CXCR4-tropic HIV-1 infection in an immunocompetent monkey model

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Current animal models of HIV-1 are either immunocompromised or rely on proxy viruses instead of HIV-1. Here, we establish an immunocompetent animal model for CXCR4-tropic HIV-1 in owl monkeys (Aotus nancymaae). Through analysis of 191 owl monkeys, genetic characterization and functional testing demonstrate that CD4 and Tetherin in this species support HIV-1 replication. Although owl monkeys do carry restrictive TRIMCyp and APOBEC3G alleles, small changes to the HIV-1 genome allow the virus to overcome these barriers. The resulting virus remains 93% wildtype HIV-1 in sequence. Fully immunocompetent owl monkeys can be infected with this virus, recapitulating key aspects of HIV-1 infection in humans: an initial surge of virus replication, subsequent establishment of a durable set point viremia, seroconversion, and the formation of a viral reservoir. The owl monkey model broadens the experimental options for HIV-1 research, and future studies will explore its utility for CCR5-tropic virus strains.

Introduction
It was determined early in the HIV/AIDS pandemic that HIV-1 does not infect any mammal other than humans and chimpanzees. However, we and others have shown that only certain chimpanzee individuals express alleles of the main HIV-1 entry receptor, CD4, that are functional for HIV-1 entry 1,2. More importantly, chimpanzees are endangered apes and are thus not a tractable research model 3. Instead of chimpanzees, all current animal models of HIV-1 infection are either immunocompromised or rely on proxy viruses instead of HIV-1. Immunocompromised models for HIV-1 include mice and pig-tailed macaques. In mouse models, mice must be genetically immunocompromised 4. In the pig-tailed macaque model, macaques are administered a CD8\alpha antibody to deplete CD8 T cells and NK cells 5, and animals produce antibodies to HIV-1 only under some conditions 6. A fundamental problem is that the development of HIV-1 cures and vaccines will require immunocompetent animal models. For four decades, the only immunocompetent model available has been macaques infected with proxy viruses, either simian immunodeficiency virus of macaque (SIVmac) or SIVmac/HIV (SHIV) hybrid viruses 7–9. Although SIVmac mimics many aspects of HIV-1 infection and immune control, it differs from HIV-1 in its gene products and is more closely related to the non-pandemic virus HIV-2 10. SHIVs are more similar in sequence to HIV-1 in that they have had incorporated an HIV-1 Envelope (Env), but still only about 30% of the SHIV genome derives from HIV-1. To address this fundamental problem, we undertook a first-principles approach to developing an immunocompetent animal model for HIV-1.

Results
Rationale for the current study

Zoonosis is the infection of humans with the viruses of animals 11. The emergence of HIV-1 into the human population was initiated by the transmission of primate lentiviruses (simian immunodeficiency viruses) first from monkeys to chimpanzees, and then from chimpanzees to humans 12. Work over decades has demonstrated that restriction factors, and the receptor CD4, are powerful barriers in the host-switching of primate lentiviruses 13,14. The premise of the present study is that the development of model organisms in which to study HIV-1 could simply be considered the reverse of zoonosis, with the same host factors dictating the process (Fig. 1A).

To identify primate research species with minimal genetic barriers to HIV-1, and because monkeys are not isogenic, we obtained blood draws from over 900 monkeys representing 14 research species (Fig. 1B). From these, we made cDNA libraries and sequenced transcripts of genes encoding CD4 15 and three powerful restriction factors of HIV-1: TRIM5\alpha 16, APOBEC3G 17, and Tetherin 18,19 (Fig. 1C). We determined the two alleles of each gene for each individual, made molecular clones of unique alleles identified, and tested them in vitro for the ability to support entry of HIV-1 (CD4) or restrict HIV-1 (restriction factors). These investigations took years and led us to the Nancy Ma’s owl monkey (Aotus nancymaae) as the focus of the present study. Here, we describe the results obtained for this species. The results of studies of other species will be reported elsewhere.

Natural genetic attributes of owl monkey (Aotus nancymaae) make it a candidate HIV-1 model organism

We had access to a large breeding colony of Aotus nancymaae owl monkeys located at the University of Texas MD Anderson Cancer Center. For 191 of these individuals, we sequenced transcripts of genes encoding CD4 and the restriction factors listed in Fig. 1C. Synonymous polymorphisms were ignored, and alleles encoding unique protein variants were catalogued (Fig. 2). All four of these genes are known to be evolving under intense positive natural selection in primates (Fig. 1C) 20–23. This mode of evolution is known to produce highly divergent proteins between species and between individuals within populations 1,2,13,20–30. Indeed, for these four genes we find many alleles circulating in the owl monkey colony: alleles encoding 18 CD4 variants, 8 Tetherin variants, 37 TRIMCyp variants (the homolog of TRIM5\alpha found in owl monkeys 31,32), and 14 APOBEC3G variants. As controls, transcripts were also sequenced for genes encoding the HIV-1 co-receptors CCR5 33 and CXCR4 34, both of which are evolving under purifying selection 35,36. Alleles encoding only one (CXCR4) or two (CCR5) variants of these proteins are present in the colony (Fig. 2).

We previously found that the CD4 receptor homologs of several owl monkey species (Aotus spp.) support entry of most strains of HIV-1 26,38, unlike the CD4 of most other primate species, including macaques 26,39. In this owl monkey colony, we found alleles encoding 18 distinct CD4 variants (Fig. 2A, Supp. Data File 1). We cloned, expressed, and tested the most common variants, all of which supported HIV-1 entry (Fig. 3). To us, this was a highly consequential finding, because many successful cross-species virus transmissions in nature hinge on gaining receptor compatibility in a new species 40–42 .

Tetherin is known to be a potent restriction factor of primate lentiviruses, with species-specific patterns of virus engagement. Tetherin interferes with HIV-1 budding by tethering virions to the cell membrane, thereby limiting their release 18,19. In the owl monkey colony, we found alleles encoding eight distinct Tetherin variants (Fig. 2B, Supp. Data File 2), none of which inhibited HIV-1 virion release (Fig. S1). This finding is consistent with previous findings, reported by the Farzan lab, for the Tetherin homolog of another owl monkey species 43. Therefore, owl monkeys naturally have two genetic advantages as a possible HIV-1 model in that both their CD4 and Tetherin are compatible with HIV-1 infection. Further, these auspicious genetics seem to pertain to most of the individuals in this large breeding colony.

Modifications required in HIV-1 to bypass owl monkey TRIMCyp and APOBEC3G

Next, we analyzed another potent, species-specific block to HIV-1 replication. TRIMCyp targets and disrupts the HIV-1 capsid during virion cytoplasmic trafficking 31,32. We found alleles encoding 37 unique TRIMCyp protein variants in colony animals (Fig. 2B, Supp. Data File 3), and with one exception (variant 8) these potently inhibit HIV-1 (Fig. 4A). Because variant 8 is rare in the colony (Fig. 2B), it was necessary for us to engineer mutations into the HIV-1 genome to bypass the restriction by TRIMCyp predicted to occur in most individuals. As shown before 31,32, a G89V point substitution in the cyclophilin A-binding loop of HIV-1 capsid enables escape from owl monkey TRIMCyp restriction (Fig. 4B). However, we found that the G89V change reduced viral fitness (Fig. S2), and we were unable to obtain significant spreading infection with this substitution in the context of our viruses. Consequently, we sought to engineer HIV-1 to escape owl monkey TRIMCyp in a different manner, by instead mining natural genetic variation in lentiviral capsid proteins found in nature. We systematically substituted the 10-amino-acid-long “cyclophilin A-binding loop” of HIV-1 with that of 25 different SIV and HIV-1 strains (Fig. S3). We identified one, from a red-capped mangabey SIV (SIVrcm) isolate, that differed from that of HIV-1 in only four positions (Fig. 4C) yet enabled HIV-1 to bypass owl monkey TRIMCyp restriction (Fig. 4B). Another consequential moment in this project came with the finding that HIV-1, with just these four point substitutions, replicates robustly in OMK (owl monkey kidney) cells, overcoming the powerful inhibition of replication otherwise observed in these cells (Fig. 4D). Apparently, all pro-viral cofactors for HIV-1 replication are sufficient in owl monkey cells, which was not an foregone conclusion to us. OMK cells derive from an owl monkey of a different Aotus species 43, but were used because there are no immortalized lines from Nancy Ma’s owl monkeys. Nonetheless, this result provided “first light” on the possibility that HIV-1 might replicate in owl monkey cells with very few modifications.

APOBEC3G, another host restriction factor, is not expressed in OMK cells 43 but is expected to inhibit HIV-1 in owl monkey T cells. Thus, lastly we investigated the activity of APOBEC3G, a restriction factor that causes hypermutation of reverse transcribed HIV-1 genomes unless it is degraded by the HIV-1-encoded virion infectivity factor (Vif) 17. We identified alleles encoding 14 protein variants of APOBEC3G in the colony animals (Fig. 2B, Supp. Data File 4). At least one of the four most common variants inhibits HIV-1 replication, even in the presence of HIV-1 Vif (Fig. 5A). Consequently, we tested 42 different HIV-1 and SIV Vifs and identified several that degrade owl monkey APOBEC3G variants (Figs. S4, S5). HIV-1 engineered to encode Vif from either SIVmne (pig-tailed macaque) or SIVmac (rhesus macaque) bypassed restriction by all tested owl monkey APOBEC3G variants (Fig. 5A).

Our systematic analysis of CD4, Tetherin, TRIMCyp, and APOBEC3G was complete. To produce HIV-1 strains potentially capable of infecting owl monkeys, we modified a plasmid encoding the full-length HIV-1 genome to introduce the four point mutations reconstituting the SIVrcm cyclophilin A-binding loop (as diagrammed in Fig. 4C) and either SIVmne or SIVmac vif in place of HIV-1 vif. Because we previously found that owl monkey CCR5 has somewhat diminished interaction affinity with HIV-1 Env 45 , we initially built these features into the CXCR4-tropic HIV-1 strain NL4-3 46. We named these two viruses HIVSnom1 and HIVnom2, respectively, following lentiviral naming conventions in which the subscript refers to Nancy Ma’s owl monkey (Fig. 5B).

HIV-1 infection of owl monkeys

Two owl monkeys were intravenously inoculated with 1.3 x 106 infectious units of HIVnom1 and another two with 2.1 x 106 infectious units of HIVnom2 (Fig. 6A). Blood was drawn weekly for up to 30 weeks, which is approximately 7 months. The “0” timepoint was taken from the leg opposite to the site of virus injection five minutes after inoculation. Blood samples were sent to the University of Washington Clinical Retrovirology Laboratory (a human clinical diagnostics lab in Seattle, WA, USA) for determination of viral load (plasma viremia) by RT-qPCR. Although virus replication in these first four animals was modest, in each animal there was one or more timepoint at which plasma viremia increased, suggesting that HIV-1 progeny virions were being produced. By week 12, viremia in all animals was reduced to a level at or below the assay limit of quantification (dotted line; 80 HIV genomes per mL of plasma). This made us wonder whether the animals had successfully cleared the virus and eliminated it from their bodies. To test this, we subcutaneously administered dexamethasone over a short course of several weeks to three of the four animals. Dexamethasone, a glucocorticoid, induces transient immunosuppression by reversibly depleting blood T lymphocytes, NK cells, and B cells, but not monocytes, in people 47 and owl monkeys (Fig. S6). In all three animals, virus rebounded into the blood to detectable levels during treatment (Fig. 6A). These data show that animals have not eliminated the virus from their bodies but rather suggest some type of low-level chronicity with control of viremia.

Of the four infected animals, animal RI (infected with HIVnom2) had the highest and longest acute phase (0–10 week) viremia, and the highest rebound viremia upon dexamethasone treatment (Fig. 6A). Frozen plasma collected from animal RI at weeks 21 and 22 was combined and transfused into a naive animal (AB). At this point, we observed a dramatically higher level of virus replication. Plasma viremia of AB five minutes after transfusion was 526 HIV genomes per mL but, within 5 weeks, expanded by five orders of magnitude to 2.1 x 107 HIV genomes per mL (Fig. 6B). Sequencing of integrated viral genomes from the PBMCs of animal AB revealed that HIVnom2 had acquired four fixed nonsynonymous point mutations that were not present in the HIVnom2 stock used for infection of source animal RI. This virus, here referred to as HIVnom3, is characterized by four new amino-acid substitutions: capsid H120R, Tat A58T, and Env (L10W in the signal peptide; D545G in the gp41 subunit) (Fig. 6C). Three of the mutations arose in animal RI, with the final mutation arising in animal AB (Fig. S7). Notably, three of these four substitutions are to amino acids common at these positions in transmitted isolates of HIV-1 (Figs. S8–S11), preserving the likeness to HIV-1. Further, these four substitutions did not cause an obvious fitness loss (or gain) in human T cells (Fig. S12). The four mutations remained fixed in all subsequent infections, and no additional fixed HIV-1 mutations were detected in any subsequent experiments.

Frozen plasma and fresh whole blood collected from animal AB at weeks 4 and 5, respectively, were mixed and transfused into one naive animal (BO). Likewise, frozen plasma and fresh whole blood collected from animal AB at weeks 7 and 8, respectively, were mixed and transfused into a second naive animal (ON). In BO, replication was modest and then decreased over time until no longer detectable (Fig. 6B). To ensure that virus persisted, we waited for one month after viremia was last detected and then treated the animal with dexamethasone, upon which virus rebounded to low detectable levels. In contrast, a significant acute viral peak was observed in the second monkey, ON. The plasma viremia of ON five minutes after transfusion was 175 HIV genomes per mL but, within six weeks, expanded by four orders of magnitude to 1.1 x 106 HIV genomes per mL. We conclude that HIVnom3 is replicating in owl monkeys: 1) it can propagate to high titers during the acute phase of infection in at least some owl monkeys, 2) it causes all animals to seroconvert (yellow stars in Fig. 6A,B; Fig. S13), 3) it causes chronic infection in all animals and, even when controlled after acute phase, rebounds upon dexamethasone-induced immunosuppression, and 4) it can be transmitted from one animal to the next through infected blood products. All investigated animals also developed autologous neutralizing antibodies except for animal ON (Fig. S14).

HIVnom3 is an owl monkey-adapted virus

We speculated that HIVnom3 may have adapted to the owl monkey host during the serial passage experiment. To test this hypothesis, we engineered the four amino-acid substitutions acquired during animal-to-animal passage into the plasmid-based molecular clone of HIVnom2. Both plasmids were then used to generate virus stocks. A 50:50 mixture of the original HIVnom2 and the adapted HIVnom3 (which differ by only four point substitutions) was intravenously injected into two male and two female owl monkeys of varying ages. The goal was to determine whether one virus would outcompete the other in vivo. In all four animals, the viruses replicated and, whenever tested, rebounded upon dexamethasone treatment (Fig. 7A). HIVnom3 proved to have higher fitness than HIVnom2, because the latter virus could no longer be detected by one week in any of the four animals (Fig 7B, Figs. S15–S18).

Increased fitness of HIVnom3 is supported by two additional observations. First, all four animals infected with HIVnom3 seroconverted more rapidly than animals infected with HIVnom1 or HIVnom2 (Fig. 7C). People with HIV-1 produce anti-HIV-1 antibodies (i.e., seroconvert) by a median of two weeks (range 1–7 weeks) after first testing positive for viral RNA 48. All infected owl monkeys seroconverted within this time range except for animal RI, which needed 24 weeks. Times to seroconversion were correlated with the presence of the four new mutations but not with the dose of inoculated viruses (Fig. S19). The second observation that suggests that HIVnom3 is owl monkey adapted is that set point viremias in two monkeys (ME: average 298 RNA copies/mL and JI: average 139 RNA copies/mL) were higher than set points observed in any previous experiment, which tended to be below the limit of quantification except when dexamethasone was administered. Sequencing of proviruses in PBMCs collected from these two animals (ME and JI) at necropsy yielded the sequence of HIVnom3 and did not reveal any additional fixed virus mutations, even though the virus was replicating in vivo for approximately seven months. Collectively, these data indicate that the four mutations acquired during animal-to-animal serial passage increase HIV-1 fitness in owl monkeys. Stocks of this virus can be generated from the HIVnom3 molecular clone, ensuring genetic stability of this owl monkey-adapted virus over time.

Optimizing dosing of HIVnom3 in the owl monkey

All of the owl monkeys above were infected using either very high doses of stock virus or via transfusion of blood products from infected animals. We noticed that the two animals that produced the most significant acute phase virus peaks (AB and ON) were infected with the lowest inoculums of virus. The plasma viremia in these two animals just after inoculation was 526 and 175 HIV genomes per mL, whereas the plasma viremia just after inoculation for all other animals was more than 100,000 HIV genomes per mL. To explore lower-dose inoculations, we next tried an infection series where we determined the minimal infectious dose of HIVnom3, through intravenous inoculation of 12 additional owl monkeys. Four owl monkeys were infused with 100 infectious units of HIVnom3, four with 1,000 infectious units, and four with 10,000 infectious units (Fig. 8). No owl monkeys became infected at the lowest dose; 1 out of 4 monkeys were infected when infused with 1,000 infectious units; and 4 out of 4 were infected when infused with 10,000 infectious units. At the highest dose, acute phase virus peaks ranged from 1.8 x 105 - 4.8 x 106 HIV genomes per mL, similar to the median acute phase virus peak in people of 5.0 x 106 HIV genomes per mL 48. Under these conditions, we observe higher and stable chronic set point viremia in most infected monkeys (Fig. 8 and ongoing experiments). In these animals, set point viremia is somewhat lower (102 - 103 copies/mL) than in untreated humans (104 copies/mL 48). In contrast, rhesus macaques infected with common research strains of SIVmac often produce set points much higher than in humans, in the order of 105–107 copies/mL 49.

HIV-1 pathogenesis in infected owl monkeys

HIV-1 pathogenesis is defined as all aspects of the disease course including virus replication dynamics, immune responses, virus tissue distribution, reservoir formation, and the onset of immunodeficiency related illnesses. We have already presented data on the first two aspects, and here we discuss data related to the latter three.

In humans, gut-associated lymphoid tissue (GALT) is one of the earliest sites of HIV-1 replication and expansion 50–52. Lymph nodes are also important sites of HIV-1 replication in humans 53. To determine where, anatomically, HIVnom3 is replicating in owl monkeys, we performed RNAscope (RNA in situ hybridization) on formalin-fixed tissue sections. These sections were stained with commercial probes that anneal to HIV-1 RNA, identifying both HIVnom3 virions and infected cells harboring transcriptionally active HIVnom3 provirus. In owl monkeys, we see significant staining in tissues of the owl monkey intestine, particularly in GALT embedded within these intestinal sections (Fig. 9A), and in lymph nodes from different anatomical locations (Fig. 9B-D, also Fig. S20). No staining was identified in pancreas of infected animals, or in lymph nodes of naive animals (Fig. 9C,D; also Fig. S21). Therefore, HIVnom3 is replicating in GALT and lymph nodes, and possibly other locations, in owl monkeys

In humans, the lymph nodes are an important anatomical location of the virus reservoir, and some HIV-1 proviruses in lymph nodes continue to produce viral transcripts even during full suppression of viremia 54–58. Four of the six owl monkeys investigated by RNAScope (AB, ON, ME, SI) were non-viremic at the time of necropsy, yet HIVnom3 transcripts were easily identified within cells of their lymph nodes (Fig. 9 C,D). Animal AB is particularly noteworthy here as this animal had been non-viremic for 37 weeks (~ 9 months) at the time of necropsy, yet HIVnom3 RNA was clearly being transcribed in lymph nodes. These RNAscope studies, in combination with rebound of plasma viremia upon dexamethasone treatment, demonstrate the presence of a virus reservoir in owl monkey lymph nodes and possibly in other locations as well. Further characterization of the reservoir will be required.

Lastly, we do not yet know if owl monkeys develop AIDS. For the duration of these experiments, there were no clinical abnormalities, laboratory changes, gross necropsy findings, or histological lesions identified that could be directly attributable to simian AIDS. This was expected given the length of our experiments. Studies from early in the pandemic demonstrate that, in untreated humans, development of AIDS typically takes 7-12 years 60. Within this population average, infections that develop CXCR4-tropic viruses proceed somewhat more quickly, but survival after first detectable CXCR4-tropic virus is still 1.5 to 3 years 61. In SIVmac models, progression to AIDS is faster still – occurring in 1-2 years, with up to 40% of macaques progressing even more quickly (<1 year) 62. It is not obvious that animal models for the development of vaccines or cures need to recapitulate AIDS. Vaccines, especially, need to act in the first days to weeks after exposure, while in humans AIDS does not begin until years later.

Methods
Owl monkeys and biological materials

Nancy Ma’s owl monkeys (Aotus nancymaae) and blood samples used in this study originated from a breeding colony housed at the MD Anderson Cancer Center, Keeling Center for Comparative Medicine and Research (KCCMR) located in Bastrop, TX, USA. Naive colony animals were socially housed in environmentally controlled indoor enclosures containing nest boxes, perches, and a variety of physical enrichment manipulanda. Commercial monkey chow and water were available ad libitum, and fresh produce and novel food enrichment items were also provided daily. The KCCMR is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. All animal work was approved by The University of Texas, MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC Protocol #s: 00001839-RN00, 0000 1839-RN00, 00000451-RN02, and 00000451-RN03) and was performed in accordance with the standards established by the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, the Office of Animal Welfare, and the United States Department of Agriculture. Experimental virus inoculations of these animals are described below. Other blood collections and clinical procedures (e.g., physical exams, dexamethasone administration, potassium administration) were performed on animals as they were briefly manually restrained, without the use of sedation.

Genetic characterization of owl monkeys

2.5 mL of blood were collected from naive colony animals in RNA preservation tubes (PAXgene Blood RNA Tube, BD Biosciences #762165) that were processed according to the manufacturer’s instructions. After blood collection, animals were immediately returned to the breeding colony. RNA was isolated using TRIzol (Invitrogen #15596018) and cDNA libraries were prepared using the SuperScript IV First-Strand Synthesis System (Invitrogen #18091200). Gene-specific primers (placed in the 5' and 3' UTRs) were then used to PCR-amplify cDNA nucleic acids encoding CD4, Tetherin, TRIMCyp, and APOBEC3G from cDNA using Q5 High-Fidelity DNA Polymerase (New England BioLabs #M0491L). All PCR products were treated with Exonuclease I (Thermo Fisher Scientific #70073) and recombinant Shrimp Alkaline Phosphatase (rSAP, Thermo Fisher Scientific #78390), and then quantified using the EZQuant dsDNA Quantitation Kit (Fluorometric, BioVision #K900-2000). These products were pooled in equimolar quantities by individual, and then barcoded before being sequenced using a MiSeq instrument (Illumina). Raw sequenced reads were processed as follows: Adapters were trimmed using Trimmomatic v0.3679 generating both paired-end and unpaired trimmed reads. FastQC v0.11.780 was used to assess the quality of the trimmed raw sequences. Sequencing reads were re-assembled into the four targeted genes using GenBank reference sequences CD4 (#XM_021670978), Tetherin (#XM_021669213), TRIMCyp (#XM_0124 56799), and APOBEC3G (#NM_001308530). An indexed reference sequence was created utilizing BWA v0.7.17 81 and Samtools v1.6 82. Mapping the trimmed, quality-controlled reads to the indexed reference sequence was accomplished using BWA-MEM v0.7.1783 to create SAM files. The conversion of SAM to the binary BAM format, subsequent sorting, and removal of duplicate reads were performed using Picard Tools v2.6.0 (https://broadinstitute.github.io/picard; Broad Institute). VCF files containing genotype likelihoods were generated from the marked BAM files with the mpileup algorithm in BCFtools v1.8 84. A variant callset was created using the multiallelic calling and rare-variant calling model in BCFtools v1.8 with a prior probability of 0.99 after which a set filter excluded sites with a QUAL<20. Using this variant callset and the FastaAlternate ReferenceMaker function in GATK v3.7.0 85 , individual FASTA files for each sample and gene were produced with homogeneous single nucleotide polymorphism sites replacing their respective reference bases while IUPAC ambiguity codes were used at heterozygous sites. FASTA files containing ambiguity codes were then trimmed to the coding region of each gene and manipulated to be merged by gene and individual using Linux command line utility commands. Pedigree-independent allelic phasing was performed on sequence data for each of the four genes of interest using DnaSP v5.10.1 86. In most cases, allelic phasing was later validated with available pedigree information from the KCCMR animal colony, and/or by amplifying them by PCR from cDNA libraries, cloning, and Sanger-sequencing individual products. Throughout this manuscript, protein “variants” were defined groups of alleles encoding the same protein; synonymous SNPs were ignored. Aotus nancymaae CXCR4 alleles identified all encoded a protein matching GenBank #NM_001308529. Aotus nancymaae mRNA gene sequences generated here are available in GenBank with accessions: PX876883-PX877262 (CD4); PX877263-PX877642 (CCR5); PX877926-PX878305 (Tetherin); PX878306-PX878685 (TRIMCyp), and PX911634-PX912013 (APPOBEC3G).

HIV-1 entry assays

HIV-1-EGFP was produced by co-transfecting 293T cells (ATCC #CRL-3216) with plasmids encoding HIV-1 (NL4-3\DeltaEnv-EGFP, BEI ARP-11100) and HIV-1 Env (isolate US1.EC6, a CCR5-using subtype B virus, GenBank #MK501602). Cf2Th cells (ATCC CRL-1430) were transduced to stably express human CCR5 alone (Genbank #NM_001394783), or with human (Genbank #NM_000616) or various owl monkey CD4 variants. Cf2Th cell lines expressing different receptor combinations were plated, exposed to an equivalent volume of HIV-1-EGFP, and 48 h later assayed for EGFP signal by flow cytometry.

Engineering HIV-1 escape from owl monkey TRIMCyp

Crandell-Rees feline kidney (CRFK) cells (ATCC #CCL-94) stably expressing various owl monkey TRIMCyp alleles (Supp. Data File 3) were established via transduction using retroviral vectors. To generate the retroviral vectors, human embryonic kidney (HEK) 293T cells (ATCC #CRL-3216) were seeded at a concentration of 1 x 106 cells/well in 6-well dishes. Each well was transfected 24 h later with 2 \mu g of pLPCX plasmid from the LRCX Retroviral Vector Set (Takara #631511; empty or encoding the TRIMCyp allele of interest), 1 \mu g of pCS2-mGP plasmid containing murine leukemia virus (MLV) gag-pol 87, and 0.2 \mu g of pC-VSV-G plasmid encoding the vesicular stomatitis Indiana virus glycoprotein (G) using the standard TransIT-293 protocol (Mirus Bio #MIR 2705). Supernatants were collected after 48 h, passed through a 0.2-\mu m filter, and used to transduce CRFK cells. After 24 h, medium containing 8 \mu g/mL of puromycin was added to select transduced cells. Cell lines were expanded and grown in puromycin for at least two weeks before expression of TRIMCyp constructs was detected by western blot.

These cell lines were then used in a screen to identify cyclophilin A-binding loop modifications that allow escape from owl monkey TRIMCyp restriction. To introduced mutations into the cyclophilin A-binding loop (Fig. S3), the pMDLg/pRRE plasmid 88 containing HIV-1 gag-pol was used as a template for site-directed mutagenesis using PfuTurbo DNA polymerase (Agilent #600254) 89. Viruses for single-cycle infection assays were packaged in 293T cells (ATCC #CRL-3216) by co-transfection of plasmids encoding viral proteins and VSV G, along with a transfer vector (pMDLg/pRRE, pRSV-Rev, pMD2.G, pRRLSIN.cPPT. PGK-GFP.WPRE; all available from Addgene). 293T cells (ATCC #CRL-3216) were seeded into 6-well plates at a concentration of 1 x 106 cells/well prior to transfection. After 48 h, supernatants containing viruses were harvested, filtered using a 0.45-micron filter, and frozen. The titer of viruses was determined on CRFK cells stably expressing human TRIM5 or owl monkey TRIMCyp by measuring percent green fluorescent protein (GFP)-positive cells along a volume gradient of virus supernatant. For infection assays, the modified CRFK cells were plated at a concentration of 7.5 x 104 cells/well in 24-well plates and exposed to HIV-1 wildtype or variants containing mutations. Two days later, cells were fixed in 2% paraformaldehyde for 15 min, washed three times with 2 mL of FACS buffer (Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 2% fetal bovine serum and 1 mM ethylenediamine tetraacetic acid (EDTA)), resuspended in 500 \mu l of FACS buffer, and analyzed by flow cytometry for expression of GFP. The percentage of each cell line that was GFP+ was calculated and normalized to the percentage of GFP+ cells in the control line (cells without TRIMCyp). All exposures were performed in triplicate using a single virus stock, and all results were confirmed using at least two experimental replicates.

Full-length HIV-1 engineered to bypass owl monkey TRIMCyp was then assessed for the ability to cause infection in two different immortalized cell lines, one from human and one from owl monkey. HIV-1 (Q23-17)90 was engineered to contain the four mutations re-constituting the SIVrcm cyclophilin A-binding loop. This plasmid (wildtype or altered as described) was transfected into human cells to produce virus stocks, the titers of which were measured on TZM-bl cells (BEI Resources #ARP-8129), which are HeLa cells that express CD4, CCR5, CXCR4, and reporters that respond to HIV-1, specifically \beta-galactosidase (\beta-gal) or firefly luciferase, under the control of the HIV-1 long terminal repeat 91,92. Then, Hut78 (human) cells stably expressing human CCR5 were seeded into a 48-well dish at 106 cells/well in a total of 600 \mu L of complete Roswell Park Memorial Institute (RPMI) medium. Three-striped owl monkey (Aotus trivirgatus) kidney OMK cells (ATCC #CRL-1556) stably expressing human CD4 and CCR5 were seeded into a 6-well dish at 200,000 cells/well in a total of 2 mL of complete DMEM. The next day, cells were exposed to virus at an MOI = 0.1 by spinoculation at 1,200 xg for 90 min in the presence of 20 ug/mL diethylaminoethyl (DEAE)-dextran. After spinoculation, cells were washed five times with 1 mL of either RPMI (Hut78 cells) or D-PBS (OMK cells). After washing, Hut78 cells were resuspended in 600 \mu L of complete RPMI in a 24-well dish, and OMK cells were provided 2 mL of complete DMEM. Supernatant aliquots were collected on 0, 3, 7, and 10 days post-exposure to quantify virus production. The ZeptoMetrix p24 Antigen ELISA kit (ZeptoMetrix #0801008) was used to quantify the amount of capsid in supernatant aliquots according to the manufacturer’s instructions. A capsid protein standard provided with the kit was used to generate a standard curve.

Engineering HIV-1 escape from owl monkey APOBEC3G

Through screening of 42 HIV-1, HIV-2, and SIV Vifs, we identified SIVmne and SIVmac Vifs as capable of degrading owl monkey APOBEC3G (Fig. S4; Supp. Data File 5). Using an infection assay, we then tested the effect of these Vifs on HIV-1 production in the presence of various APOBEC3Gs. Human APOBEC3G corresponds to GenBank #NM_021822. Nucleic acids encoding the four major owl monkey APOBEC3G alleles were PCR-amplified from owl monkey cDNA isolated from blood. 293T cells (ATCC #CRL-3216) were plated in 24-well dishes at a density of 200,000 cells/well. Twenty-four hours later, cells were co-transfected with a plasmid encoding APOBEC3G (human at 20 ng and owl monkey at 200 ng) and 300 ng of a plasmid encoding HIV-1 (NL4-3) provirus or variants thereof: \Deltavif, SIVmne vif or SIVmac vif. Forty-eight hours post-transfection, virion-containing supernatants were collected and spun at 1,200 xg for 5 min to remove cell debris. Equivalent volumes of supernatants were added to TZM-bl cells, which had been plated one day before in 96-well plates at 10,000 cells/well. Forty-eight hours post-exposure, cells were washed with D-PBS and resuspended in Passive Lysis 5X Buffer (Promega #E194A) and incubated at room temperature for 15 min. Luciferase signal was activated using the Promega luciferase assay system (Promega #E1501) and detected using an Agilent BioTek Synergy HTX multi-mode microplate reader (Agilent #S1LFA). We did not test whether the SIV Vifs antagonized other APOBEC3 proteins, but successful in vivo infections of owl monkeys suggests that they antagonize all relevant APOBEC3 proteins in this species.

Generation of HIV-1 molecular clones and variants

Viruses for owl monkeys were produced through manipulation of the pNL4-3 plasmid (BEI Resources #ARP-114), which encodes the full-length HIV-1 (NL4-3) provirus. Gibson cloning was performed to construct a vif-deleted version of pNL4-3, using the strategy shown in Fig. S22, and to replace the vif open reading frame with the vif from SIVmne (GenBank #U79412) or SIVmac (GenBank #M19499), using the cloning strategy shown in Fig. S23. To replace the cyclophilin A-binding loop with that of SIVrcm, we used a gene block (gBlock) of 440-bp length roughly centered in the region encoding the cyclophilin A-binding loop. The pNL4-3 proviruses with the inserted SIVmne or SIVmac vif were used as templates for PCR amplification of Gibson fragments. These PCR products overlapped with the 5' and 3' ends of the SIVrcm cyclophilin A-binding loop-encoding gBlock and the ampicillin resistance gene on the plasmid. The HIVnom2 plasmid was used to construct a derivative clone (HIVnom3) containing mutations reconstituting the four substitutions that arose during in vivo adaptation in owl monkeys. This clone was made through four consecutive site-directed mutagenesis reactions using primers designed using NEBaseChanger (https://nebasechanger.neb.com) and the standard protocol of the Q5 Site-Directed Mutagenesis Kit (New England Biolabs #E0554S). The sequences of HIVnom1, HIVnom2, and HIVnom3 can be found at GenBank with accession numbers PX925169, PX925170, and PX925171, respectively. The plasmid-based molecular clones encoding HIVnom1, HIVnom2, and HIVnom3 have been deposited at ATCC.

HIVnom stock production

Nancy Ma’s owl monkeys were exposed to three viruses that were produced from molecular clones: HIVnom1, HIVnom2, or HIVnom3. To generate these stocks, 13 x 106 293T cells (ATCC #CRL-3216) were plated in 15-cm tissue culture dishes in media without antibiotics. The following day, 20 \mu g of plasmid was transfected using the standard Trans-IT 293 transfection reagent protocol. Six hours post-transfection, media were removed and replaced with complete DMEM containing 3% fetal bovine serum. Forty-eight hours later, media were removed and spun at 1,200 xg for 5 min to remove cell debris. Virion-containing supernatants were clarified through 0.45-\mu m cellulose acetate syringe filters followed by concentration through 100-kDa Amicon columns (Millipore UFC910024). Concentrated virions were further purified through a 20% sucrose cushion at 21,000 xg for 90 min at 4°C. Virus pellets were resuspended in 1 mL of D-PBS and 100-mL aliquots were frozen for future use. To determine virus titers, one frozen aliquot was removed for testing. Virus titers were determined on TZM-bl cells using a \beta-galactosidase assay 93 using the protocol associated with NIH HIV Reagent Program entry #ARP-1470. (We did not use TCID50, as is common in the macaque/SIVmac field. However, we typically see a ratio of 15-25:1 for \beta-galactosidase:TCID50, meaning that \beta-galactosidase gives a titer about 15-25 times higher per ml.) To test replication of modified viruses, human SUP-T1 T-lymphoblasts (ATCC #CRL-1942) expressing both CD4 and CCR5 (and which naturally express CXCR4) were exposed at a MOI of 0.1 via spinoculation (500 xg for 90 min at 30°C). Cell supernatants were collected every other day for 12–14 days and frozen. Viruses in supernatants were quantified using both an SG-PERT assay as previously described 94 and via exposure of TZM-bl cells followed by measuring firefly luciferase luminescence.

Owl monkey challenge studies

In total, 23 Nancy Ma’s owl monkeys were used in virus exposure studies. Details regarding the age and sex of individual animals used in each exposure study are provided in the body of the paper and/or the figure legends. All studies balanced animal age and sex to the greatest extent possible. Animals were either pair-housed or single-housed in a designated study room outside of the main KCCMR colony but were otherwise maintained similarly to colony animals. Prior to beginning the study, animals were given complete physical examinations and had blood work performed to establish baseline data and ensure their health. Blood work included an in-house CBC analysis (Advia 120, Siemens Medical Solutions), serum chemistry analysis (Beckman Coulter #AU680), and flow cytometry analysis along with a baseline HIV-1 RNA quantification and serological screening assay, both performed by the University of Washington Retrovirology Laboratory.

Twenty of the animals were infused with individual aliquots of stock virus that had been maintained in liquid nitrogen vapor following production in tissue culture. Two animals (CA and SO) were administered 1.27 x 106 infectious units of HIVnom1, two animals (KI and RI) were administered 2.07 x 106 infectious units of HIVnom2, and the remaining four animals (ME, SI, ST, and JI) were administered 9.3 x 105 infectious units of HIVnom2premixed with 9.1 x 105 infectious units of HIVnom3 as part of the virus competition experiment. For the minimal dose study, four animals (#86956, 4 year-old male; #86957, 4 year-old male; #87031, 4 year-old female; # 87033, 4 year-old female) were administered 1.67 x 102 infectious units of HIVnom3; four animals (#86945, 4 year-old male; #87053, 2 year-old male; #86955 4 year-old female; #86990, 3 year-old female) were administered 1.06 x 103 infectious units of HIVnom3; four animals (#87054, 3 year-old male; #87089, 3 year-old male; #8 6092 5 year-old female; #87067, 3 year-old female) were administered 1.04 x 104 infectious units of HIVnom3.

The remaining three animals (AB, BO, and ON) were transfused with either citrate-phosphate-dextrose solution with adenine (CPDA)-1 preserved plasma, or a mixture of CPDA-1 preserved plasma and fresh CPDA-1 preserved whole blood obtained from previously exposed animals. Prior to the transfusion experiments, a major and minor crossmatch was performed by the KCCMR diagnostic laboratory on blood from the donor and recipient animals. No adverse reactions were identified between the donor and recipient animals in any of the crossmatch assays. Animal AB was transfused with a mixture of two frozen plasma aliquots from animal RI collected at week 21 and week 22 post-infection. The week-21 aliquot was 0.6 mL (viral RNA [vRNA] = 2,760 genomes per mL) and the week-22 aliquot was 2.4 mL (vRNA = 18,220 genomes per mL). Animals BO and ON were both transfused using a mixture of frozen plasma and fresh whole blood from animal AB. Animal BO received a 0.6-mL frozen plasma aliquot from week 4 post-infection (vRNA = 895,304 genomes per mL) mixed with a 1.0-mL fresh whole blood sample from week 5 post-infection (vRNA = > 21,000,000 genomes per mL). Animal ON received a 0.6-mL frozen plasma aliquot from week 7 post-infection (vRNA = 11,104 genomes per mL) mixed with a 1.5-mL fresh whole blood sample from week 8 post-infection (vRNA = 344 genomes per mL).

On the day of virus administration (day 0), each of the virus exposure animals was anesthetized with a combination of xylazine (Akorn 20 mg/mL, Gurnee, IL, USA) and ketamine (Dechra 100 mg/mL, Overland Park, KS, USA) at 2.3 mg/kg and 20 mg/kg total body weight, respectively. Aliquots of the virus stocks and the CPDA-1 preserved plasma were thawed on ice and maintained cold until administered. CPDA-1 preserved whole blood samples were administered within 1 h of collection. Viral solutions were slowly intravenously infused into one of the femoral veins of an anesthetized animal. Approximately 5 min after completion of the infusion, a blood sample was collected from the contralateral femoral vein into a K2 EDTA tube (BD Biosciences #367856 or #365974) for “day 0” vRNA quantification. Animals were monitored until fully recovered from anesthesia and then were observed a minimum of twice daily thereafter for the remainder of the study. Physical examinations and blood work analyses for health monitoring were performed on a periodic basis throughout the remainder of the study. The CBC data obtained for each study animal are provided in Supp. Data File 6.

On the final day of the study, animals were anesthetized using xylazine and ketamine and a blood sample was collected. The animals were then humanely euthanized using an overdose of pentobarbital and phenytoin (Euthasol, Virbac, Fort Worth, TX, USA), consistent with the regulatory guidelines of the American Veterinary Medical Association. Full diagnostic necropsies and tissue collections were performed for each animal.

Blood sampling from infected animals

A maximum of 3 mL of blood was collected per week over the study from animals included in the virus exposure studies and deposited in: K2 EDTA tubes (BD Vacutainer, BD Biosciences #367856 or BD Microtainer, BD Biosciences #365974); serum clot tubes (BD Vacutainer, BD Biosciences #367812); or microcentrifuge tubes (Fisher Scientific #02-681-343) containing CPDA-1 anticoagulant (citric acid, sodium citrate, monobasic sodium phosphate, dextrose, and adenine) at a concentration of 0.14 mL of CPDA-1/mL of whole blood. The CPDA-1 anticoagulant was acquired aseptically from a commercial veterinary blood transfusion bag (IV Blood Transfusion bag, Jorgensen Laboratories #J0520X, Loveland, CO, USA). EDTA preserved blood was utilized for complete blood counts (CBC), to obtain plasma for vRNA and antibody assays and for the acquisition of peripheral blood mononuclear cells (PBMCs). Standard Ficoll (Ficoll-Paque Plus, GE Healthcare #17-1440-03) processing was used to obtain PBMCs. These cells were preserved in fetal bovine serum with 10% dimethyl sulfoxide (DMSO) at a density of 106–107 cells/mL and were maintained in liquid nitrogen vapor in cryogenic vials (Corning #431417) until needed. Sera derived from clot tubes were used for serum chemistry analyses. CPDA-1 preserved whole blood was used on the same day it was collected for blood transfusion experiments or the CPDA-1 plasma from these samples was collected and preserved in liquid nitrogen vapor in cryogenic vials (Corning #431417) until needed.

Dexamethasone treatment

Dexamethasone sodium phosphate (Hikma 10 mg/mL #00641036725, Eatontown, NJ, USA) was administered to immunosuppress some of the study animals. Treatment consisted of a once-daily subcutaneous injection. Initially, animals were administered dexamethasone at 1.5 mg/kg with the dose gradually increasing to 6 mg/kg over a 2-week period. Study animals were maintained at 6 mg/kg for an additional 1–2 weeks. Two of the study animals (SO and SI) were euthanized at the point when the dexamethasone dose was at its highest peak. The remaining study animals underwent a gradual withdrawal from dexamethasone over a 2–3-week period. Two study animals (ST and JI) were euthanized at the time point when dexamethasone dose was reduced to 1.5 mg/kg, just prior to full withdrawal from the drug. The remaining three animals (KI, RI, and BO) were maintained on study for several weeks following withdrawal of the dexamethasone. A 0.75-mL dose of oral potassium gluconate gel supplement (Renal K+, Vetoquinol, Ft Worth, TX) was administered daily to all animals on dexamethasone treatment to counteract the mild hypokalemia noted to occur in some animals.

Determination of viral loads and seroconversion

Plasma vRNA quantification was performed by the commercial human diagnostic services at the University of Washington Retrovirology Laboratory (UWRL; Department of Laboratory Medicine, Seattle, WA, USA) using the Abbott RealTime HIV-1 RNA assay (Abbot #06L18-090) in conjunction with RT-qPCR instrumentation, practices, and procedures standard to that laboratory. Briefly, 500 \mu L of frozen EDTA owl monkey plasma were submitted for analysis and then diluted to 1 mL by the laboratory prior to processing. The range of vRNA quantitation for the assay is 40–10,000,000 HIV genomes per mL when using undiluted samples, although the range of vRNA reported in this study was 80–20,000,000 HIV genomes per mL due to the dilution required for the animal samples. The laboratory reported four possible outcomes: 1) virus detected and a vRNA quantification in HIV genomes per mL; 2) virus detected but numbers were below the lower limit of quantification (<80 HIV genomes per mL); 3) virus detected but the numbers were above the upper limit of quantification (>20 million HIV genomes per mL); 4) virus not detected. The vRNA assay was performed for each animal prior to the start of the study, on the day of exposure (day 0), and on multiple days throughout the study (Supp. Data File 6). For the minimal dose study, plasma vRNA quantification was performed by the diagnostic services available through Duke University, using the Roche Cobas HIV-1 test on the Cobas 6800 System.

The initial HIV-1 seroconversion analyses in this study were also performed by UWRL through their two-step protocol for HIV-1/HIV-2 antibody detection. A 500-\mu L frozen plasma sample first underwent qualitative detection of HIV-1 capsid or HIV-1/HIV-2 antibodies in the plasma using the Architect HIV Ag/Ab Combo (Abbott Diagnostics #873766). Samples identified to be reactive to the first assay then underwent further analysis using the Bio-Rad Geenius HIV1/2 system (Life Science). This second assay confirmed the presence of HIV-1 antibodies and also identified the specific type of antibodies (anti-p24 (CA), -p31 (IN), -gp41, and -gp160) present in reactive samples. Given that only 3 mL of blood could be collected from each animal per week and that there were numerous blood-based assessments desired for each of the study animals, we sought other means to document time-to-seroconversion that might require less than 500 \mu L of plasma. Through retrospective testing of plasma samples that had been previously analyzed by UWRL, one human point-of-care HIV assay (Determine HIV-1/2 Ag/Ab Combo test, Abbott Laboratories #7D2648) was identified to correlate well with the UWRL results. This assay was subsequently used to confirm the UWRL results. As this point-of-care assay requires only 25 \mu L of plasma, it has also been used to retrospectively determine the earliest date of seroconversion for all study animals. The results of all UWRL and point-of-care assays performed in this study are provided in Supp. Data 5.

Detection of neutralizing antibodies

Owl monkey plasma samples were obtained at different weeks post-exposure. Plasma samples were received frozen and heat-inactivated at 56°C for 30 min. These samples were then mixed 1:5 in DMEM containing 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin, and 2-fold serially diluted. Dilutions were mixed 1:1 with HIVnom3 (4,000 infectious units/well) and incubated at 37°C for 1 h. TZM-bl cell culture media (cells plated the day before at 1.1 x 104 cells/well in a 96-well plate) were removed and replaced with virus/plasma mixtures. TZM-bl cells contain an integrated Tat-inducible firefly luciferase gene. The samples were incubated for 48 h, after which the cells were lysed using the Luciferase Assay System (Promega #E1500) and relative light units (RLUs) were measured using a luminometer. Uninfected cells were used to correct for background firefly luciferase activity. Samples were normalized to cells exposed to virus but not exposed to plasma. Infectivity surpassing 100% in some samples suggests incomplete heat inactivation of virus in samples with very high viral loads.

Histopathological and in situ hybridization tissue analyses

Tissues collected at necropsy were immediately placed in 10% neutral buffered formalin and then processed into paraffin blocks within 72 hours of collection. Each study animal had 5 \mu m sections prepared from a minimum of 44 unique tissues. These sections were hematoxylin and eosin (H&E) stained by the KCCMR Histology Lab using instruments, practices, and procedures standard to that lab. The resultant slides were reviewed by a boarded veterinary pathologist (GKW) for pathologic lesions, and specifically for any changes typical of lentiviral infections or immune dysfunction.

Additional lymph node (axial, inguinal, mandibular, and mesenteric) and pancreas sections were obtained from animals (AB, ON, ME, SI, ST, and JI) for analysis through RNA in situ hybridization. Negative and positive control samples were obtained for optimization of the RNA in situ hybridization assay and for comparison to the infected owl monkey lymph nodes. As pancreas is not generally considered a primary reservoir tissue in chronic HIV-1 95,96, we expected HIV-1 pancreatic staining to be negligible in infected owl monkeys and processed this tissue as a potential internal negative control. Lymph node sections were also obtained from uninfected (naive) owl monkeys and served as an external negative control. TZMbl cells stably expressing human CD4, CCR5, and CXCR4 were infected with varying MOIs (0, 0.1, 1, 10) of HIVnom3 and these were used as both negative (MOI 0) and positive (MOIs 0.1, 1, 10) controls. In brief, following infection, the cells underwent 48 hours of incubation and were then collected, washed, pelleted, suspended in agarose gel (HistoGel™ # 22-110-678, Fischer Scientific), and processed into paraffin blocks for sectioning. As a final positive control, we obtained two paraffin-embedded lymph node blocks derived from human patients chronically infected with HIV-1. These tissues were obtained through a commercial biobank (Aurora Diagnostics, Palm Beach, FL, USA; formerly Aurora Research Institute, LLC, Las Vegas, NV, USA). The lymph nodes were collected through approved IRB protocols and all tissues had undergone de-identification of personal health information in accordance with the HIPAA Privacy Rule prior to being provided to the KCCMR.

Serial 5 \mu m sections were collected from the experimental and control tissue blocks for use in the RNA in situ hybridization assays.

RNA in situ hybridization processing of the experimental and control tissues was performed by the Pathology Services Core at the University of North Carolina at Chapel Hill using instruments, practices, and procedures standard to that lab. In brief, RNA in situ hybridization processing utilized a fully automated system (Leica BOND RX, Leica biosystems) and a commercially available detection reagent kit (#322100 RNAscope® 2.5 LS RNA in situ hybridization singleplex, chromogenic-brown assay, Bio-Techne). One RNA probe (#313908; RNAscope® 2.5 LS Positive Control Probe Hs-PPIB, Bio-Techne) was applied to one of the serial sections to assess RNA viability in the samples through analysis of the housekeeping gene peptidylprolyl isomerase B (PPIB). A second RNA probe (#312038 RNAscope® 2.5 LS Negative Control Probe Bacillus subtilis strain dihydrodipicolinate reductase (dapB)) was applied to another serial section to identify background/nonspecific staining. A third RNA probe (#416118 RNAscope® 2.5 LS Probe - V-HIV1-CladeB - HIV-1 NL4-3 complete sequence) was applied to additional serial sections for the detection of HIV-1 RNA.

Sequencing of HIVnom proviruses

Virus evolution was monitored throughout animal studies. PBMC samples were obtained from owl monkeys at multiple timepoints of infection, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN #69504). Twelve primer pairs were designed for nested PCR to amplify six separate overlapping amplicons (900–2,500 bp each) from integrated pro-viral DNA representing the entire HIVnom genome. PCR was performed using the Q5 High-Fidelity 2X Master Mix (NEB #M0492S). The first of each nested set of amplicons were gel-extracted using the Wizard SV Gel and PCR Clean-Up System (Promega #A9281) and the resulting products were diluted 1:100 in nuclease-free water before being used as templates for a second round of PCR. Both strands of the second amplicons were Sanger-sequenced (Quintara Biosciences (https://www.quintarabio.com)). Single nucleotide polymorphisms (SNPs) that had reached fixation in the proviral pool were flagged at each time point in each animal.

For dual infections with HIVnom2+HIVnom3, Sanger sequencing was used to determine the presence of each virus over time. Genomic DNA was prepared from PBMCs at various timepoints after exposure, and then used as template for nested PCR and Sanger-sequencing of proviral DNA as described above. Samples were selectively sequenced at the four sites that differ between the original and adapted virus. The complete virus genomes were sequenced from gDNA isolated at time of necropsy to identify any new mutations that may have arisen during infection.

Biosafety

Unlike SIV and SHIV studies, this study utilized viruses that are presumably infectious to people, because they replicate on a human T cell line (Fig. S12). All procedures instituted for risk minimization and management of human virus exposures during in vitro work were overseen and approved by Environmental Health and Safety and the Institutional Biosafety Committee (IBC Approval #BA18-MCDB-SAW-01) at the University of Colorado, Boulder, Colorado, USA. Virus stocks and biologic samples from infected animals were handled with biosafety level 3 operating procedures within a biosafety level 2 physical facility. Prior to animal testing, all virus stocks were first confirmed by a third-party laboratory (VELA Diagnostics) to be susceptible to the drugs used in standard post-exposure prophylaxis treatment, typically tenofovir, emtricitabine, and raltegravir 97. This ensured that any research personnel, in case of accidental exposure to viruses or virus-exposed animals, could be successfully treated with standard post-exposure protocols. All procedures instituted for risk minimization and management of human virus exposures during in vivo experimentation were overseen by Environmental Health and Safety and were approved by the Institutional Biosafety Committee (IBC Approval #s: RM00002884-RN00 and RM00005639-RN00) at The University of Texas, MD Anderson Cancer Center.

Study compliance

All animal studies were performed at the Keeling Center for Comparative Medicine and Research (KCCMR) located in Bastrop, TX. The KCCMR is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International and the KCCMR personnel and veterinary staff are highly experienced with owl monkey husbandry and manipulations. All animal work was approved by The University of Texas, MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC Protocols: 00001839-RN00, 00001839-RN00, 00000451-RN02, and 00000451-RN03) and was performed in accordance with the standards established by the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, the Office of Animal Welfare, and the United States Department of Agriculture.

Data Availability

All data are available within this manuscript or its supporting files. Sequencing data generated in this study have been deposited in the GenBank database under accession numbers PX876883 - PX877642, PX877926-PX878685, PX911634 - PX912013, PX925169 - PX925171. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Meyerson and colleagues aim to establish Nancy Ma’s owl monkeys as an animal model for HIV-1 infection. By genetically characterizing this species, test the impact of some potentially restrictive owl monkey genes, and replacing the viral vif gene together with a limited number of point-mutations, they generated derivatives of the T-cell line adapted CXCR4-tropic HIV-1 NL4-3 strain that replicate at least to some extent in Owl monkeys.\n\nStudies of SIV or SHIVs in macaque species are valuable but also have significant limitations. Thus, an Owl monkey model for studies on key features of human HIV-1 infection would be highly valuable. The present study makes significant steps towards it and, for most part, the results are clearly presented. However, as the authors mention they “are just at the beginning of characterizing the owl monkey model”. As outlined below, the results suggest that in its current state the model has several limitations that should be clearly mentioned and discussed. While the study represents a significance advance, evidence that owl monkeys can also be infected with analogous derivatives of more relevant CCR5-tropic HIV-1 strains would greatly increase its significance.\n\nMajor points\n1. The study used i.v. injection with high doses of derivatives of the T cell line adapted CXCR4-tropic HIV-1 NL4-3 molecular clone. While the owl monkeys show some evidence for HIV-1 replication during the acute phase, the virus seems to be rapidly and efficiently controlled in most animals. Thus, the value of this model for studies on virus transmission and pathogenesis, as well as the development of antiviral drugs or vaccines seems limited. While the authors mention some limitations, including the limited relevance of NL4-3 for primary HIV-1 infection, these other limitations should also be clearly stated.\n2. Evidence that owl monkeys provide a model for CCR5-tropic HIV-1 strains would greatly increase the significance of the study. While this might be beyond the scope of the present study that is already data-rich, the authors note that they “have now identified HIV-1 strains that can enter cells via owl monkey CCR5 and these viruses are in development and in in vivo studies. They should consider to add some of these data to provide a perspective of their model for better mimicking HIV-1 infection in humans.\n\nMinor points\n3. While Figure 1 illustrates that the authors analyzed an enormous number of samples from various monkey species, the results of these analyses that led to the focus on Nancy Ma’s owl monkeys are not reported. Either some results of these analyses should be reported or Fig. 1a and 1b omitted and 1c be included in the current Fig. 2. Currently it adds little to the present study.\n4. The authors write in the 1st sentences of the abstract and introduction that “HIV-1 does not infect any mammal other than chimpanzees”. Suggest to modify since humans are also a mammalian species. Of limited relevance here (since not suitable as animal model) but perhaps noteworthy: Gorillas may also be susceptible to HIV-1 M as they represent the original host of HIV-1 O and P.\n5. Fig. 2: Shouldn’t it be “4” instead of “2” in the circle underneath 104 in panel B? Are the sequences generated in this study available?\n6. Fig. 3: The features of the HIV-1 Env from isolate US1.EC6 and the rational for why it was selected for analysis should be briefly provided in the text.\n7. Fig. 4: Why was variant 30 selected for analysis in panel B?\n8. Fig. 6: the period of undetectable viral loads prior to dexa-methasome treatment was very short. Thus, conclusions about viral persistence should be cautioned.\n9. Do the authors have an idea why TRIMCyp variants 8 does not restrict HIV-1? Would it make sense to breed animals harboring this variant for future study? It is stated “Because variant 8 is rare in the colony…”. Specific information on its frequency (hetero- and homozygous) should be provided.\n10. The authors write “HIVnom3, is characterized by four new amino-acid substitutions: in p24(CA) (H120R), Tat (A58T), and Env (L10W in the signal peptide; D545G in the gp41 subunit)”. They should briefly discuss why these changes may be advantageous for viral replication in owl monkeys and which of these substitutions is uncommon in circulating HIV-1 strains.\n11. Were the TCID50`s determined in the transfusion experiments? If yes, they should be provided.", "reviewer_2": "This study describes the initial characterization of Nancy Ma owl monkeys as a primate model for HIV-1 infection This work includes targeted modification of HIV-1 to overcome cellular restriction factors in owl monkeys. The authors surveyed common primate research species for models that have viral entry and restriction factor genes permissible to HIV-1 infection. Blocks to HIV-1 infection by TRIMCyp and APOBEC3G were overcome by modifying HIV-1 to evade, or degrade those factors based on characterization of related SIV proteins that can overcome these restriction factors. Four additional mutations arose in one owl monkey that were shown in in vivo competition infections to confer increased fitness. Owl monkeys infected with this modified HIV-1 recapitulated some features of HIV-1 infection in humans. The strengths of this study are the evolution inspired design of modifications of HIV-1 to escape owl monkey restriction factors TRIMCyp and APOBEC3G and the in vivo competition experiment demonstrating the improved fitness of an in vivo selected virus. Additional discussion and consideration of how this model compares to existing, long-studied rhesus and pig-tailed macaque primate models is merited. Furthermore, the manuscript would benefit from more explicit discussion on how this model in its current or potentially further-developed form would address practical limitations in HIV/AIDS research and vaccine/therapeutic development. This new primate model of HIV infection does not recapitulate all aspects of HIV pathogenesis observed in humans. It does appear that some aspects of immune responses could be modeled in this system but further development of the model may be required.\n\nMajor Revisions:\nThe authors provide comparisons of HIV in the owl monkey model to HIV infection of humans. Missing from the discussion is explicit comparison of this model to the rhesus and pig-tailed macaque infection models. This should be addressed.\n\nMinor Revisions:\nCD8 T cells are a major component of the immune response to HIV infection and provide a level of immune control in PWH. At a minimum some discussion of expectations of the role of CD8 T cells in controlling infection in this model is warranted.\n“Although SIVs and SHIVs mimic many aspects of HIV-1 infection and immune control, they differ from HIV-1 in most gene products” – it is not made clear why this is so important. For example, one could argue that having an HIV-like genome is most important for characterizing CD8 T cell responses to infection (which is not evaluated or described in any detail here) as this would include epitopes for MHC presentation in the model that are the same as HIV-1 in humans.\nAPOBEC3G is chosen as the primary APOBEC3 family member to explore and test in this manuscript. However, other APOBEC3 paralogs may be important lentiviral restrictors in owl monkeys. These other APOBEC3s may also be antagonized the SIV Vifs used in “HIVnom1” and “HIVnom2”. This should be acknowledged in the text.\n“Animal-to-animal variation was observed in peripheral CD4+ T cell decline during the acute phase (Fig. S8)” is FigS6 the data showing variation in CD4 T cell decline? Or is there additional data from additional monkeys? It seems that drawing conclusions from three monkeys is not warranted. Instead, it does not seem that CD4 levels in this model have at this point been rigorously evaluated.\nThe discussion of “set point” viremias is odd. It’s not clear if these set points represent spurious, low level viral production or ongoing low-level replication in CD4+ cells for example. The “set points” are all essentially at the limit of detection of the assays making them difficult to interpret. The low viremias observed in the more adapted virus here is quite low and it’s unclear if this represents immune control (by CD8s for example) or simply inefficient, low level replication in these monkeys. Additional discussion should be added to clarify what the observed “set point” does and does not mean given the data presented.\nThe sequencing data for all genes from Figure 1 should be deposited and made available to the broader research community.\nFigure 4B –a brief explanation in the main text of the V86A mutation included in this Figure would be helpful\nFigure 8A – this is unclear – what is being shown?\nSup Figure S14 -16 –change Figure S15 to Figure S13\nSup Figure S21 – change TMB-bl cells to TZM-bl cells\nPage 5-6 “This mode of evolution is known to produce highly divergent proteins between species and between individuals within populations 2,3,25-33” reference 32 does not appear to support this statement.\nPage 20 line 3 – “we have now identified HIV-1 strains that can enter cells via owl monkey CCR5”, inclusion of this data should be considered", "reviewer_3": "Meyerson et al reported here infection in Owl monkeys (Aotus nancumaae) with adapted HIV-1 providing thus insights in molecular determinants of susceptibility to HIV replication. The group has analyzed the sequences of 191 Owl monkeys in regions encoding the CD4, the CCR5, and the CXCR4, receptors as well as three HIV-1 restriction factors. They found alleles encoding highly divergent proteins between species and individuals that may account for HIV-1 restriction of replication. The owl monkey CD4 and Tetherin support HIV-1 replication but TRIMCyp and APOBECC3G protein variants inhibit HIV-1. Engineered HIV-1 to encode four substitutions present in TRIMCyp red-capped mangabey and replacement of vif gene by SIV SIVmne (pigtailed macaque) or SIVmac (rhesus macaque) sequences bypassed restriction in cell culture assays.\n\nThe authors then produced HIV-1 genetically modified clones (HIVnom1 and HIVnom2) encoding the full-length HIV-1 genome of NL4-3 strain with the four point mutations reconstituting the SIVrcm cyclophilin A-binding loop and SIVmne/SIVmac vif gene. First and second passage in Owl monkey following IV infusion and plasma transfer, respectively, result in poor in vivo replication but persistent infection since viral rebound could be induced by Dexamethasone treatment. The second passage isolated sequence (HIVnom3) presented four non-synonymous mutations in P24, TAT, ENV (two mutations) probably accounting for Owl monkey adaptation. Third in vivo passage results in increase viremia peaking between week five and six in one of the transfused animals. The viral RNA copies quantification indicated low level of virus replication in lymph nodes. Infection induced specific binding antibodies and T cell responses but no signs of immunodeficiency (CD4 counts decrease) or simian AIDS were observed.\n\nThere is no strong novelty of the work except that infection of new world monkeys can be achieved with HIV-1 providing engineered adaptation of the virus through mutations affecting TRIMCyp and APOBEC3G induced restriction and acquisition of several mutations in tat, gag and env genes. Infection with HIV-1 have previously being achieved in pigtailed macaques with similar low levels of replication. Therefore, this is not the first successful attempt to adapt HIV-1 to non-human primates’ infection. Levels of viremia to the level of HIV-1 infection in non-treated humans could not be achieved, neither disease was observed, although at least these two parameters are critical to use the model for pathogenesis studies and assessment of vaccine and therapeutic strategies.\n\nMinor comments:\n1. We may consider the concept of “reverse zoonosis” a bit abusive. The term zoonosis is already including spillover from human to other animal species. In addition, zoonosis or “reverse zoonosis” implies regional colocalization of the species and/or the vector. Aotus monkeys are new world primates and would have thus never contribute to cross HIV/SIV emergence.\n2. The sentence “One of the most exciting moments” is not appropriate. Authors should make a “factual” description of the work.\n3. Same comment for “exiting results” mentioned two lines later. Only readers can make this statement.\n4. The indication that HIV-1 Env cannot use CD4 in other primate species including macaques is not true. Macaques CD4 support entry of many SHIV viruses made of an SIV backbone in which env gene is replaced by the HIV-1 gene. Restriction usually occurs after viral entry and not at CD4 and coreceptors level.\n5. It is not true to say that NL4-3 strain of HIV-1 is the most well studied strain. It may not even be the most relevant due to its capacity to bypass the use of CD4 by entry through CXCR4 co-receptor engagement only."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "Meyerson and colleagues revised the manuscript and added new data to support the conclusion that owl monkeys recapitulate features of human HIV-1 pathogenesis, including viral replication in GALT and lymph nodes, chronic low-level viral transcription in reservoirs, and development of HIV-1–specific and neutralizing antibodies. They also added a new section with RNAscope tissue analyses as well as experiments indicating that lower-dose inoculation may result in more sustained chronic viremia. The Introduction and Discussion were expanded to more explicitly compare their model to SIV/SHIV and pig-tailed macaque systems.\n\nThe manuscript contains a large amount of data and an immune-competent primate model for HIV-1 infection would be of significant interest. However, although the authors made an effort to address previous concerns the value of this model for most part remain speculative and some results should be presented and discussed in a more critical manner.\n\nSpecific comments\n1. The authors report that the model captures essential features of acute HIV-1 infection. However, it remains unclear whether some characteristics, such as transient CD4 decline and acute IFN induction, were observed.\n2. As previously mentioned, all results were obtained using X4-tropic HIV-1, while the vast majority of infections in humans is caused by R5-tropic HIV-1. As mentioned by the authors, AIDS progression in humans usually takes 6 to 10 years in absence of ART. However, median survival is only about 1–3 years after X4-HIV-1 variants first become detectable (DOI: 10.7326/0003-4819-118-9-199305010-00004; 10.1086/591623). In addition, rare X4-tropic SIV strains cause rapid CD4 T cell depletion in other primate species (DOI: 10.4049/jimmunol.179.5.3047). Since the authors used X4 HIV-1 it seems more appropriate to compare it to X4 and not R5 HIV-1 in humans.\n3. The results of the dosing experiments show that at least in some animals, virus is still detectable after about 20 weeks (Fig. 8) albeit at about 10- to 100-fold lower levels compared to HIV-1 infected humans. Whether or not this will lead to disease remains a matter of speculation and again it needs to be considered that X4 HIV-1, which is typically associated with rapid disease progression in humans was used.\n4. Due to the CXCR4 tropism of HIVnom3 and intravenous inoculation, the model does not reproduce early events characteristic of mucosal CCR5-tropic HIV-1 transmission, including preferential CCR5⁺ memory CD4⁺ depletion. As previously noted: while the present X4 HIV-1 model may have some value some supportive evidence that R5-tropic HIV-1 replicates in this model would greatly increase its significance.\n5. Given that follow-up is still relatively short and infection well controlled in most animals, the potential value for vaccine and cure studies should be presented in a more critical manner.", "reviewer_2": "The authors have adequately addressed the concerns raised in our initial review either through inclusion of new data or in discussion. This revised manuscript more appropriately places this work in the context of existing rhesus and pig-tailed macaque primate models and highlights the potential advantages that an HIV owl monkey model may provide in regard to understanding T cell mediated immunity. The new viral dose experiments included suggest that higher viral doses may in fact result in chronic viremia at a detectable set point that more closely mirrors what is observed during HIV infection in humans. There does seem to be some variability still in these data and the significance of the lower set point is difficult to gauge. Overall, this is a significant body of work developing a novel, immunocompetent model of HIV infection.", "reviewer_3": "The authors have included new data and proposed changes that partially respond to the reviewers’ comments. In particular, new convincing results indicate persistence of replicative virus in lymph nodes and GALT. In addition, infection with higher doses (10,000 Infectious units) of HIVnom3 induce persistent detectable viremia in several animals.\n\nHowever, many remarks and conclusions remain inaccurate. The authors should also avoid any sense of self-satisfaction or triumphalism, and such comments should receive particular attention from the editor.\n\nThe studies presented are informative regarding the mechanisms of HIV infection and suggest that providing significant improvement (e.g., CCR5-tropic viruses, CD4 count decline, quality of humoral and T-cell responses) the model could potentially enable the preclinical evaluation of preventive or therapeutic strategies. However, the results do not support the model's relevance at its current stage of development.\n\nBelow are some examples of responses to reviewers are erroneous and changes in the manuscript that should be corrected or removed:\n- The fact that 4/6 monkeys infected with HIVnom3 “were non-viremic because they had been infected at high doses and had developed the elite controller phenotype that we see under such conditions” is not supported by the literature in the field. Higher challenge doses result in most of the cases in higher level of infection and rapid progression to disease.\n- We cannot agree with the comment: “We now show that owl monkeys should be a rich model for studying HIV-1 pathogenesis – they correctly recapitulate the viral replication kinetics, tissue distribution, immune responses, reservoir formation, and the slow onset of immunodeficiency related illnesses that are observed in humans. So far, our model does actually recapitulate all tested aspects of HIV pathogenesis observed in humans.” The paper do not bring evidences on many of these aspects, significant improvement still required.\n- The following response to reviewers is inaccurate: “Since CXCR4-tropic viruses will be critical targets for successful cure strategies in humans, the modeling of these viruses in owl monkeys may be one key area where new advances could be made over those possible in current SIV macaque models.” There is not evidence today that CXCR4-tropic viruses are more relevant for cure that CCR5 tropic viruses.\n- We may not agree with the following sentence in the introduction section. “Our major accomplishment here is the closing of this critical gap in HIV-1research.” Readers should make the own conclusion and may strongly disagree.\nRoger Le Grand"}, "author_response": "Dear reviewers:\nWe respectfully ask you to reconsider our revised manuscript on the owl monkey model for HIV-1. We have\nadded critical new data to the paper"}]

Decision letter