Indian-origin Rhesus Macaques Research Articles

Quantitative and Qualitative Distinctions between HIV-1 and SIV Reservoirs: Implications for HIV-1 Cure-Related Studies.

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Defining genetic diversity of rhesus macaque Fcγ receptors with long-read RNA sequencing.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Hematological and biochemical reference intervals of wild-caught and inhouse adult Indian rhesus macaques (Macaca mulatta)

BackgroundNonhuman primates are used for research purposes such as studying diseases and drug discovery and development programs. Various clinical pathology parameters are used as biomarkers of disease conditions in biomedical research. Detailed reports of these parameters are not available for Indian-origin rhesus macaques. To meet the increasing need for information, we conducted this study on 121 adult Indian rhesus macaques (57 wild-sourced and 64 inhouse animals, aged 3–7 years). A total of 18 hematology and 18 biochemistry parameters were evaluated and reported in this study. Data from these parameters were statistically evaluated for significance amongst inhouse and wild-born animals and for differences amongst sexes. The reference range was calculated according to C28-A3 guidelines for reporting reference intervals of clinical laboratory parameters.ResultsSource of the animals and sex appeared to have statistically significant effects on reference values and range. Wild-born animals reported higher WBC, platelets, neutrophils, RBC, hemoglobin, HCT, MCV, and total protein values in comparison to inhouse monkeys. Sex-based differences were observed for parameters such as RBCs, hemoglobin, HCT, creatinine, calcium, phosphorus, albumin, and total protein amongst others.ConclusionsThrough this study, we have established a comprehensive data set of reference values and intervals for certain hematological and biochemical parameters which will help researchers in planning, conducting, and interpreting various aspects of biomedical research employing Indian-origin rhesus monkeys.

Isoniazid and rifapentine treatment effectively reduces persistent M. tuberculosis infection in macaque lungs.

A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.

Abstract 1325: Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency

Abstract Background Non-human primates (NHP) such as rhesus macaques with naturally occurring cancers are a proposed model for translational cancer immunotherapy (CIT) research and have generated relevant proof-of-mechanism evidence for 3 different CIT agents. NHP spontaneously develop cancers with progression patterns, histology, and clinical symptoms similar to humans. Gene suppression by DNA hypermethylation in the promoter region is the major characteristics of the CpG-island-methylator-phenotype (CIMP) described in human CRC patients but information in rhesus macaques is scarce. To further validate these animals as translational models for CIT, we conducted a deep molecular characterization of NHP colorectal cancers and established novel qPCR panels to assess DNA methylation of marker genes published for humans. Methods Our cohort (n=16) consisted of Indian-origin rhesus macaques (Macaca mulatta) with naturally occurring CRC (n=16, female=11). Clinical examination, imaging (contrast-enhanced CT, FDG-PET) and biopsy to confirm cancer histology were performed. Molecular characterization was done by IHC for CRC-associated mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 and by PCR/electrophoresis for microsatellite instability. Ultimately, we designed DNA methylation- and rhesus-specific qPCR probes (Methylight) targeting corresponding regions as published in human patients, including CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1 as parts of the CIMP panel and BMP3, NDRG4, and SEPTIN9 as probed for human CRC-screening. Results MLH1 deficiency by IHC, in conjunction with PMS2 absence, is observed in all NHP CRC cases, clearly exceeding frequencies reported in human CRCs (ranging from 2-15%). Moreover, we have documented microsatellite instable cases in some NHP CRCs, analog to human CRCs. DNA methylation of the MLH1 promoter region was significantly elevated in CRCs (100% of CRCs >2-fold, p<0.0001) compared to healthy colon. We hypothesized that this elevation would suppress MLH1 mRNA expression. This hypothesis of epigenetic suppression is corroborated by both qPCR and RNA sequencing which demonstrate significantly downregulated levels of MLH1 mRNA. DNA methylation of the other markers is less consistent but revealed CIMP positive and CIMP negative cases in our NHP CRC cohort. Conclusions Transcriptional suppression of MLH1 by promoter hypermethylation is a major and widespread driver of genetic instability and carcinogenesis in rhesus macaque colorectal cancer. Differential DNA methylation in the promoter regions as observed in NHP CRCs can provide a screening target for liquid biopsies. This work highlights the possible translatability of naturally occurring NHP cancers for human cancer immunotherapy research and can be further explored in future tumor-bearing monkey trials. Citation Format: Simon Deycmar, Brendan Johnson, Declan Ryan, Shane Sills, David Caudell, Greg Dugan, George Schaaf, Christopher Whitlow, Kiran Kumar Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Karina Ray, Cassandra Cullin, Brandy Dozier, Armando Burgos, Michael Hettich, Bruno Gomes, Jehad Charo, Maurizio Ceppi, Mark Cline. Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1325.

Cooperation Between Systemic and Mucosal Antibodies Induced by Virosomal Vaccines Targeting HIV-1 Env: Protection of Indian Rhesus Macaques Against Low-Dose Intravaginal SHIV Challenges.

A virosomal vaccine inducing systemic/mucosal anti-HIV-1 gp41 IgG/IgA had previously protected Chinese-origin rhesus macaques (RMs) against vaginal SHIVSF162P3 challenges. Here, we assessed its efficacy in Indian-origin RMs by intramuscular priming/intranasal boosting (n=12/group). Group K received virosome-P1-peptide alone (harboring the Membrane Proximal External Region), Group L combined virosome-rgp41 plus virosome-P1, and Group M placebo virosomes. Vaccination induced plasma binding but no neutralizing antibodies. Five weeks after boosting, all RMs were challenged intravaginally with low-dose SHIVSF162P3 until persistent systemic infection developed. After SHIV challenge #7, six controls were persistently infected versus only one Group L animal (vaccine efficacy 87%; P=0.0319); Group K was not protected. After a 50% SHIV dose increase starting with challenge #8, protection in Group L was lost. Plasmas/sera were analyzed for IgG phenotypes and effector functions; the former revealed that protection in Group L was significantly associated with increased binding to FcγR2/3(A/B) across several time-points, as were some IgG measurements. Vaginal washes contained low-level anti-gp41 IgGs and IgAs, representing a 1-to-5-fold excess over the SHIV inoculum’s gp41 content, possibly explaining loss of protection after the increase in challenge-virus dose. Virosomal gp41-vaccine efficacy was confirmed during the initial seven SHIV challenges in Indian-origin RMs when the SHIV inoculum had at least 100-fold more HIV RNA than acutely infected men’s semen. Vaccine protection by virosome-induced IgG and IgA parallels the cooperation between systemically administered IgG1 and mucosally applied dimeric IgA2 monoclonal antibodies that as single-agents provided no/low protection – but when combined, prevented mucosal SHIV transmission in all passively immunized RMs.

VDJ Gene Usage in IgM Repertoires of Rhesus and Cynomolgus Macaques.

Macaques are frequently used to evaluate candidate vaccines and to study infection-induced antibody responses, requiring an improved understanding of their naïve immunoglobulin (IG) repertoires. Baseline gene usage frequencies contextualize studies of antigen-specific immune responses, providing information about how easily one may stimulate a response with a particular VDJ recombination. Studies of human IgM repertoires have shown that IG VDJ gene frequencies vary several orders of magnitude between the most and least utilized genes in a manner that is consistent across many individuals but to date similar analyses are lacking for macaque IgM repertoires. Here, we quantified VDJ gene usage levels in unmutated IgM repertoires of 45 macaques, belonging to two species and four commonly used subgroups: Indian and Chinese origin rhesus macaques and Indonesian and Mauritian origin cynomolgus macaques. We show that VDJ gene frequencies differed greatly between the most and least used genes, with similar overall patterns observed in macaque subgroups and individuals. However, there were also clear differences affecting the use of specific V, D and J genes. Furthermore, in contrast to humans, macaques of both species utilized IGHV4 family genes to a much higher extent and showed evidence of evolutionary expansion of genes of this family. Finally, we used the results to inform the analysis of a broadly neutralizing HIV-1 antibody elicited in SHIV-infected rhesus macaques, RHA1.V2.01, which binds the apex of the Env trimer in a manner that mimics the binding mode of PGT145. We discuss the likelihood that similar antibodies could be elicited in different macaque subgroups.

#20: Modeling Zika virus tissue tropism in rhesus macaques to define the risk of donor derived transmission

Abstract Background Almost 115,000 people in the United States are currently on a transplant waitlist, which vastly exceeds the number of organ donors every year. This discrepancy emphasizes the need for retention of all possible donors. Those who have recently traveled to an area with an active outbreak of Zika virus (ZIKV) are often disqualified as a donor because immunosuppressed recipients would be at risk of a donor-derived ZIKV infection. Therefore, we define ZIKV tissue tropism and the risk of donor derived transmission. Methods We subcutaneously inoculated 15 Indian-origin rhesus macaques (RM) with a Puerto Rican isolate of ZIKV (PRVABC59). All RMs were inoculated in mid to early gestation.We inoculated during pregnancy because plasma viremia is typically prolonged in pregnancy and we wanted to model tissue tropism for donor derived transmission in the worst scenario of prolonged viremia. At 30, 65, and 105 days post-infection (dpi), the animals were euthanized and comprehensive necropsies were performed, which evaluated a minimum of 60 tissues per animal. ZIKV RNA was quantified in tissues via qRT-PCR. Results Plasma viremia duration was >10 days in 13 of 15 RMs. ZIKV RNA was most commonly detected in lymph nodes, with 19/45 lymph nodes that were vRNA positive in 5 RMs at 30 dpi. There were 15/45 vRNA positive lymph nodes at 60 dpi and 8/38 at 105 dpi. Reproductive and maternal fetal-interface (MFI) tissues were the second most commonly positive tissues. Twenty-five MFI tissues, including the amniotic/chorionic membrane, decidua, placenta, uterus, and placental bed, were positive, with 10/53 positive at 30 dpi, 14/24 positive at 60 dpi and 1/47 positive at 105 dpi. Other vRNA positive tissues included the primary bronchus, femoral vein, kidney, thyroid, lung, colon, mammary gland, pericardium, hand nerve, and sciatic nerve in 1–2 RMs at one of the three timepoints. Conclusions We found ZIKV RNA most frequently within lymph nodes. Lymph nodes are included in lung and small bowel transplants, indicating that these transplants could pose a risk of donor-derived ZIKV transmission. Virus detection within other commonly transplanted tissues, such as the kidney and blood vessels was much less common. We did not determine what fraction of vRNA comes from replication-competent virus in each tissue; some tissues with vRNA might not contain virions that could initiate new infections. Donor-derived Zika virus transmission from other commonly transplanted organs, such as liver, seems unlikely since no viral RNA was detected in this organ.

#79: Modeling Zika Virus Tissue Tropism in Rhesus Macaques to Define the Risk of Donor-derived Transmission

Abstract Background Almost 115,000 people in the United States are currently on a transplant waitlist, which vastly exceeds the number of organ donors every year. This discrepancy emphasizes the need for retention of all possible donors. Those who have recently traveled to an area with an active outbreak of Zika virus (ZIKV) are often disqualified as a donor because immunosuppressed recipients would be at risk of a donor-derived ZIKV infection. Therefore, we define ZIKV tissue tropism and the risk of donor-derived transmission. Methods We subcutaneously inoculated 15 Indian-origin rhesus macaques (RM) with a Puerto Rican isolate of ZIKV (PRVABC59). All RMs were inoculated in mid to early gestation. We inoculated during pregnancy because plasma viremia is typically prolonged in pregnancy and we wanted to model tissue tropism for donor-derived transmission in the worst scenario of prolonged viremia. At 30, 65, and 105 days post-infection (dpi), the animals were euthanized and comprehensive necropsies were performed, which evaluated a minimum of 60 tissues per animal. ZIKV RNA was quantified in tissues via qRT-PCR. Results Plasma viremia duration was >10 days in 13 of 15 RMs. ZIKV RNA was most commonly detected in lymph nodes, with 19/45 lymph nodes that were vRNA positive in 5 RMs at 30 dpi. There were 15/45 vRNA positive lymph nodes at 60 dpi and 8/38 at 105 dpi. Reproductive and maternal fetal-interface (MFI) tissues were the second most commonly positive tissues. Twenty-five MFI tissues, including the amniotic/chorionic membrane, decidua, placenta, uterus, and placental bed, were positive, with 10/53 positive at 30 dpi, 14/24 positive at 60 dpi and 1/47 positive at 105 dpi. Other vRNA positive tissues included the primary bronchus, femoral vein, kidney, thyroid, lung, colon, mammary gland, pericardium, hand nerve, and sciatic nerve in 1–2 RMs at one of the three timepoints. Conclusions We found ZIKV RNA most frequently within lymph nodes. Lymph nodes are included in lung and small bowel transplants, indicating that these transplants could pose a risk of donor-derived ZIKV transmission. Virus detection within other commonly transplanted tissues, such as the kidney and blood vessels was much less common. We did not determine what fraction of vRNA comes from replication-competent virus in each tissue; some tissues with vRNA might not contain virions that could initiate new infections. Donor-derived Zika virus transmission from other commonly transplanted organs, such as the liver, seems unlikely since no viral RNA was detected in this organ.

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles

Definition of the specific germline immunoglobulin (Ig) alleles present in an individual is a critical first step to delineate the ontogeny and evolution of antigen-specific antibody responses. Rhesus and cynomolgus macaques are important animal models for pre-clinical studies, with four main sub-groups being used: Indian- and Chinese-origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. We applied the (Ig) gene inference tool IgDiscover and performed extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. There was allelic overlap between Chinese- and Indian-origin rhesus macaques and also between the two macaque species, which is consistent with substantial admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet maintained high individual allelic diversity. These comprehensive databases of germline IGH alleles for rhesus and cynomolgus macaques provide a resource toward the study of B cell responses in these important pre-clinical models.

Reduced meiotic recombination in rhesus macaques and the origin of the human recombination landscape.

Characterizing meiotic recombination rates across the genomes of nonhuman primates is important for understanding the genetics of primate populations, performing genetic analyses of phenotypic variation and reconstructing the evolution of human recombination. Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primates in biomedical research. We constructed a high-resolution genetic map of the rhesus genome based on whole genome sequence data from Indian-origin rhesus macaques. The genetic markers used were approximately 18 million SNPs, with marker density 6.93 per kb across the autosomes. We report that the genome-wide recombination rate in rhesus macaques is significantly lower than rates observed in apes or humans, while the distribution of recombination across the macaque genome is more uniform. These observations provide new comparative information regarding the evolution of recombination in primates.

A Zika virus primary isolate induces neuroinflammation, compromises the blood-brain barrier and upregulates CXCL12 in adult macaques.

Zika virus (ZIKV) is a flavivirus that can cause neuropathogenesis in adults and fetal neurologic malformation following the infection of pregnant women. We used a nonhuman primate model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks of ZIKV-induced adult neuropathology. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. ZIKV infection resulted in specific short- and long-term augmented expression of the chemokine CXCL12 in the central nervous system (CNS)of adult IRMs. Moreover, CXCL12 expression persists long after the initial viral infection is apparently cleared. CXCL12 plays a key role both in regulating lymphocyte trafficking through the BBB to the CNS and in mediating repair of damaged neural tissue including remyelination. Understanding how CXCL12 expression is controlled will likely be of central importance in the definition of ZIKV-associated neuropathology in adults.

Rhesus and Cynomolgus IGH Genotyping Yields Comprehensive Databases of Germline VDJ Alleles for In-Depth Immunological Studies

Rhesus and cynomolgus macaques are important animal models for the pre-clinical assessment of candidate drugs and vaccines. Four main sub-groups are used: Indian and Chinese origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. Here, we applied the IG gene inference tool IgDiscover and extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. We observed a significant allelic overlap between Chinese and Indian origin rhesus macaques and, surprisingly, also between the two macaque species, consistent with significant levels of admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet they maintained high individual allelic diversity. Our analysis resulted in comprehensive DBs of germline IGH alleles for rhesus and cynomolgus macaques, of which 64% and 79% of alleles respectively were not previously described, thereby facilitating high-quality B cell response studies in these species.

Comparison of immunogenicity and safety outcomes of a malaria vaccine FMP013/ALFQ in rhesus macaques (Macaca mulatta) of Indian and Chinese origin

BackgroundIndian-origin rhesus (InR) are preferred for research, but strict export restrictions continue to limit their use. Chinese-origin rhesus (ChR), although easier to procure, are genetically distinct from InR and differ in their immune response to infectious agents, such as the Simian Immunodeficiency Virus. The most advanced malaria vaccine, RTS,S (GlaxoSmithKline), is based on the circumsporozoite protein (CSP) of Plasmodium falciparum. The efficacy of RTS,S vaccine in the field remains low and short-lived; efforts are underway to improve CSP-based vaccines. Rhesus models can accelerate preclinical down-selection of the next generation of malaria vaccines. This study was used to determine if the safety and immunogenicity outcomes following vaccination with a CSP vaccine would differ in the InR and ChR models, given the genetic differences between the two sub-populations of rhesus.MethodsThe FMP013 vaccine, was composed of nearly full-length soluble P. falciparum CSP produced in Escherichia coli and was adjuvanted with the Army liposomal formulation (ALFQ). Three doses of the vaccine were administered in InR and ChR (n = 6) at 1-month intervals and the antibody and T cell responses were assessed.ResultsLocal and systemic toxicity profile of FMP013 vaccine in InR and ChR were similar and they revealed that the FMP013 vaccine was safe and caused only mild and transient inflammatory adverse reactions. Following the first 2 vaccines, there was a slower acquisition of antibodies to the CSP repeat region in ChR. However after the 3rd vaccination the titers in the two models were comparable. The ChR group repeat-specific antibodies had higher avidity and ChR group showed higher inhibition of liver stage development activity compared to InR. There was no difference in T-cell responses to the FMP013 vaccine between the two models.ConclusionsA difference in the quality of serological responses was detected between the two sub-populations of rhesus. However, both models confirmed that FMP013/ALFQ vaccine was safe, highly immunogenic, elicited functional antibodies and T-cell responses. Overall, the data suggests that rhesus of Indian and Chinese origins can be interchangeably used to compare the safety and immunogenicity of next-generation of malaria vaccines and adjuvants.

Rational design and in vivo selection of SHIVs encoding transmitted/founder subtype C HIV-1 envelopes.

Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 103 to 107 vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs.

Neutrophil progenitor populations of rhesus macaques.

Captive-bred rhesus macaques of Indian origin represent one of the most important large animal models for infectious disease, solid organ transplantation, and stem cell research. There is a dearth of information defining hematopoietic development, including neutrophil leukocyte differentiation in this species using multicolor flow cytometry. In the current study, we sought to identify cell surface markers that delineate neutrophil progenitor populations with characteristic immunophenotypes. We defined four different postmitotic populations based on their CD11b and CD87 expression pattern, and further refined their immunophenotypes using CD32, CD64, lactoferrin, and myeloperoxidase as antigenic markers. The four subsets contained myelocyte, metamyelocyte, band, and segmented neutrophil populations. We compared our flow cytometry-based classification with the classical nuclear morphology-based classification. We found overlap of immunological phenotype between populations of different nuclear morphology and identified phenotypically different subsets within populations of similar nuclear morphology. We assessed the responsiveness of these populations to stimulatory signals, such as LPS, fMLP, or PMA, and demonstrated significant differences between human and rhesus macaque neutrophil progenitors. In this study, we provided evidence for species-specific features of granulopoiesis that ultimately manifested in the divergent immunophenotypes of the fully differentiated segmented neutrophils of humans and rhesus macaques. Additionally, we found functional markers that can be used to accurately quantify neutrophil progenitors by flow cytometry. Although these markers do not coincide with the classical nuclear-morphology-based grading, they enable us to perform functional studies monitoring immunophenotypic markers.

Intragastric Administration of Lactobacillus plantarum and 2,2'-Dithiodipyridine-Inactivated Simian Immunodeficiency Virus (SIV) Does Not Protect Indian Rhesus Macaques from Intrarectal SIV Challenge or Reduce Virus Replication after Transmission.

A major obstacle to development of an effective AIDS vaccine is that along with the intended beneficial responses, the immunization regimen may activate CD4+ T cells that can facilitate acquisition of human immunodeficiency virus (HIV) by serving as target cells for the virus. Lu et al. (W. Lu et al., Cell Rep 2:1736-1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIVmac239 and Lactobacillus plantarum (iSIV-L. plantarum) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIVmac239 challenge at 3 months postimmunization. They attributed the observed protection to induction of immune tolerance, mediated by "MHC-Ib/E-restricted CD8+ regulatory T cells that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T." J.-M. Andrieu et al. (Front Immunol 5:297, 2014, https://doi.org/10.3389/fimmu.2014.00297) subsequently reported protection from infection in 23/24 RMs immunized intragastrically or intravaginally with iSIV and Mycobacterium bovis BCG, L. plantarum, or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our coauthors, we conducted an immunization and challenge experiment with 54 Indian RMs and included control groups receiving iSIV only or L. plantarum only as well as unvaccinated animals. Intrarectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV-L. plantarum-vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or L. plantarum only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV-L. plantarum administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.IMPORTANCE Despite an increased understanding of immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4+ T cells that may act as target cells for acquisition of HIV. An alternative strategy may involve induction of a tolerance-inducing response that limits the availability of activated CD4+ T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained for Chinese-origin rhesus macaques by using a "tolerogenic" vaccine, consisting of intragastric administration of Lactobacillus plantarum and 2,2'-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV-L. plantarum to Indian-origin rhesus macaques and failed to observe any protective effect on virus acquisition in this experimental setting. This work is important because it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on iSIV-L. plantarum.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.

In Vitro and In Vivo Models of HIV Latency.

Latently infected cells are very infrequent in CD4+ T cells from antiretroviral (ARV) treated individuals, with only approximately one in a million infected CD4+ T cells in blood. Given the low frequency of infected cells in vivo, multiple in vitro latency models have been developed to facilitate investigations into mechanisms of HIV latency, as well as to enable the evaluation of pharmacological and immunological interventions aimed at depleting latently infected cells. These in vitro models include clones of transformed cell lines with integrated HIV proviruses or primary CD4+ T cells from uninfected donors that have been infected with HIV in particular conditions. This chapter presents a description of these various in vitro models, along with an overview of their advantages and limitations.Preclinical animal models represent a critical bridge between in vitro studies and human clinical trials. Simian immunodeficiency virus (SIV) infection of Indian origin rhesus macaques has been well established as an informative model of HIV infection. Recent years have seen breakthroughs in ARVs that permit the potent suppression of SIV replication, enabling studies of latency and putative curative interventions in this model. Small animal models of HIV infection can be generated by engrafting immunodeficient mice with human immune cells. These "humanized mice" have provided valuable insights into HIV pathogenesis and are under development as models for studying HIV latency. We summarize both the promise of these models and outstanding challenges that remain to be overcome to realize their potential to inform efforts to cure HIV infection.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.