Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences

Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.


Author Summary
In this study, we generated and evaluated novel simian-human immunodeficiency virus (SHIV) challenge stocks expressing env sequences from HIV-1 strains from Thailand. The lack of such SHIV challenge stocks has been a major unmet need for the field and has hindered progress to evaluate strategies aimed at preventing HIV-1 infection for this region of the world. The challenge stocks described in this manuscript should prove useful for

Introduction
The generation and evaluation of simian-human immunodeficiency virus (SHIV) challenge stocks are important for preclinical testing of vaccines, microbicides, monoclonal antibodies, and other interventions aimed at preventing HIV-1 infection. SHIVs are typically constructed with an SIV backbone and HIV-1 env, tat, rev, and vpu [1][2][3]. The predominant HIV-1 subtype in the United States and Europe is subtype B [4]. In contrast, circulating recombinant form 01_AE (CRF01_AE) viruses are predominant in Southeast Asia, including Thailand [5]. The RV144 study in Thailand has been the only partially successful vaccine clinical efficacy trial to date [6]. A SHIV challenge stock expressing CRF01_AE env would therefore be critical for testing concepts related to the protection observed in RV144. Studies involving correlates of risk and molecular sieve analyses have raised important hypotheses for how the RV144 vaccine might have worked [7,8], but mechanistic studies have been limited by the lack of an available CRF01_AE SHIV challenge stock. We therefore sought to produce mucosally transmissible SHIVs containing early CRF01_AE env sequences.
HIV-1 transmission across a mucosal barrier is a relatively inefficient process and typically results in propagation of only a single transmitted/founder (T/F) virus [9]. A single T/F virus has been deemed responsible for propagation of HIV-1 in 60-80% of mucosal infections [10,11]. SHIVs encoding early or acute HIV-1 envs might therefore optimally model acute infection and thus be relevant for testing vaccines and other interventions aimed at preventing mucosal infection.
Three challenge stocks, SHIV-AE6, SHIV-AE6RM, and SHIV-AE16, were generated in human or rhesus PBMC without in vivo passaging. These viruses displayed CCR5 tropism and a neutralization phenotype consistent with tier 2 primary isolates. They established mucosal infection in rhesus monkeys, exhibited robust acute viral replication, and maintained chronic viremia in a subset of animals challenged by the intrarectal (i.r.) route. These novel CRF01_AE SHIV challenge stocks should facilitate preclinical mechanistic and efficacy studies involving interventions aimed at preventing infection with CRF01_AE HIV-1.

Materials and Methods Animals
Indian origin rhesus monkeys (Macaca mulatta) were used in the current study. Eight Mamu-A Ã 01-negative adult male and female animals were housed at Bioqual (Rockville, MD), and 16 Mamu-A Ã 01-positive adult male and female animals were housed at the New England Primate Research Center (Southborough, MA).

Ethics
All animals were maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animals with approvals from the relevant Institutional Animal Care and Use Committees (IACUC). The Harvard Medical School Standing Committee on Animals (#03970) and the Bioqual IACUC (14-3131-62P) approved these studies. The animal protocols adhered to NIH standards set forth in the "Policy on Humane Care of Vertebrate Animals Used in Testing, Research, and Training" and the "Guidelines for the Care and Use of Laboratory Animals" (DHHS publication #NIH 85-23). Animal welfare was maintained by the NEPRC and Bioqual animal management programs, which are accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) and meet all applicable federal and institutional standards for standard housing, standard monkey diet, water ad libitum, social enrichments, and steps intended to minimize suffering, such as the use of anesthesia for all procedures and analgesia for invasive procedures such as biopsies. Animals were euthanized by ketamine followed by sodium pentobarbital, which is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Cells
Human and rhesus monkey peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient purification, followed by stimulation with concanavalin A (Con A; 6.25 μg/ml) and human interleukin-2 (20 units/ml IL-2; AIDS Research and Reference Reagent Program) overnight. Human PBMCs were purchased commercially (Research Blood Components). Cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 20 units/ml of IL-2. TZM-bl cells (JC53-bl clone 13 cells) are derived from a HeLa cell line (JC.53) that stably expressed CD4 and HIV coreceptors, as well as luciferase and β-galactosidase under the control of the HIV-1 long terminal repeat. TZM-bl cells and GHOST cells expressed CD4 alone or with different chemokine receptors and were obtained from the NIH AIDS Research and Reference Reagent Program.
Lymph node and colorectal biopsies were collected at week 12 and incubated in RPMI 1640 containing 10% FBS supplemented with 200 U/ml of type IV collagenase (Sigma-Aldrich) and 30 U/ml of DNase I (Sigma-Aldrich) at 37°C with rocking for 30 min. The digested biopsy tissues were then homogenized, and the solution was strained with a 70-μm (pore size) cell strainer (BD Falcon). The cell suspension was then centrifuged at 695 × g for 25 min on a 35% Percoll (Sigma-Aldrich) gradient. Pellets containing lymphocytes were collected and processed for assays. Proviral DNA assays were conducted using the QIAcube HT (Qiagen) performed as previously described [12].

Construction of SHIV molecular clones
Twenty-nine HIV-1 CRF01_AE early env sequences (AE1 -AE29) derived from acutely HIV-1-infected participants in either the RV217 Early Capture HIV Cohort Study in East Africa and Thailand (Merlin Robb, AIDS Vaccine 2012) or the RV254/SEARCH study in Thailand [13] were used to generate SHIV molecular clones ( Table 1). The env sequences were synthesized by GeneArt (GeneArt, Invitrogen) and inserted into the KB9-AC plasmid as described [14]. Plasmids were sequenced and transfected into 293T cells using LipoD293 (SignaGen Laboratories). Cell culture supernatants were collected after 48 h and clarified through a 0.45 μm filter.

Generation of large-scale SHIV stocks
Eight infectious molecular clones (AE3, AE4, AE6, AE15, AE16, AE17, AE21, AE22) that replicated to high levels in human PBMC were selected to generate large-scale challenge stocks. Each virus stock was produced from PBMC isolated from 120 ml of human or rhesus monkey whole blood. Cell culture supernatants harvested from transiently transfected 293T cells were used to inoculate ConA-stimulated PBMC in the presence of 20 U/ml human IL-2 (AIDS Research and Reference Reagent Program). SHIV-AE6RM was prepared by propagation of the human SHIV-AE6 stock in rhesus monkey PBMC. The media was replaced and collected in 3-day increments over the course of 12-15 days. Virus was quantified by SIV p27 enzyme-linked immunosorbent assay (ELISA; Zeptometrix), and the 50% tissue culture infectious does (TCID 50 ) was determined in TZM-bl cells.

Coreceptor studies
The inhibitors TAK-779 and AMD-3100 were obtained from the AIDS Research and Reference Reagent Program and were diluted per manufacturer instructions. TZM-bl cells were seeded at 3 X 10 4 cells/well in flat-bottom plates. The cells were incubated for 1 h in 10-fold dilutions of TAK-779 ranging from 5 to 500,000 ng/ml or 10-fold dilutions of AMD-3100 ranging from 5 to 50,000 ng/ml or mock infected with supernatant from cultured PBMC without viral infection. After 1 h, 100 TCID 50 of the SHIV stocks were added to the wells, and 48 h later the cells were lysed and analyzed by Steady-Glo (Promega) luciferase assay with luminescence measured on a Victor 3 luminometer (Perkin-Elmer). GHOST cells expressing coreceptors CXCR4, CCR5, and CCR5/CXCR4 and the parental strain were obtained from the AIDS Research and Reference Reagent Program. These were maintained in Dulbecco modified Eagle medium with 10% fetal calf serum and penicillin-streptomycin and supplemented with 500 μg/ ml G418, 100 μg/ml hygromycin, and 1 μg/ml puromycin. Puromycin was excluded for culture of the parental cell line. Cells were plated at 10 4 cells per ml in 96-well round-bottom wells in medium supplemented with 20 μg/ml Polybrene. One hundred TCID 50 of each virus stock was added to each well, followed by incubation overnight. After being washed with phosphate-buffered saline (PBS) and replacement with fresh culture medium, cell culture supernatants were harvested 4 days after incubation and assessed for SIV p27 ELISA (Zeptometrix).

Neutralization assay
Neutralizing antibody titers against SHIV isolates were determined using a luciferase-based assay in TZM.bl cells as previously described [15,16]. This assay measures a decrease in luciferase reporter gene expression following single-round viral infection of TZM.bl cells. Briefly, 3-fold serial dilutions mAb reagents were performed in duplicate and incubated with primary SHIV isolates for 1 hour at 37°C. TZM.bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/ml, and assay plates incubated for 48 hours at 37°C, 5% CO 2 . Luciferase reporter gene expression was measured using Bright-Glo luciferase reagent (Promega) and a Victor 3 luminometer (Perkin Elmer

Intrarectal inoculation of CRF01_AE SHIVs
Animals received a single intrarectal (i.r.) inoculation with 1 ml of undiluted SHIV-AE6, SHI-V-AE6RM, or SHIVAE-16 stocks. For the SHIV-AE16 in vivo titration, six i.r. challenges with 1 ml of 1:1 or 1:10 diluted virus stock were performed with two week intervals from week 0 to week 10. All animals were monitored for viral loads and CD4 + T cell counts.

CD4+ T cell counts
EDTA-anticoagulated whole blood, PBMC, and lymph node mononuclear cells (LNMC) were stained using anti-CD3-APC, anti-CD4-PE, and anti-CD8-FITC MAbs and analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, Ca). Cell counts were determined using the BD True Count tubes according to the manufacturer's instructions (BD Biosciences).

Measurement of plasma viral RNA levels
Viral RNA was isolated from cell-free plasma using the QIAamp viral RNA isolation kit (Qiagen) and quantified as previously described [4,14,17]. Quantitative PCR and viral load assays were performed as previously described [6,12,14]. All viral loads were run at the end of each study to maintain objectivity and ensure that all animals were handled identically.
Single-genome amplification (SGA) and sequencing Viral RNA was extracted using a QIAamp viral RNA minikit (Qiagen). RNA was eluted and immediately subjected to cDNA synthesis. Reverse transcription of RNA to single-stranded cDNA was performed using SuperScript III reverse transcriptase (Invitrogen) as described [9,14].

Nucleotide sequence accession numbers
The env sequences of the amplicons were deposited in GenBank under accession numbers KT581024-KT581117 as detailed in Table 1.

Generation of CRF01_AE SHIV stocks
Twenty-nine CRF01_AE early HIV-1 env sequences from acutely HIV-1 infected individuals from Thailand (Table 1) were cloned into the KB9-AC backbone, essentially as described [14]. Plasmids were used for transfections in 293T cells to generate virus, and virus infectivity titers were determined (Table 2). Culture supernatants were used to assess viral growth in human and rhesus PBMC. As is the case with other SHIVs [10,11,14], these viruses replicated well in human PBMC but inefficiently in rhesus PBMC (Table 2). Interestingly, SHIV-AE22 was identified as the most infectious virus in this set of SHIVs, but this virus was confirmed to be subtype B rather than CRF01_AE (Table 1). We selected eight viruses (AE3, AE4, AE6, AE15, AE16, AE17, AE21, AE22) that replicated to high levels in human PBMC for the generation of large-scale challenge stocks by inoculation of the 293T transfection-derived supernatants into primary human PBMC. The large-scale challenge stocks ranged in infectivity from 6.4 X 10 2 to 7.0 X 10 8 TCID 50 /ml, and viral loads ranged from 3.2 X 10 8 to 2.1 X 10 9 RNA copies/ml ( Table 3). The challenge stocks were then assessed for their ability to replicate in rhesus PBMC. Only two stocks, SHIV-AE6 and SHI-V-AE16, replicated at detectable levels in rhesus PBMC, and SHIV-AE6 exhibited the highest SIV p27 level as determined by ELISA (Table 3). This stock was therefore chosen to produce a large-scale challenge stock in primary rhesus PBMC, which was designated SHIV-AE6RM. The rhesus-derived SHIV-AE6RM stock replicated 114-fold more efficiently than did the human-derived SHIV-AE6 stock in rhesus PBMC (Table 3). We next performed sequencing by single genome amplification (SGA) [11] to analyze the env sequences in the SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 challenge stocks as compared to the original HIV-1 env sequences from the RV217 and the RV254/SEARCH studies in Thailand. We evaluated 31 SGA-derived sequences obtained for each challenge stock (Fig 1). The SHIV-AE6 stock had one to five random amino acid differences in 10 amplicons (Fig 1A), while the SHIV-AE16 stock had one to three intermittent amino acid differences in 16 amplicons (Fig 1B). The rest of the amplicons for both stocks was identical to the parent sequence. For the rhesus PBMC-derived SHIV-AE6RM challenge stock, 30/31 amplicons contained at least one amino acid difference as compared to the original HIV-1 env sequence, and 20/31 amplicons contained a consistent D168H mutation in the V2 loop ( Fig 1C). These data show that these SHIV stocks expressed envs that were >99% identical to the parental HIV-1 env sequences and only contained rare and sporadic amino acid changes, except for the D168H mutation in SHIV-AE6RM.

Coreceptor usage analysis of the SHIV-AE stocks
We examined coreceptor usage of the early CRF01_AE SHIV stocks using TZM-bl cells in the presence of the coreceptor inhibitors TAK779 (CCR5) and AMD3100 (CXCR4). All of the SHIV stocks were potently inhibited by the CCR5 TAK-779 inhibitor (Fig 2A) but were not inhibited by the CXCR4 inhibitor AMD3100 (Fig 2B). We then tested the replication ability of SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 stocks in GHOST cells expressing alternate coreceptors and then measured the SIV p27 production of each SHIV stock in the culture supernatant (Fig 2C). The assay verified that the SHIV stocks replicated in the R5 or dual X4/R5

Antigenic characterization of the SHIV-AE stocks
We next examined the neutralization sensitivity of each SHIV stock in TZM-bl cells with a panel of broadly reactive mAbs (Table 4) targeting the major neutralization epitopes. The SHIV stocks all displayed neutralization profiles consistent with tier 2 neutralization resistant phenotypes, although SHIV-AE6RM showed sensitivity to soluble CD4.

Mucosal infectivity of SHIV-AE6, SHIV-AE6RM, SHIV-AE16 stocks in rhesus monkeys
We next assessed the capacity of SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 stocks that displayed detectable levels of replication in rhesus PBMC for their ability to infect rhesus monkeys in vivo by the i.r. route. Twelve adult rhesus monkeys were challenged with a single i.r. inoculation of 1 ml of undiluted virus for each stock (n = 4/group). For the SHIV-AE6 stock, 3/4 animals exhibited peak viral loads ranging from 6.9 to 7.3 log RNA copies/ml at 2 to 3 weeks post inoculation, but minimal CD4 + T cell decline ranging from 10 to 14% during acute infection (Fig 3A), and 2/4 animals were still viremic at week 10. All 4/4 animals challenged with the SHIV-AE6RM stock became infected, and 2/4 animals were viremic at week 10 ( Fig 3B). For the SHIV-AE16 challenge, 2/4 animals displayed robust peak viral loads up to 7.2 log RNA copies/ml and detectable viremia at week 10, while the other two animals had low or undetectable levels of viremia (Fig 3C).

Intrarectal titration of the SHIV-AE16 stock in rhesus monkeys
SHIV-AE16 contained V2 sequences that match target epitopes of V2-specific mAbs isolated from the RV144 study [14,18]. Thus, SHIV-AE16 was chosen for further study. We conducted an in vivo titration experiment to determine the infectivity of SHIV-AE16 in a larger group of animals, as well as its infectivity following repetitive lower dose inoculations. Twelve adult rhesus monkeys received six repetitive i.r. challenges with either a 1:1 dilution (n = 6) or a 1:10  dilution (n = 6) of the SHIV-AE16 stock. 6/6 animals that received the 1:1 dilution were productively infected after the first challenge. These data suggest that this dose may be useful for single, high-dose challenge studies. Peak viral loads ranged between 6.1 to 7.3 log RNA copies/ ml by week 2-6 and resulted in modest 6-14% declines in CD4 + T cell levels during acute Production of CRF01_AE SHIV Challenge Stocks infection ( Fig 4A). Viral loads declined but were still detectable in 6/6 animals at week 10 and in 3/6 animals by week 20. In the 1:10 dilution group, 3/6 animals became infected after the first challenge, while the remaining three animals became infected after the second or third challenge (Fig 4B). Animals established peak viral loads ranging from 5.8 to 7.4 log RNA copies/ml, and viral loads were detectable in 3/6 animals by week 20. These data suggest that the 1:10 dilution may be useful for repetitive, low-dose challenge studies. In addition to viral RNA, we also confirmed the presence of viral DNA in peripheral blood mononuclear cells (PBMC), lymph node mononuclear cells (LNMC), and colorectal cells from these animals at week 12 (Fig 5), indicating viral infection in tissues. Taken together, these data show that the 1:1 and 1:10 dilutions of SHIV-AE16 stock led to productive infection in rhesus monkeys.

Discussion
The development of CRF01_AE SHIV challenge stocks has to date remained elusive. This has been an unmet need for the HIV-1 research field, as such viruses are critical for preclinical testing of vaccines, antibodies, and other interventions aimed at preventing CRF01_AE HIV-1 infection. In this manuscript, we describe the generation of mucosally transmissible CRF01_AE SHIV challenge stocks. These challenge stocks will likely prove useful for preclinical testing of vaccines and mAbs related to the RV144 vaccine study, which showed a modest level of protection in Thailand [6].
Our goal was to generate mucosally transmissible CRF01_AE SHIV challenge stocks expressing early HIV-1 env CRF01_AE sequences. SHIV-AE6, SHIV-AE6RM, and SHI-V-AE16 encoded env sequences that closely matched the original sequences (>99%) from  (Table 5), displayed robust acute viral replication, modest CD4 + T cell declines, and chronic viremia in 17/24 (71%) animals at week 10 and in 6/12 (50%) animals at week 20 (Figs 3 and 4). Previous research has shown that in vivo passaged SHIVs accumulate mutations in HIV-1 env that subsequently increase the pathogenicity of passaged viruses [14,19]. In an effort to prevent mutations and divergence from the parental HIV-1 env sequences, we generated our challenge stocks without in vivo passaging. Despite these attempts, SHIV-AE6RM developed a single D168H mutation in the V2 loop in 20/31 amplicons (Fig 1). Mutations in the V2 loop are common in the generation of SHIVs [3], and other studies have suggested that laboratory adapted HIV-1 strains acquire positively charged amino acid mutations in variable loops that confer increased sensitivity to sCD4 (Table 4) [20]. Further studies will be needed to determine if the D168H mutation is critical for conferring adaptation in rhesus PBMC.
It is desirable for a SHIV challenge stock to be titrated for both single, high-dose challenges as well as repetitive, low-dose challenges. Vaccine studies in rhesus monkeys are often assessed by repetitive, low-dose challenges, whereas passive protection studies with monoclonal antibodies typically utilize single, high-dose challenges [21]. To develop a CRF01_AE SHIV stock that would be broadly useful, we titrated the SHIV-AE16 challenge stock in vivo (Fig 4). These data suggest the potential utility of SHIV-AE16 at 1:1 dilution for single, high-dose challenges and at 1:10 dilution for repetitive, low-dose challenges. In particular, this CRF01_AE SHIV challenge stock may prove useful for preclinical evaluations of vaccine regimens or V2-specific mAbs [7] related to the RV144 study to evaluate proposed mechanistic immune correlates of protection.
The infectivity and pathogenicity of our CRF01_AE SHIVs appear lower than our previously published clade B and C SHIVs [14,22]. These CRF01_AE SHIV stocks will therefore likely prove useful for challenge studies in which protection from acquisition of infection is the endpoint, but for studies of chronic virus pathogenesis, in vivo adapted virus stocks will likely be required. We note that there was higher infectivity of the SHIV-AE16 challenge stock at 1:1 dilution (6/6 animals; Fig 4A) as compared with undiluted virus (2/4 animals; Fig 3C), but the implications of this observation remain unclear given the small numbers of animals utilized in the initial study. In summary, we report the generation of novel CRF01_AE SHIV challenge stocks. These viruses did not require in vivo passaging and were titrated for use in rhesus monkeys. These CRF01_AE SHIV challenge stocks should prove useful to test vaccines, antibodies, and other interventions targeted to CRF01_AE HIV-1.  Table 5. Summary of in vivo infectivity of SHIVs -AE6, -AE6RM, and -AE16 stocks in rhesus monkeys.