen with compartments of different sizes (compare Fig. 7c andf). Thus, both scenarios suggest the gut must be more suscep-tible to infection than the other tissues in order to be depletedfirst. However, failure to contribute to the peak viral load mayarise either because of a small number of susceptible cells inthe gut or because gut CD4� T cells produce less virus perinfected cell than other tissues. We note that the latter expla-nation would also be compatible with a high level of “bystanderdeath” of uninfected CD4� T cells in the gut during acute SIVinfection, as suggested by Li et al. (10).

The experimental studies demonstrated directly that thepeak rate of depletion of CD4� T cells in the gut occurssignificantly before the peak in the viral load. From analysis ofthe modeling, we have shown that the timing of the maximumCD4� loss rate is a direct indicator of the peak of infected cellsin a chosen compartment (equation 4). Because of the extremelyhigh clearance rate of the free virus, the peak of infected cellsnearly coincides with the peak production of virus in the com-partment (Fig. 8).

The maximum CD4� loss rate in the gut precedes the peakviral load by several days both in rhesus macaques and in sootymangabeys (Fig. 4). This means that virus production by in-

FIG. 7. Modeling the peak viral load and CD4� T-cell depletion in compartments of different sizes or with different virus production rates. Thedashed black lines represent viral loads; the colored lines are total observed (uninfected and infected; T � I) CD4� T-cell numbers. The circlesmark the fastest CD4� T-cell depletion in each compartment. We first modeled infection in compartments differing in size and either susceptibilityto infection or death rate (using equations 1 to 3). Before infection, the largest (red) compartment contained 900 cells, the medium-sizecompartment (blue) had 90 cells, and the smallest compartment (green) had 9 cells. (a) When all three compartments have same susceptibility toinfection (kL � kM � kS � 5 � 10�8 ml/copy/day), they are depleted synchronously. (b) If the largest compartment has the highest susceptibility(kL � 10�7 ml/copy/day � 10kM � 100kS), then it is infected rapidly, driving the peak viral load. In the less susceptible compartments, maximumdepletion occurs around the peak viral load, and thus depletion occurs almost synchronously in all compartments. (c) If the largest compartmenthas the lowest susceptibility (kL � 10�8 ml/copy/day � 0.1kM � 0.01kS), then the smaller compartments are depleted first, but the peak viral loadis not reached until the large compartment is infected. In panels a to c, the other parameters are as follows: � � 1/day, p � 106 copies/cell/day,and c � 20/day. (d) Early depletion cannot be driven by high death rates. If the compartments have the same susceptibility to infection but thedeath rates of infected cells vary, this has relatively little effect on driving early depletion. All compartments are infected synchronously (as in panela), and differences in death rates do not affect the timing of depletion greatly. The largest compartment has the highest death rate of infected cells(�L � 2/day, �M � 1/day, and �S � 0.5/day). The other parameters are as follows: k � 10�7 ml/copy/day, p � 5 � 105 copies/cell/day, and c � 20/day.Early depletion can also be seen if all compartments are the same size (333 cells) but differ in susceptibility to infection and the rate at whichinfected cells produce virus. (e) If compartments differ only in susceptibilities, with the first (red) compartment the least susceptible (k1 � 10�8

ml/copy/day � 0.1k2 � 0.01k3), but with the same viral production in each compartment (p � 5 � 105 copies/cell/day), then they differ in the timingof depletion, but with the most susceptible compartment still depleting around the peak of virus (not before it). (f) If compartments differ in virusproduction rates, as well as susceptibilities, then the highest-producing compartment (red) (producing a p1 of 10 copies/cell/day, which is 10-foldmore than the medium-producing [blue] and 100-fold more than the low-producing [green] compartment) drives the peak viral load. If thehighest-producing, red, compartment is the least susceptible, then the other compartments are depleted earlier. Thus, low production and smallcompartment size in the gut are both compatible with the observed kinetics in vivo. The other parameters are as follows: � � 1/day, and c � 20/day.

VOL. 83, 2009 IS THE GUT THE MAJOR VIRUS SOURCE IN EARLY SIV INFECTION? 7521

fected cells in the gut peaks approximately 4 days before thepeak in the viral load and that it becomes negligible comparedto the contributions of the other compartments at the peak ofacute infection.

DISCUSSION

The intestinal mucosa is an anatomic compartment thatplays a major role in the interaction between primate lentivi-ruses and the host immune system. This is due to severalfactors, including the presence of both high levels of activatedCD4� CCR5� T cells that are preferential targets for HIV/SIVand strong antiviral cellular immune responses (8). In thecurrent study, we have observed that CD4� T cells in RBspecimens are often depleted several days before the peak inthe plasma viral load occurs. Since the time of maximum deathof rectal CD4� T cell occurs before the peak of virus in theblood, the rapid death of these CD4� T cells cannot be con-nected with the peak of virus replication during acute SIVinfection. We used a modeling approach to analyze how thesizes and susceptibilities to infection of different anatomicalcompartments affect the timing and extent of CD4� T-celldepletion relative to the peak viral loads. This analysis suggeststhat the maximum loss rate of CD4� T cells in the largestcompartment will always coincide with the peak viral load.Other compartments may be depleted before the peak viralload if they are more susceptible to infection than the majorcompartment. If a compartment is observed to deplete sepa-rately from the peak, it cannot be the driver of the peak viralload. However, just because a compartment is depleted at thesame time as the peak viral load does not indicate that it drivesthe peak—it may just have similar susceptibility to infection.The scenario most similar to the experimental data is that seenin Fig. 7c. There, a small compartment of highly susceptiblecells (in the rectum) is depleted early. A compartment withintermediate susceptibility is also partially depleted at this timeand goes on to similar levels of peak depletion (as seen in theBAL specimens [Fig. 2]). A similar scenario is seen if theproduction rate of virus per infected cell, rather than the num-ber of cells, is what differentiates compartments (Fig. 7f). Thus,it is possible that the number of cells in the gut is large but thatthey do not produce much virus per infected cell. Regardless of

the mechanisms, the timing of depletion of CD4� T cells in therectal mucosa indicates that this cannot be the major source ofvirus, and it is likely just a very susceptible population of cellsthat is rapidly targeted and depleted. However, the coinci-dence of CD4� T-cell depletion in LNs, BAL specimens, andPB with peak plasma viremia does not guarantee that one orall of these is driving peak viremia. They may just have sus-ceptibilities to infection similar to that of the major compart-ment(s).

The results of this study appear at first to directly contradicta number of earlier studies. One major difference between thisstudy and previous ones is the serial biopsies of individualanimals. In earlier studies, only a necropsy sample or onebiopsy sample plus a necropsy sample were obtained duringearly infection (10, 13). These were staggered at various stagesin early infection, so that the “average viral peak” and “averagedepletion” were obtained by combining data from differentanimals. Thus, these studies correctly concluded that the viralpeak and CD4 depletion occurred around the same time, butthey lacked the resolution to distinguish a difference of 4 to 5days in this timing.

Our study analyzed RB samples as a representative region ofthe gut. We note that previous studies have not described anymajor differences between the upper and lower gastrointestinaltract (10, 13). However, we cannot exclude the possibility thatthe dynamics may vary at different sites. Similarly, we haveanalyzed the dynamics of total CD4� T cells in the gut. Thus,we cannot exclude the possibility of “special cells” that are avery small minority population of CD4� T cells in the gut butsomehow produce all of the virus. However, we note that thereis no more reason to suspect that “special cells” exist in the gutthan in any other site. Finally, our conclusion that the dynamicsof CD4� T-cell loss in the gut are inconsistent with it being amajor source of virus does not exclude a role for the gut inindirectly causing high viral production elsewhere; HIV infec-tion and CD4� T-cell loss in the gut have been associated withthe influx of microbial products into the systemic circulation (2,11). These microbial products may play a role in activating Tcells in other sites, leading to increased susceptibility to infec-tion and increased viral production elsewhere. It is possiblethat the peak in the viral load immediately following T-celldepletion in the gut is a consequence of this activation in othersites.

The rapid early depletion of CD4� T cells from the gut hassuggested an important role of this anatomic compartment inthe pathogenesis of HIV infection. Our results suggest thatthese features may be explained by an increased susceptibilityof gut cells to infection but that the gut does not provide amajor source of virus (at least during acute infection). Thus,the gut is a “passenger” and not a “driver” in the dramaticinteraction between HIV/SIV and the host immune systemthat takes place during acute infection. The peak viral load inacute infection is a major determinant of the extent of CD4�

T-cell depletion in simian/human immunodeficiency virus andSIV infection, and the extent of depletion can be reduced byvaccines that effectively control the peak viral load (1, 4, 12, 17,23). Identifying which tissues are the major drivers of the peakviral load provides an opportunity for interventions aimed atreducing infection and/or immune activation at these sites. Theapproach of comparing the timing of infection and depletion of

FIG. 8. The peak depletion rate of CD4� T cells coincides with thepeak production of virus in the compartment. Shown is modeling ofCD4� T-cell depletion using the same parameters as in Fig. 7c butshowing viral production from each compartment. The solid linesrepresent CD4� T cells in each compartment, with circles at themaximum rates of loss of CD4� T cells. The dotted lines represent thecontributions of each compartment to the total viral load.

7522 LAY ET AL. J. VIROL.

CD4� T cells in different tissues is one way of identifying themajor source of virus during infection and identifying potentialanatomical sites for immune control.

ACKNOWLEDGMENTS

We thank Meritxel Genesca for helpful discussions on the role oflipopolysaccharide in SIV infection.

This work is supported by the James S. McDonnell Foundation 21stCentury Research Award/Studying Complex Systems, the NationalHealth and Medical Research Council (Australia), and the NIH (grantR01-AI66998 P51-RR00165, U19 AI074078, and U19 AI76174 toG.S.). M.P.D. is supported by a Sylvia and Charles Viertel CharitableFoundation Senior Medical Research Fellowship.

REFERENCES

1. Barouch, D. H., S. Santra, J. E. Schmitz, M. J. Kuroda, T. M. Fu, W.Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R. Krivulka, K. Beaudry, M. A.Lifton, C. E. Nickerson, W. L. Trigona, K. Punt, D. C. Freed, L. M. Guan, S.Dubey, D. Casimiro, A. Simon, M. E. Davies, M. Chastain, T. B. Strom, R. S.Gelman, D. C. Montefiori, et al. 2000. Control of viremia and prevention ofclinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.Science 290:486–492.

2. Brenchley, J. M., D. A. Price, T. W. Schacker, T. E. Asher, G. Silvestri, S.Rao, Z. Kazzaz, E. Bornstein, O. Lambotte, D. Altmann, B. R. Blazar, B.Rodriguez, L. Teixeira-Johnson, A. Landay, J. N. Martin, F. M. Hecht, L. J.Picker, M. M. Lederman, S. G. Deeks, and D. C. Douek. 2006. Microbialtranslocation is a cause of systemic immune activation in chronic HIV in-fection. Nat. Med. 12:1365–1371.

3. Brenchley, J. M., T. W. Schacker, L. E. Ruff, D. A. Price, J. H. Taylor, G. J.Beilman, P. L. Nguyen, A. Khoruts, M. Larson, A. T. Haase, and D. C.Douek. 2004. CD4� T cell depletion during all stages of HIV disease occurspredominantly in the gastrointestinal tract. J. Exp. Med. 200:749–759.

4. Davenport, M. P., L. Zhang, J. W. Shiver, D. R. Casmiro, R. M. Ribeiro, andA. S. Perelson. 2006. Influence of peak viral load on the extent of CD4�

T-cell depletion in simian HIV infection. J. Acquir. Immune Defic. Syndr.41:259–265.

4a.Engram, J. C., R. M. Dunham, G. Makedonas, T. H. Vanderford, B.Sumpter, N. R. Klatt, S. J. Ratcliffe, S. Garg, M. Paiardini, M. McQuoid,J. D. Altman, S. I. Staprans, M. R. Betts, D. A. Garber, M. B. Feinberg, andG. Silvestri. Vaccine-induced, SIV-specific CD8� T-cells reduce virus rep-lication but do not protect from SIV disease progression. J. Immunol., inpress.

5. Ganusov, V. V., and R. J. De Boer. 2007. Do most lymphocytes in humansreally reside in the gut? Trends Immunol. 28:514–518.

6. Garber, D. A., G. Silvestri, A. P. Barry, A. Fedanov, N. Kozyr, H. McClure,D. C. Montefiori, C. P. Larsen, J. D. Altman, S. I. Staprans, and M. B.Feinberg. 2004. Blockade of T cell costimulation reveals interrelated actionsof CD4� and CD8� T cells in control of SIV replication. J. Clin. Investig.113:836–845.

7. Gordon, S. N., N. R. Klatt, S. E. Bosinger, J. M. Brenchley, J. M. Milush,J. C. Engram, R. M. Dunham, M. Paiardini, S. Klucking, A. Danesh, E. A.Strobert, C. Apetrei, I. V. Pandrea, D. Kelvin, D. C. Douek, S. I. Staprans,D. L. Sodora, and G. Silvestri. 2007. Severe depletion of mucosal CD4� Tcells in AIDS-free simian immunodeficiency virus-infected sooty mangabeys.J. Immunol. 179:3026–3034.

8. Haase, A. T. 2005. Perils at mucosal front lines for HIV and SIV and theirhosts. Nat. Rev. Immunol. 5:783–792.

9. Ho, D. D., A. U. Neumann, A. S. Perelson, W. Chen, J. M. Leonard, and M.Markowitz. 1995. Rapid turnover of plasma virions and CD4 lymphocytes inHIV-1 infection. Nature 373:123–126.

10. Li, Q., L. Duan, J. D. Estes, Z. M. Ma, T. Rourke, Y. Wang, C. Reilly,J. Carlis, C. J. Miller, and A. T. Haase. 2005. Peak SIV replication in restingmemory CD4� T cells depletes gut lamina propria CD4� T cells. Nature434:1148–1152.

11. Li, Q., J. D. Estes, L. Duan, J. Jessurun, S. Pambuccian, C. Forster, S.Wietgrefe, M. Zupancic, T. Schacker, C. Reilly, J. V. Carlis, and A. T. Haase.2008. Simian immunodeficiency virus-induced intestinal cell apoptosis is theunderlying mechanism of the regenerative enteropathy of early infection.J. Infect. Dis. 197:420–429.

12. Mattapallil, J. J., D. C. Douek, A. Buckler-White, D. Montefiori, N. L.Letvin, G. J. Nabel, and M. Roederer. 2006. Vaccination preserves CD4memory T cells during acute simian immunodeficiency virus challenge. J.Exp. Med. 203:1533–1541.

13. Mattapallil, J. J., D. C. Douek, B. Hill, Y. Nishimura, M. Martin, and M.Roederer. 2005. Massive infection and loss of memory CD4� T cells inmultiple tissues during acute SIV infection. Nature 434:1093–1097.

14. Perelson, A. S. 2002. Modelling viral and immune system dynamics. Nat.Rev. Immunol. 2:28–36.

15. Picker, L. J. 2006. Immunopathogenesis of acute AIDS virus infection. Curr.Opin. Immunol. 18:399–405.

16. Ramratnam, B., S. Bonhoeffer, J. Binley, A. Hurley, L. Zhang, J. E. Mittler,M. Markowitz, J. P. Moore, A. S. Perelson, and D. D. Ho. 1999. Rapidproduction and clearance of HIV-1 and hepatitis C virus assessed by largevolume plasma apheresis. Lancet 354:1782–1785.

17. Shiver, J. W., T. M. Fu, L. Chen, D. R. Casimiro, M. E. Davies, R. K. Evans,Z. Q. Zhang, A. J. Simon, W. L. Trigona, S. A. Dubey, L. Huang, V. A. Harris,R. S. Long, X. Liang, L. Handt, W. A. Schleif, L. Zhu, D. C. Freed, N. V.Persaud, L. Guan, K. S. Punt, A. Tang, M. Chen, K. A. Wilson, K. B. Collins,G. J. Heidecker, V. R. Fernandez, H. C. Perry, J. G. Joyce, K. M. Grimm,J. C. Cook, P. M. Keller, D. S. Kresock, H. Mach, R. D. Troutman, L. A.Isopi, D. M. Williams, Z. Xu, K. E. Bohannon, D. B. Volkin, D. C. Monte-fiori, A. Miura, G. R. Krivulka, M. A. Lifton, M. J. Kuroda, J. E. Schmitz,N. L. Letvin, M. J. Caulfield, A. J. Bett, R. Youil, D. C. Kaslow, and E. A.Emini. 2002. Replication-incompetent adenoviral vaccine vector elicits ef-fective anti-immunodeficiency-virus immunity. Nature 415:331–335.

18. Silvestri, G., D. L. Sodora, R. A. Koup, M. Paiardini, S. P. O’Neil, H. M.McClure, S. I. Staprans, and M. B. Feinberg. 2003. Nonpathogenic SIVinfection of sooty mangabeys is characterized by limited bystander immuno-pathology despite chronic high-level viremia. Immunity 18:441–452.

19. Smit-McBride, Z., J. J. Mattapallil, M. McChesney, D. Ferrick, and S.Dandekar. 1998. Gastrointestinal T lymphocytes retain high potential forcytokine responses but have severe CD4� T-cell depletion at all stages ofsimian immunodeficiency virus infection compared to peripheral lympho-cytes. J. Virol. 72:6646–6656.

20. van’t Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-vanLent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema,and H. Schuitemaker. 1994. Macrophage-tropic variants initiate human im-munodeficiency virus type 1 infection after sexual, parenteral, and verticaltransmission. J. Clin. Investig. 94:2060–2067.

21. Veazey, R. S., M. Demaria, L. V. Chalifoux, D. E. Shvetz, D. R. Pauley, H. L.Knight, M. Rosenzweig, R. P. Johnson, R. C. Desrosiers, and A. A. Lackner.1998. Gastrointestinal tract as a major site of Cd4� T cell depletion and viralreplication in SIV infection. Science 280:427–431.

22. Veazey, R. S., and A. A. Lackner. 2004. Getting to the guts of HIV patho-genesis. J. Exp. Med. 200:697–700.

23. Wilson, D. P., J. J. Mattapallil, M. D. Lay, L. Zhang, M. Roederer, and M. P.Davenport. 2007. Estimating the infectivity of CCR5-tropic simian immuno-deficiency virus SIVmac251 in the gut. J. Virol. 81:8025–8029.

VOL. 83, 2009 IS THE GUT THE MAJOR VIRUS SOURCE IN EARLY SIV INFECTION? 7523