Long-acting capsid inhibitor protects macaques from repeat SHIV challenges

Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian–human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg−1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans. A single dose of a small-molecule HIV capsid inhibitor provides long-term protection from repeated simian–human immunodeficiency virus challenges in macaques and might serve as a novel strategy for HIV prevention in humans.

The HIV pandemic is a leading cause of morbidity and mortality worldwide 5 . Current strategies for HIV prevention include public health measures as well as vaccine development and improved pre-exposure prophylaxis (PrEP) uptake. Studies conducted by the Centre for the AIDS Programme of Research in South Africa (CAPRISA; trial 004) 6 , Pre-exposure Prophylaxis Initiative (iPrEx) 7 and Partners PrEP 8 have shown that tenofovir-based PrEP can reduce HIV transmission. Recent real-world data confirm a significant population-level reduction in HIV-1 incidence in areas in which PrEP uptake is high 9,10 . However, PrEP strategies reliant on frequent drug administration are limited by adherence, which reduces their real-world impact on HIV transmission [11][12][13] . Long-acting PrEP agents may reduce the barriers associated with daily drug administration, frequent healthcare interactions and the stigma surrounding sexually transmitted infections including HIV 3 . As part of this approach, a long-acting formulation of the integrase strand-transfer inhibitor cabotegravir (CAB-LA), which is injected subcutaneously every 2 months, was shown to reduce HIV transmission in an HIV Prevention Trials Network (HPTN) study (HPTN 083) 14 .
The HIV capsid protein has multiple essential roles in the early and late stages of the viral replication cycle, making it an attractive target for antiretrovirals (ARVs) 15 . Lenacapavir (LEN, formerly GS-6207) is the first clinically validated HIV capsid inhibitor and displays picomolar antiviral activity against both wild-type virus and variants resistant to current ARVs 16 . LEN binds at a highly conserved interface between capsid protein monomers, which causes defects in capsid nuclear import, reduced virion production and aberrant capsid assembly. A long-acting formulation of LEN has been shown to have potent antiviral activity with a maximum 2.3 log 10 decline in HIV-1 RNA after 9 days of monotherapy 17 and the potential for twice-yearly subcutaneous dosing in a phase 1b study 18 . GS-CA1, a structural analogue of LEN, has the same capsid-dependent multistage mechanism of action, similar binding affinity for different forms of HIV capsid (i.e., precursor, monomer, pentamer and hexamer), similar potency against both HIV and simian immunodeficiency virus (SIV), and a similar resistance profile. In addition, it has previously been demonstrated to have high preclinical efficacy in a humanized mouse model of HIV-1 treatment (Extended Data Table 1) 4 . However, long-term prophylactic efficacy of LEN or GS-CA1 has not previously been demonstrated. In this study, we assess the potential of a single dose of long-acting capsid inhibitor to offer protection against repeated challenges with simian-human immunodeficiency virus (SHIV) in rhesus macaques. GS-CA1 was chosen for this analysis because of its predicted higher rate of metabolic clearance in comparison to LEN and the associated accelerated washout phase after dose administration, which enables timely evaluation of the prophylactic efficacy of this compound class over a wide range of exposures.

GS-CA1 inhibits SHIV in macaque cells
GS-CA1 displayed potent in vitro anti-SHIV activity in peripheral blood mononuclear cells (PBMCs) isolated from three individual rhesus macaques of Indian origin (Macaca mulatta), with a mean 50% effective concentration (EC 50 ) of 0.72 nM ( Fig. 1a and Extended Data Table 2). This compound also showed a mean Hill slope value of 3.0 ± 0.7 in high-density antiviral dose-response curves measured against the HIV-1 IIIb strain in MT-4 cells, yielding a calculated 95% effective concentration (EC 95 ) of 1.91 nM when applied to the EC 50 measured in SHIV-infected rhesus PBMCs. In vivo application showed that a large portion of the subcutaneously administered GS-CA1 became bound by plasma proteins, leaving less free drug available for antiviral effects. Competitive equilibrium dialysis was thus used to account for rhesus plasma protein binding to GS-CA1, resulting in a projected 15.8-fold decrease in free GS-CA1 concentration in vivo and yielding a rhesus protein-adjusted EC 95 (paEC 95 ) value of 30.2 nM.

GS-CA1 shows long-acting plasma exposure
Low hepatic clearance is an essential attribute for a long-acting agent. Titration with 3 H-labelled GS-CA1 was necessary to accurately measure the low turnover of GS-CA1 in primary rhesus hepatocytes and showed a predicted rate of hepatic clearance of 0.07 l h −1 kg −1 , or 2.9% of the hepatic extraction. These in vitro data suggest that GS-CA1 has the potential to sustain long-acting plasma exposure in rhesus macaques. To test this hypothesis and to select an appropriate GS-CA1 dose for the rhesus efficacy studies, we performed a pilot pharmacokinetic study with a single subcutaneous administration of a GS-CA1 formulation at two dose levels predicted to cover a broad range of plasma exposures over the projected length of the study. GS-CA1 was administered at 100 mg kg −1 and 300 mg kg −1 to two and three naive rhesus macaques of Indian origin, respectively, and its levels were monitored for 18 weeks. Plasma drug levels peaked in the concentration range of 1-3 µM by day 1 after dose administration, before decreasing to 0.4-1.1 µM by day 7 after dose administration. After this, GS-CA1 levels were maintained in excess of the rhesus paEC 95 value for at least 8 weeks and 14 weeks and in excess of six times the rhesus paEC 95 value for at least 1 week and 8 weeks for the 100 mg kg −1 and 300 mg kg −1 doses, respectively (Fig. 1b). Given that the mean target clinical exposure of LEN for HIV treatment is six times its paEC 95 in humans 18 , GS-CA1 doses of 300 mg kg −1 and 150 mg kg −1 were selected for the repeat SHIV challenge study to assess the reduction in transmission risk across rhesus-equivalent GS-CA1 exposures in excess of, equal to and below this clinically relevant target concentration.

GS-CA1 provides protection from SHIV challenges
We next conducted a study to evaluate the protective efficacy of a single administration of GS-CA1 against repeated, escalating-dose rectal SHIV-SF162P3 challenges in rhesus macaques (Fig. 2a). For this challenge study, 24 rhesus macaques of Indian origin were divided into 3 groups of 8 monkeys each with balanced sex and weight distributions. All animals received a single subcutaneous administration at week 0 in the scapular region. Animals in group 1 received the vehicle control, whereas those in groups 2 and 3 received GS-CA1 doses of 150 mg kg −1 and 300 mg kg −1 , respectively. Consistent with the pilot study, a single administration of GS-CA1 at both 150 mg kg −1 and 300 mg kg −1 achieved long-acting exposure. Specifically, peaks were reached at plasma GS-CA1 concentrations of 3.0 µM and 5.5 µM approximately 24 h and 42 h after dose administration, respectively, and GS-CA1 levels decreased slowly thereafter with a mean half-life of 287-317 h ( Fig. 2b and Extended Data Table 3). The group receiving a dose of 300 mg kg −1 remained above the rhesus paEC 95 and six times above the rhesus paEC 95 for 14-16 weeks and 5-8 weeks, respectively, whereas the group receiving a dose of 150 mg kg −1 remained above these target concentrations for 8-15 weeks and 3-7 weeks, respectively. The variance in GS-CA1 pharmacokinetic parameters across animals was comparable to that observed with 900 mg of LEN in humans 18 , whereas the half-life and length of exposure above the corresponding 6× paEC 95 threshold were lower than expected.
To define the protective efficacy of GS-CA1, all animals received 15 weekly intrarectal SHIV-SF162P3 challenges in a dose-escalation protocol beginning at week 1 following GS-CA1 administration. Infection was assessed by quantifying viral gag RNA levels in the plasma by Article quantitative PCR with reverse transcription (RT-qPCR). To define the drug levels required for protection, we monitored plasma viraemia up to week 24 as GS-CA1 levels declined below therapeutic concentrations. Eight weekly intrarectal challenges at 30 times the median tissue culture infectious dose (TCID 50 ) resulted in infection of five of the eight vehicle-treated animals ( Fig. 2c, Table 1 and Extended Data Fig. 1). Viral challenge dose escalation to 100 TCID 50 for 2 weeks yielded no additional infections, whereas further escalation to 300 TCID 50 resulted in infection of the remaining three control animals by week 15.
In contrast to the vehicle-treated group, the group receiving 300 mg kg −1 GS-CA1 showed no viraemia until week 17 with this escalating-dose challenge protocol, when three of the eight monkeys became SHIV positive (Fig. 2c, Table 1 and Extended Data Fig. 1). The other five monkeys remained aviraemic until the end of the study, which translates to a 97% per-exposure risk reduction with the 300 mg kg −1 dose (P = 0.0001, Cox proportional hazard regression analysis). The group that received a single administration of GS-CA1 at the reduced dose of 150 mg kg −1 also exhibited fewer and delayed infections relative to the vehicle-treated control group. Specifically, no viraemia was detected until week 11, and two of the eight monkeys remained aviraemic until the end of the study, which represents an 87% per-exposure risk reduction (P = 0.0038). The median time to viraemia was 7.5 weeks in the vehicle-treated group and 16 weeks in the group receiving 150 mg kg −1 GS-CA1; this point was not reached in the group receiving 300 mg kg −1 GS-CA1 owing to an insufficient number of infections. The peak viral loads were significantly lower and the viral loads measured 7 weeks after infection showed a trend towards lower levels among the GS-CA1-treated animals as compared with the vehicle-treated control animals. This result could reflect a residual antiviral effect from subtherapeutic inhibitor levels (Extended Data Fig. 2).
We next performed immunological analyses in the GS-CA1-treated rhesus monkeys. First, we assessed the development of humoral immune responses against the SHIV envelope (Env) glycoprotein by enzyme-linked immunosorbent assay (ELISA). We detected binding antibody responses to Env at week 24 in all animals with SHIV viraemia but none in those without SHIV viraemia (Extended Data Fig. 3a). Second, we assessed the development of cellular immune responses against SHIV Gag polyprotein by enzyme-linked immune absorbent spot (ELISPOT) assay. We detected T cell responses to the Gag protein at week 19 in all but one animal with SHIV viraemia and in none of the animals without SHIV viraemia (Extended Data Fig. 3b). These immunologic data suggest that the GS-CA1-treated animals that remained aviraemic during the study period were in fact protected from SHIV challenge.
Finally, we performed additional studies to confirm that our plasma viraemia measurements provided early detection of initial infection in the setting of GS-CA1 prophylaxis.
First, we performed intact proviral DNA analysis (IPDA) to detect integrated SHIV in a subset of infected animals treated with vehicle control or GS-CA1 with available PBMCs. Intact SHIV proviruses became detectable at the same time points at which plasma viraemia was detected in all cases except for a single GS-CA1-treated animal (K394) that showed very low-level intact provirus 1 week before detection of viraemia (Extended Data Table 4). Second, we determined the Env ELISA seroconversion time points for all infected animals and observed that viraemia preceded seroconversion in all cases (Extended Data Table 5). The median time to seroconversion from the onset of viraemia was 2.5 weeks (range, 1 to 13 weeks) in vehicle-treated control animals and 4 weeks (range, 1 to 5 weeks) in GS-CA1-treated animals, although the difference between the groups was not significant (P = 0.80, Mann-Whitney U test).

Protective levels of GS-CA1 in macaques
We next investigated the relationship between GS-CA1 plasma concentrations and protection against SHIV challenge. To facilitate exposure comparisons with other antivirals including LEN, we converted the concentrations for GS-CA1 to multiples of its rhesus paEC 95 . To estimate the protective levels for GS-CA1, we focused on six animals that developed viraemia in the group receiving a dose of 150 mg kg −1 and three animals that developed viraemia in the group receiving a dose of 300 mg kg −1 . Assuming a 2-week delay between rectal SHIV infection and detectable peripheral blood viraemia, we averaged the GS-CA1 exposure values 2 weeks before the first detectable viraemia among the infected animals. We estimated that mucosal infection occurred in the presence of 31.4 nM GS-CA1 on average, which is equal to the concentration of rhesus paEC 95 at a multiple of 1.0 (range, 0.4-1.6; Fig. 3a, b). Therefore, we estimated that, overall, all animals were fully protected in this model when the GS-CA1 plasma concentrations were more than twice the rhesus paEC 95 value for GS-CA1.

Plasma virus resistance analysis
To evaluate potential development of resistance against GS-CA1, particularly during the washout phase, we conducted longitudinal population-level sequence analysis of the SHIV gag region encoding capsid in rhesus plasma (Extended Data Fig. 4). As expected, plasma virus obtained from placebo-treated control animals encoded only wild-type capsid protein. Of the plasma virus samples analysed from the nine viraemic animals dosed with GS-CA1, 34 of 35 (97%) produced high-quality sequence reads, with wild-type capsid detected in all nine animals at the end of the study. Animal K342 in the low-dose (150 mg kg −1 GS-CA1) group showed transient prevalence of a capsid variant with substitution to alanine at Val11 (V11A; the numbering used here follows the HIV-1 HXB2 reference sequence) between study weeks 13 and 21. This V11A substitution, which is located far outside the GS-CA1-binding site in the capsid, disappeared by week 22 in this animal and is not associated with GS-CA1 resistance 4 . Thus, no study animal showed emergence of variants associated with GS-CA1 resistance for the duration of this 24-week efficacy study.

Discussion
Long-acting PrEP regimens might overcome some of the current barriers to PrEP implementation, including the need for daily administration and frequent healthcare interaction. The HPTN 083 study recently demonstrated that long-acting ARV monotherapy agents such as CAB-LA can effectively reduce the rate of HIV-1 acquisition 14 .
In the present study, we investigated whether a single administration of the capsid inhibitor GS-CA1 could provide long-term protection from repeated SHIV challenges in rhesus macaques. GS-CA1 provided significant protection from rectal infection after 15 repeat challenges, with complete protection achieved when GS-CA1 levels were more than twice the rhesus paEC 95 .
Given the similar structural, mechanistic and long-acting properties of GS-CA1 and LEN, the preclinical GS-CA1 data in this study may inform the clinical development of LEN for PrEP. Our data show protection against rectal SHIV acquisition in macaques at plasma GS-CA1 exposures more than twice the rhesus paEC 95 , which suggests that the current clinical formulation of LEN, conferring a mean exposure of more than six times human paEC 95 for at least 6 months after a single subcutaneous dose, might be sufficient for PrEP in humans. Nonetheless, the exposure threshold for prophylaxis against HIV acquisition with LEN in humans cannot be directly inferred from this escalating-dose preclinical study and will need to be defined in adequately powered clinical studies. Moreover, animal studies may underestimate exposures required for protection, as evident from the 4 of 12 incident infections among 2,243 analysed participants in the HPTN 083 study that occurred even though the target CAB-LA exposures were predicted preclinically to be efficacious 19,20 . In future work, it will be important to evaluate the efficacy of long-acting capsid inhibitors by other routes of transmission (e.g., vaginal) to establish broader relevance to the populations at risk. Furthermore, a study incorporating mucosal biopsies could establish the pharmacokinetic relationship between plasma and tissue levels of GS-CA1 at the sites of infection. The emergence of resistance mutations may be an important consideration during the implementation of any single-agent long-acting PrEP strategy. In the phase 3 HPTN 083 trial, 16 HIV seroconversions occurred among participants randomized to CAB-LA, including 4 baseline infections 20 . Genotyping in 14 of these 16 cases revealed integrase mutations among 5 participants, although no resistance was observed among infections presumed to have occurred during the pharmacokinetic tail. Our preclinical study revealed no resistance mutations in the capsid protein among nine rhesus macaques that were treated with GS-CA1 and became infected. However, the low-level resistance mutation Q67H in capsid (conferring a sixfold decrease in susceptibility to LEN 16 ) was detected in 2 of 29 participants randomized to long-acting LEN monotherapy in a recent phase 1b study 21 . This mutation was detected 9 days after dose administration in at least one study participant in two of the LEN treatment arms among the five total receiving the lowest doses (20 mg and 50 mg) when the average LEN plasma concentrations measured 0.7 and 1.1 times the human paEC 95 , respectively; both concentrations are considered to be subtherapeutic for HIV-1 treatment. These data suggest that large clinical studies will likely be necessary to fully characterize the potential emergence of resistance mutations among long-acting PrEP recipients.
In summary, our data demonstrate that a single subcutaneous administration of the capsid inhibitor GS-CA1 provides long-term protection against SHIV infection in rhesus macaques. Together with recent studies showing the potency and pharmacokinetics of LEN in people living with HIV, these data suggest that long-acting capsid inhibitors might offer an important and novel strategy for HIV prevention. A phase 3 clinical study to assess the safety and effectiveness of LEN for HIV PrEP has therefore been initiated (NCT04925752).

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-021-04279-4. The bottom dotted line represents the assay LOD (1 nM). The bottom and top dashed lines represent one and two times the rhesus paEC 95 , respectively. b, Same data as in a for rhesus macaques treated with GS-CA1 at a dose of 300 mg kg −1 . No signal above the assay LOD was observed among the eight placebo-treated control animals throughout the study.

Drug and formulation
GS-CA1 and a generic internal small-molecule standard used for liquid chromatography coupled with mass spectrometry (LC-MS) experiments were both synthesized at Gilead Sciences and were subjected to standard quality control analysis. For the animal dosing studies, GS-CA1 was dissolved in vehicle (58.03% PEG 300, 27.1% water, 6.78% ethanol, 6.61% poloxamer 188, 1.48% sodium hydroxide) at 300 mg ml −1 , producing a clear, yellow-orange solution. The solution was stored at ambient temperature protected from light until dosing.

Metabolic stability of [ 3 H]GS-CA1 in primary rhesus hepatocytes
A 500-µl suspension of human hepatocytes (1 × 10 6 cells per ml) and 0.25 µM [ 3 H]GS-CA1 was prepared in Krebs-Henseleit buffer (KHB) medium and was incubated in a humidified incubator at 37 °C with 5% CO 2 in duplicate wells of a 24-well plate. Propranolol (1 µM final), a compound known to be efficiently metabolized by hepatocytes by oxidation and conjugation, was used as a positive control. A cell-free control was incubated in parallel as a negative control. Aliquots (100 µl) were removed after 0 h, 1 h, 3 h and 6 h and were then mixed with 200 µl quenching solution, placed on a shaker for 10 min and centrifuged at 3,000g for 60 min. The supernatant was transferred to a new plate, diluted with 100 µl water and placed on a shaker for 10 min. Quantification of [ 3 H]GS-CA1 and its metabolites was performed by radio flow chromatography using a PerkinElmer Radiomatic 625TR flow scintillation analyser with a 500-µl flow cell coupled to a Dionex Ultimate 3000 high-performance liquid chromatography (HPLC) system. PerkinElmer Ultima-Flo was used as the scintillation cocktail, which was mixed with the HPLC effluent at a ratio of 1:1. Sample (100 µl) was injected using a Leap Technologies CTC PAL autosampler. Separation was achieved on a Phenomenex Synergi Fusion-RP 80-Å pore size, 4-µm particle size, 150 × 4.6-mm column maintained at 32 °C. Mobile phase A consisted of 95% water, 5% acetonitrile and 0.1% trifluoroacetic acid (TFA). Mobile phase B consisted of 95% acetonitrile, 5% water and 0.1% TFA. Elution was achieved at a flow rate of 1 ml min −1 by linear gradients: the initial condition was 2% B at 0 min, which was increased to 75% B over 45 min, held for 4 min at 75% B and then returned to the initial conditions. The column was allowed to re-equilibrate for 12 min between injections. Quantification was based on the radiochromatographic peak area using Dionex (Thermo Scientific) Chromeleon 6.8 software.

Anti-SHIV antiviral assay in rhesus PBMCs
PHA/IL-2-stimulated rhesus PBMCs were infected in bulk culture with SHIV-SF162P3 at a concentration of 130 pg of p27 equivalent per million PBMCs. The cells were maintained in suspension by gently rocking the cultures mixed with virus inoculum for 3 h at 37 °C. The cells were then pelleted by centrifugation at 500g for 5 min, washed twice with complete RPMI to remove any unadsorbed virus and seeded into 96-well plates at a cell density of 2 × 10 5 cells per well in 100 µl. Eight-point threefold serial dilutions of GS-CA1 were made in complete RPMI containing 50 U ml −1 IL-2 and were added in triplicate to wells containing cells (100 µl per well). The cultures were incubated in a 5% CO 2 incubator at 37 °C for 7 days. Cell-free supernatants derived from the PBMC cultures were harvested 7 days after infection, the amount of SHIV present was quantified using an SIV p27 antigen-capture ELISA assay (5436, Advanced Bioscience Laboratories) performed according to the manufacturer's protocol, and the data were acquired using SoftMax Pro 6.3.1 software (Molecular Devices). The mean EC 50 value for GS-CA1, which was determined from a total of seven assays performed in triplicate, was calculated from the doseresponse curves using XLfit 5.5.0.5 software (IDBS) and was expressed graphically using GraphPad Prism 8.1.2 software. The Hill coefficient (n) for GS-CA1 was measured from the slope of the dose-response curves (n = 3.03 ± 0.69) and was used to derive the EC 95 value using the following equation: EC 95 = EC 50 × (95/5) 1/n .

Equilibrium dialysis shift assay
Rhesus plasma protein binding to GS-CA1 was determined by competitive equilibrium dialysis. Rhesus plasma (10%) was spiked with GS-CA1 (2 µM), and this mixture and blank RPMI cell culture medium containing 10% FBS (CCM) were placed into opposite sides of assembled dialysis cells; afterwards, incubations were performed in triplicate. After a 24-h equilibration period at 37 °C, samples were quenched with 4 volumes of 90% (vol/vol) acetonitrile and 10% (vol/vol) methanol containing internal standard. Then, they were quantified using the AB Sciex API 4000 LC-MS/MS system (GenTech Scientific) with electrospray ionization in positive mode and multiple-reaction monitoring and were analysed using Analyst 1.6.1 software. The fold change value in 100% rhesus plasma was then calculated using the plasma/CCM ratio after correcting for the sample dilution factor and the percentage of free fraction in the matrix. This shift value was multiplied by the calculated anti-SHIV EC 95 value for GS-CA1 to derive the corresponding rhesus plasma paEC 95 .

Animals, drug administration and viral stocks
Studies involving the evaluation of GS-CA1 pharmacokinetics in naive male rhesus macaques of Indian origin were conducted at Covance Laboratories in a Laboratory Animal Care-accredited facility and were performed in strict compliance with all relevant ethical regulations. All study protocols were reviewed and approved by the Covance Laboratories Institutional Animal Care and Use Committee (IACUC). On day 0 of the study, rhesus monkeys were administered GS-CA1 dosed at 100 mg kg −1 (n = 2) or 300 mg kg −1 (n = 3) in the scapular region by subcutaneous injection using a syringe equipped with a 22-gauge needle. The GS-CA1 was prepared in a stock solution of 300 mg ml −1 ; a maximum of 2 ml of the solution was injected into a single subcutaneous site. Whole blood was collected from each animal at designated time points, processed into plasma and then stored frozen at −80 °C for bioanalysis of GS-CA1 levels.
For the challenge study, 24 outbred rhesus macaques of Indian origin were assigned to the three study groups with even sex and weight distributions. All animals were housed at Alpha Genesis (Yemassee, SC), and all procedures were conducted in compliance with all relevant local, state and federal regulations and were approved by the Alpha Genesis IACUC. On week 0, the three groups were administered vehicle control, GS-CA1 dosed at 300 mg kg −1 or GS-CA1 dosed at 150 mg kg −1 in the scapular region by subcutaneous injection using a syringe equipped with a 22-gauge needle. The GS-CA1 was prepared in a stock solution of 300 mg ml −1 ; a maximum of 2 ml of solution was injected into a single subcutaneous site. The injection sites were monitored daily by veterinary staff for 3 days. Beginning in week 1, animals in all three groups were challenged by the intrarectal route with 1 ml of RPMI containing the specified dilution of a 3.62 × 10 3 TCID 50 SHIV-SF162P3 stock. At each respective time point, the challenges were performed on the same day, using the same virus stock and inoculation method across the three groups. Whole blood was collected and processed