Rapid Clearance of Simian Immunodeficiency Virus Particles from Plasma of Rhesus Macaques

Perturbation of the equilibrium between human immunodeficiency virus type 1 (HIV-1) and the infected host by administering antiretroviral agents has provided fundamental insights into the dynamic nature of this viral infection (7, 16-18, 28). The seemingly stable levels of plasma viremia in infected individuals in fact represent a balance between two equally rapid processes, the production and clearance of HIV-1 (7, 28). Careful studies of the decay of HIV-1 RNA in plasma after antiretroviral treatment have revealed that the clearance of cell-free virions and the loss of productively infected CD4⁺ T lymphocytes both occur quickly. The estimated mean half-life (t_1/2) of productively infected CD4⁺T lymphocytes is approximately 1.1 to 1.6 days, suggesting that about half of all virus-producing cells die daily and that an equivalent number of CD4⁺ T lymphocytes must become newly infected within the same time frame (6, 16-18). However, the best estimate to date for the t_1/2 of virions in plasma is <6 h, implying that a total of >10¹⁰ virions must be produced each day to maintain the dynamic equilibrium in a typical infected person (18). A more accurate measurement of virion clearance would, therefore, provide a more precise definition of the magnitude of HIV-1 replication in vivo.

In this study, we used the simian immunodeficiency virus (SIV)-rhesus macaque model to obtain a more accurate estimate of virion clearance. First, we generated a stock of SIV particles by transfecting 293 cells with a molecular clone of SIVmac239. Cell supernatant was harvested 48 h later, and viral particles were concentrated and purified by sucrose gradient centrifugation. The purified virions recovered from the 20 to 40% sucrose interface contained all of the major structural proteins (gp120, gp41, p66, p55, and p27) as determined by a Western immunoblot assay (data not shown). The viral stock was resuspended in phosphate-buffered saline, quantified for virion concentration by a branched DNA (bDNA) amplification assay for SIV RNA, and stored at −80°C until use.

Two different approaches were used to study virion clearance in rhesus macaques. One was to follow the disappearance of viral particles from plasma after intravenous bolus injection of viral particles over a ∼5-min period. The other was to track the level of plasma viremia during and following a constant intravenous infusion of SIV particles. Four animals were used in the first approach, and three animals were used in the second. Both approaches included infected and uninfected animals (Table 1). Prior to each experiment, each animal was anesthetized with an intramuscular injection of ketamine HCl (10 to 15 mg/kg of body weight), followed by gaseous anesthesia with halothane (50 to 70 cc/min) and nitrous oxide (1,000 cc/min) throughout the course of the study. The SIV particles were infused into the monkeys via a cannula inserted into one femoral vein; this was followed by periodic blood collection from the opposite femoral vein. For the first bolus injection experiment (animal no. 1368), 3-ml blood samples were collected every 5 min in the first hour and every 15 min in the next 3 h. In the next two bolus injection experiments (animals 1336 and 537), the same amount of blood was collected every minute for the first 5 min, every 5 min for the next hour, every 15 min for the following hour, and every half hour for the subsequent 2 h. For the steady-state infusion experiments, 3-ml blood samples were collected every minute for the first 10 min, every 5 min for the next hour, and every 15 min for the following 2 h. Immediately after blood collection, plasma samples were separated and stored at −80°C. At the completion of the study, four (1336, 537, 531, and 1378) of the six animals were euthanized by an overdose of sodium pentobarbital (100 to 120 mg/kg). Various tissues, including those of lymph nodes, spleen, thymus, tonsil, heart, liver, muscle, kidney, lung, pancreas, brain, and gastrointestinal tract, as well as cerebrospinal fluid (CSF) and urine, were collected and frozen immediately in liquid nitrogen. The virion concentration in tissue homogenates or bodily fluids was also determined by the SIV bDNA assay.

Figure 1A shows the changes in plasma viral RNA during and after the bolus injection of SIV particles into three rhesus macaques. Animals 1368 and 1336 were uninfected, whereas animal 537 was infected with SIVmac251 but without detectable plasma viremia (<10,000 RNA copies/ml). During the bolus injection, a sharp increase in plasma viral RNA in each animal was observed. The peak levels of plasma viremia were variable because of differences in the number of virions injected and in body weights (Table 1) as well as variations in the rates of bolus injections. Upon completion of injection, however, a very rapid exponential decline in plasma viremia in all three animals was consistently observed. This decay appeared to be monophasic except in animal 1368. All the data points reported in this study are the geometric means of quadruplicate measurements by the bDNA assay. Data points in the mid-portion of the exponential decay were used to obtain the most accurate estimates of clearance rate constants, c, for virions (i.e., the slope of the linear regression line). Regardless of SIV infection status, the rate constants were found to be within a narrow range of 0.15 to 0.21 per minute, which in turn yielded a decay t_1/2 of 3.3 to 4.6 min (Table 1 and Fig. 1A). These values are ∼100-fold lower than our previous minimum estimate made for HIV-1 clearance in infected patients (18).

We also performed steady-state infusion experiments to confirm the above observations. The viral stock was infused into each of three monkeys through a Harvard pump at the constant rate of 250 μl per min, which is equivalent to an infusion rate of 1.4 × 10⁸ to 1.9 × 10⁸ copies of SIV RNA per minute (Table 1). Animal 531 was uninfected, whereas animals 1378 and 1306 were infected by SIVmac251 with plasma viremia levels of <10⁴ and 7.9 × 10⁴ copies/ml, respectively. Figure 1B shows the changes in plasma viremia during and after the steady-state infusion. Initially, a sharp increase in viral RNA was observed in all three animals. Within approximately 5 min, an apparent equilibrium was reached, which then persisted throughout the course of infusion. Upon stopping infusion, a rapid exponential (monophasic) decline in plasma viral RNA in all three animals was again observed (Fig. 1B). Calculating virion clearance in the same way as described above, decay t_1/2s (1.3 to 4.3 min) (Table 1) were found to be quite similar to those following the bolus injections. The consistent results obtained from two independent experiments strongly suggest that the half-life of virions in plasma is indeed substantially shorter than the previous estimate of <6 h (18). Should the rapid clearance of virions estimated here be applicable to infected patients, then HIV-1 production, and thus the number of productively infected CD4⁺ T lymphocytes or the viral burst size, must be proportionally higher than previous minimal estimates (6, 16, 18).

During the period of apparent steady state (Fig. 1B), the rate of viral clearance in vivo must equal the rate of viral infusion set experimentally, or cV_ss × D = infusion rate, where c is the clearance rate constant, V_ss is the steady-state level of viremia, and D is the volume of distribution of viral particles. As is evident from Fig. 1B, V_ss ranged between 1.0 × 10⁶ and 2.5 × 10⁶ RNA copies per ml of plasma. Thus, the volume in which SIV particles are distributed could be directly determined for each monkey. As summarized in Table 1, the total volumes of distribution were 250 to 875 ml, or only about 1.3- to 2.1-fold larger than the calculated plasma volume (21). This demonstrates that the in vivo volume of distribution of exogenously infused viral particles is substantially smaller (0.2 to 0.3) than the total extracellular fluid volume.

The results of both the bolus injection and steady-state infusion experiments are consistent with a model in which the input virions are mainly in a single compartment with distribution volume D from which they are removed with clearance rate constant c. The virion concentration in the compartment can thus be described by the equation d V /d t = I/D−cV when 0 ≤ t < t_I or d V /d t = −cV when t > t_I, where t is time, t_I is the time at which infusion ends, and I is the rate of virion infusion. Figure 1C shows one example of how well the solution of this equation compares to the experimental data.

That there were no significant differences in the t_1/2s of SIV particles in uninfected and infected macaques was unexpected, since SIV-directed antibodies in the infected animals were expected to enhance clearance through Fc-mediated mechanisms (12, 19, 25, 26). Thus, we performed an additional experiment to specifically address this point. An additional uninfected monkey (1350) was given two bolus injections of SIV particles 1 h apart. The first was administered as previously described, but the second injection was given after the viral stock was premixed in vitro with antibodies, primarily immunoglobulin G, directed against SIVmac239. The amount of antibodies used was 2 mg, which, based on in vitro binding results, was sufficient to capture all SIV particles (8.0 × 10¹⁰) present in the inoculum (data not shown). Figure 1D shows the changes in plasma viremia during and after each bolus injection. Again, plasma viral RNA increased dramatically and then decayed exponentially with similarly rapid kinetics (t_1/2 = 4.6 min) after both injections. Thus, we have been unable to demonstrate a facilitating effect of specific antibodies in virion clearance. However, this result should be viewed as preliminary and should not be interpreted to mean that antibodies of greater binding affinity or neutralizing activity than we used could not aid the clearance of virions in vivo. Careful studies to examine the effect of specific antibodies on viral clearance are warranted.

The observations above clearly demonstrate that the half-life of exogenously infused SIV particles in plasma is exceedingly short. Are the virions actually being eliminated from blood or are they simply degraded within plasma or adherent to cells in the circulation? Studies were done to address this specific question. Experiments were carried out to study serial blood samples drawn from a monkey (1378) that underwent SIV particle infusion and to examine the sequential changes after mixing SIV particles with freshly drawn monkey (539 and 1466) blood ex vivo. Serial blood samples from animal 1378 were collected and fractionated using a standard Ficoll-Hypaque gradient centrifugation into plasma, erythrocytes, peripheral blood mononuclear cells (PBMC), granulocytes, and platelets. As shown in Fig. 2, the plasma viremia declines quickly without any appreciable increase in the number of virions associated with any cellular component. This finding demonstrates that virion adsorption onto circulating cells is not a quantitatively significant mechanism of viral clearance from plasma. In the ex vivo mixing experiments, 20-ml samples of blood from two uninfected macaques were each inoculated with 1.3 × 10⁹ SIV particles and each was then subdivided into four 5-ml aliquots, which were incubated at 37°C for various periods of time (5, 10, 20, or 30 min). Each aliquot was promptly subjected to fractionation to yield plasma, erythrocytes, PBMC, granulocytes, and platelets. As shown in Fig. 2, 95 to 97% of total viral inoculum was found in the plasma fraction, regardless of the length of incubation time, thereby showing that there is no evidence of virion degradation within plasma ex vivo. Furthermore, there was again no evidence of any significant virion binding to cellular components or any increase in cell adherence over time. Taken together, these results suggest that the observed rapid viral clearance from plasma is not mediated by rapid degradation within plasma or by adsorption of viruses onto cellular components in blood. Clearance is therefore mostly due to the physical removal of particles from circulation. Nevertheless, this observation does not necessarily dismiss the roles that Fc receptors and complement receptors play in the clearance or destruction of virions (2, 12, 19, 23-25).

To identify potential sites of viral clearance, four animals, two from bolus injection (1336 and 537) and two from steady-state infusion (531 and 1378) experiments, were euthanized immediately after the completion of particle infusion experiments. Various organs, including lymph nodes, spleen, liver, kidney, lung, pancreas, and heart, were collected and weighed. Pieces of tissue from each organ were then homogenized for SIV RNA measurements by the bDNA assay. Results of testing with 304 tissue or fluid samples from the four monkeys are summarized in Table2. The copy number of SIV RNA in each organ was then estimated by multiplying the mean of the measured RNA per milligram of tissue homogenate by the total organ weight. It was not feasible to measure the total lymphoid mass; therefore, an upper bound of 1% of body weight was used as the total mass of lymph nodes (5, 29) for these calculations. In all four animals studied, detectable levels of SIV RNA were principally found in lymph nodes, but smaller quantities were also present in spleen, lungs, and liver. This set of findings was not surprising since these tissues are known to have high numbers of CD4⁺ lymphocytes and macrophages to which SIV particles could bind. Moreover, liver and spleen have been shown to be the major sites of clearance for many different viruses (4, 8-11, 13, 14, 22). In contrast, viral RNA was not detected in tissues such as those from thymus, tonsil, heart, muscle, kidney, pancreas, brain, and various parts of the gastrointestinal tract, nor in CSF and urine. However, only ∼1 to 10% or less of the infused virions were accounted for by this thorough tissue sampling (Table 2), indicating that the vast majority of the infused particles must have been degraded prior to tissue sampling. Alternatively, although less likely, the low percentage of recoverable virus could also be due to the fact that the current SIV bDNA technique is not sufficiently sensitive (10,000 copies/ml) or that virions are just simply present in some other unsampled sites.

In summary, using the SIV-rhesus macaque model, we performed a series of studies to obtain more accurate estimates of viral clearance in vivo. The most consistent result emerging from the multiple approaches is that virion half-life in blood is extremely short, on the order of minutes (a mean of 3.3 min). This new estimate is ∼100-fold lower than the upper bound of 6 h previously reported for HIV-1 in infected humans (18). Since the dynamics of the decline in plasma viremia after antiretroviral treatment are similar between SIV-infected macaques and HIV-1-infected humans (15, 27), the clearance rate determined here for SIV is likely to reflect that of HIV-1 in vivo.

At first glance, a virion t_1/2 in plasma of only a few minutes seems surprisingly short. However, this rapid rate of clearance is in fact very similar to those defined long ago for many different viruses (Langat flavivirus, ectromelia virus, Rift Valley fever virus, influenza virus, vaccinia virus, Murray Valley encephalitis virus, tobacco mosaic virus, vesicular stomatitis virus, Newcastle disease virus, and poliovirus), bacteria, and bacteriophages in various animal models (mice, rats, dogs, and monkeys) (4, 8-11, 13, 14, 22). Collectively, these results suggest that particle clearance from the circulation is likely mediated by a common, nonspecific mechanism, such as phagocytosis by cells in the reticuloendothelial system (2, 25, 26). The rapid clearance and degradation of exogenously infused virions could pose a major obstacle for those pursuing gene therapy with viral vectors unless strategies to overcome the rapid in vivo elimination of these particles are developed.

A few caveats are in order. First, it is conceivable that the clearance of endogenously produced virions might be different from that of exogenously infused virions. Second, our SIV stock used for the experiments was prepared in human cells (293 cells); therefore, it is possible that human molecules on the virion surface might have resulted in faster clearance in monkeys due to xeno-specific effects. Third, the actual clearance rate in infected individuals may be slower if the described defects in the reticuloendothelial system prove to be functionally important (1-3, 20). Future studies to address these uncertainties are required.

The exact mechanism underlying the rapid clearance of SIV particles remains ill defined. Our current results convincingly show that virions are physically removed from the circulation with great rapidity and without any evidence of degradation within plasma or significant viral adsorption onto blood cells. As expected, detectable levels of the infused virions were found in tissues rich in CD4⁺ lymphocytes and macrophages. The detection of SIV RNA in liver and spleen was not surprising since these organs constitute the bulk of the reticuloendothelial system, which is believed to be important in the removal of particulate matter from the circulation. Indeed, prior virus-infusion studies using radioisotope- or fluorescein-labeled viruses have shown that clearance is primarily mediated by liver and spleen (4, 8-11, 13, 14, 22). Similar studies using SIV or HIV-1 are in order.

In this study, we have also found that the effective in vivo volume of distribution (D) of exogenously infused SIV particles in vivo is only slightly larger than the plasma volume and substantially less than the total extracellular fluid volume. We can only speculate that the movement of infused virions from the circulation to the rest of the extracellular fluid space must be slow compared to the time scale of the present study. On the other hand, viral particles produced under the condition of natural infection must be able to move freely from lymphoid tissues to the circulation, probably via the efferent lymphatic system and the thoracic duct, in order to maintain the dynamic equilibrium seen in chronic HIV-1 infection. The estimate of D presented here should be quite useful in calculating dynamic parameters associated with HIV-1 or SIV replication in vivo.

A precise definition of the virion clearance in vivo is not merely an academic exercise. To maintain a dynamic equilibrium in an infected host, the rapid clearance has to be matched by equally rapid production. Thus, given the mean virion half-life in plasma of 3.3 min, one can picture the circulation as a conduit through which virions flow rapidly from producer cells to either target cells or sites of elimination. Circulating virions represent particles that have come out of infected cells only in the preceding minutes. If the current estimate of t_1/2 for SIV clearance allows us to estimate the daily production of HIV-1 in a typical patient not on antiretroviral therapy, then there would be ∼10¹²particles produced per day, which is about 100-fold higher than previously thought (6, 18). Thus, importantly, the total number of productively infected CD4⁺ lymphocytes in an infected person or the viral burst size has to be proportionally higher than prior estimates (6, 17, 18).