Enhancement of Microbiota in Healthy Macaques Results in Beneficial Modulation of Mucosal and Systemic Immune Function.

Given the critical role of mucosal surfaces in susceptibility to infection, it is imperative that effective mucosal responses are induced when developing efficacious vaccines and prevention strategies for infection. Modulating the microbiota in the gastrointestinal (GI) tract through the use of probiotics (PBio) is a safe and well-tolerated approach to enhance mucosal and overall health. We assessed the longitudinal impact of daily treatment with the VSL#3 probiotic on cellular and humoral immunity and inflammation in healthy macaques. PBio therapy resulted in significantly increased frequencies of B cells expressing IgA in the colon and lymph node (LN), likely because of significantly increased LN T follicular helper cell frequencies and LN follicles. Increased frequencies of IL-23(+) APCs in the colon were found post-PBio treatment, which correlated with LN T follicular helper cells. Finally, VSL#3 significantly downmodulated the response of TLR2-, TLR3-, TLR4-, and TLR9-expressing HEK293 cells to stimulation with Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and ODN2006, respectively. These data provide a mechanism for the beneficial impact of PBio on mucosal health and implicates the use of PBio therapy in the context of vaccination or preventative approaches to enhance protection from mucosal infection by improving immune defenses at the mucosal portal of entry.

Effects of Fecal Microbial Transplantation on Microbiome and Immunity in Simian Immunodeficiency Virus-Infected Macaques.

UNLABELLED: An altered intestinal microbiome during chronic human immunodeficiency virus (HIV) infection is associated with mucosal dysfunction, inflammation, and disease progression. We performed a preclinical evaluation of the safety and efficacy of fecal microbiota transplantation (FMT) as a potential therapeutic in HIV-infected individuals. Antiretroviral-treated, chronically simian immunodeficiency virus (SIV)-infected rhesus macaques received antibiotics followed by FMT. The greatest microbiota shift was observed after antibiotic treatment. The bacterial community composition at 2 weeks post-FMT resembled the pre-FMT community structure, although differences in the abundances of minor bacterial populations remained. Immunologically, we observed significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4(+) T cell activation in gastrointestinal tissues post-FMT. Importantly, the transplant was well tolerated with no negative clinical side effects. Although this pilot study did not control for the differential contributions of antibiotic treatment and FMT to the observed results, the data suggest that FMT may have beneficial effects that should be further evaluated in larger studies. IMPORTANCE: Due to the immunodeficiency and chronic inflammation that occurs during HIV infection, determination of the safety of FMT is crucial to prevent deleterious consequences if it is to be used as a treatment in the future. Here we used the macaque model of HIV infection and performed FMT on six chronically SIV-infected rhesus macaques on antiretroviral treatment. In addition to providing a preclinical demonstration of the safety of FMT in primates infected with a lentivirus, this study provided a unique opportunity to examine the relationships between alterations to the microbiome and immunological parameters. In this study, we found increased numbers of Th17 and Th22 cells as well as decreased activation of CD4(+) T cells post-FMT, and these changes correlated most strongly across all sampling time points with lower-abundance taxonomic groups and other taxonomic groups in the colon. Overall, these data provide evidence that changes in the microbiome, particularly in terms of diversity and changes in minor populations, can enhance immunity and do not have adverse consequences.

Emerging diagnostic challenges and characteristics of simian betaretrovirus infections in captive macaque colonies.

To better understand Simian betaretrovirus (SRV) seropositivity in virus-negative macaques, we transfused blood from SRV-infected or suspect donors into immunosuppressed naive recipients. Our results do not support typical SRV1-5 infection as the cause, but provide evidence for several possibilities including serological artifact, new/different SRV, or an endogenous virus.

Deep transcriptional sequencing of mucosal challenge compartment from rhesus macaques acutely infected with simian immunodeficiency virus implicates loss of cell adhesion preceding immune activation.

Pathology resulting from human immunodeficiency virus (HIV) infection is driven by protracted inflammation; the primary loss of CD4(+) T cells is caused by activation-driven apoptosis. Recent studies of nonhuman primates (NHPs) have suggested that during the acute phase of infection, antiviral mucosal immunity restricts viral replication in the primary infection compartment. These studies imply that HIV achieves systemic infection as a consequence of a failure in host antiviral immunity. Here, we used high-dose intrarectal inoculation of rhesus macaques with simian immunodeficiency virus (SIV) SIVmac251 to examine how the mucosal immune system is overcome by SIV during acute infection. The host response in rectal mucosa was characterized by deep mRNA sequencing (mRNA-seq) at 3 and 12 days postinoculation (dpi) in 4 animals for each time point. While we observed a strong host transcriptional response at 3 dpi, functions relating to antiviral immunity were absent. Instead, we observed a significant number of differentially expressed genes relating to cell adhesion and reorganization of the cytoskeleton. We also observed downregulation of genes encoding members of the claudin family of cell adhesion molecules, which are coexpressed with genes associated with pathology in the colorectal mucosa, and a large number of noncoding transcripts. In contrast, at 12 dpi the differentially expressed genes were enriched in those involved with immune system functions, in particular, functions relating to T cells, B cells, and NK cells. Our findings indicate that host responses that negatively affect mucosal integrity occur before inflammation. Consequently, when inflammation is activated at peak viremia, mucosal integrity is already compromised, potentially enabling rapid tissue damage, driving further inflammation. Importance: The HIV pandemic is one of the major threats to human health, causing over a million deaths per year. Recent studies have suggested that mucosal antiviral immune responses play an important role in preventing systemic infection after exposure to the virus. Yet, despite their potential role in decreasing transmission rates between individuals, these antiviral mechanisms are poorly understood. Here, we carried out the first deep mRNA sequencing analysis of mucosal host responses in the primary infection compartment during acute SIV infection. We found that during acute infection, a significant host response was mounted in the mucosa before inflammation was triggered. Our analysis indicated that the response has a detrimental effect on tissue integrity, causing increased permeability, tissue damage, and recruitment of SIV target cells. These results emphasize the importance of mucosal host responses preceding immune activation in preventing systemic SIV infection.

Correction: Antibiotic-induced microbiome perturbations are associated with significant alterations to colonic mucosal immunity in rhesus macaques.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibiotic-induced microbiome perturbations are associated with significant alterations to colonic mucosal immunity in rhesus macaques.

The diverse bacterial communities that colonize the gastrointestinal tract play an essential role in maintaining immune homeostasis through the production of critical metabolites such as short-chain fatty acids (SCFAs) and this can be disrupted by antibiotic use. However, few studies have addressed the effects of specific antibiotics longitudinally on the microbiome and immunity. We evaluated the effects of four specific antibiotics: enrofloxacin, cephalexin, paromomycin, and clindamycin, in healthy female rhesus macaques. All antibiotics disrupted the microbiome, including reduced abundances of fermentative bacteria and increased abundances of potentially pathogenic bacteria, including Enterobacteriaceae in the stool, and decreased Helicobacteraceae in the colon. This was associated with decreased SCFAs, indicating altered bacterial metabolism. Importantly, antibiotic use also substantially altered local immune responses, including increased neutrophils and Th17 cells in the colon. Furthermore, we observed increased soluble CD14 in plasma, indicating microbial translocation. These data provide a longitudinal evaluation of antibiotic-induced changes to the composition and function of colonic bacterial communities associated with specific alterations in mucosal and systemic immunity.

Intestinal damage precedes mucosal immune dysfunction in SIV infection.

HIV and pathogenic SIV infection are characterized by mucosal dysfunction including epithelial barrier damage, loss of Th17 cells, neutrophil infiltration, and microbial translocation with accompanying inflammation. However, it is unclear how and when these contributing factors occur relative to one another. In order to determine whether any of these features initiates the cycle of damage, we longitudinally evaluated the kinetics of mucosal and systemic T-cell activation, microbial translocation, and Th17 cell and neutrophil frequencies following intrarectal SIV infection of rhesus macaques. We additionally assessed the colon proteome to elucidate molecular pathways altered early after infection. We demonstrate increased T-cell activation (HLA-DR+) beginning 3-14 days post-SIV challenge, reduced peripheral zonulin 3-14 days post-SIV, and evidence of microbial translocation 14 days post-SIV. The onset of mucosal dysfunction preceded peripheral and mucosal Th17 depletion, which occurred 14-28 days post-SIV, and gut neutrophil accumulation was not observed. Proteins involved in epithelial structure were downregulated 3 days post-SIV followed by an upregulation of immune proteins 14 days post-SIV. These data demonstrate that immune perturbations such as Th17 loss and neutrophil infiltration occur after alterations to epithelial structural protein pathways, suggesting that epithelial damage occurs prior to widespread immune dysfunction.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction.

OBJECTIVE: Ex vivo expansion of primitive hematopoietic cells remains of interest for gene therapy and transplantation. Previous studies reported loss of repopulating activity following culture of cells for more than 4-7 days in the presence of cytokines or stromal cells. In the current study, we investigated whether prolonged culture and transduction in the presence of the carboxy-terminal portion of fibronectin (FN) could maintain or expand retrovirally transduced repopulating hematopoietic stem cells (HSCs). METHODS: The impact of culture and transduction on rhesus macaque CD34+ peripheral blood stem cells (PBSCs) was assessed in the presence of FN and stimulatory cytokines. A competitive repopulation design using up to three retroviral vectors allowed direct comparison of repopulating activity between cells transduced and cultured for 4 days vs 10 days. RESULTS: In the first animal, all cells were cultured and transduced for 10 days, with one vector used on days 0-4 and a second on days 4-10. There was stable long-term marking from both vectors, indicating that cells cycling both early and late could engraft. In three animals, we compared cells that were cryopreserved following a 4-day transduction to cells that were continued in culture for an additional 6 days. Total marking derived from the 10-day expanded cells was significantly higher than marking from the 4-day cultured cells. CONCLUSIONS: These results suggest that culture on FN support allows prolonged ex vivo maintenance and even expansion of transduced repopulating stem cells.

Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates.

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34(+) cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34(+) cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF-mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

Direct comparison of RD114-pseudotyped versus amphotropic-pseudotyped retroviral vectors for transduction of rhesus macaque long-term repopulating cells.

Recently, RD114 (feline endogenous retrovirus envelope protein)-pseudotyped retroviral particles have been shown to transduce human NOD/SCID repopulating cells efficiently. In this study, we compared directly transduction of repopulating cells with RD114-pseudotyped vector to that with standard amphotropic vector in the rhesus macaque model. G-CSF/SCF-mobilized CD34(+) rhesus peripheral blood cells were cultured in the presence of SCF, Flt-3 ligand, and MGDF on Retronectin-coated flasks. To assess directly the ability of the two pseudotypes to transduce primitive cells, both vectors were added simultaneously to the target cells every 24 h, for a total of four exposures in 96 h. The cells were reinfused after the animals received 1000 cGy total body irradiation. At the end of transduction, gene marking efficiency of CFU was higher with amphotropic LNL6 vector (mean 88.4%) vs RD114-G1Na vector (mean 18.5%). After long-term engraftment in three animals, total neo gene marking levels were 4-5% in PBMNCs and 1.5-4% in granulocytes. The RD114-G1Na marking levels were consistently higher in granulocytes than in mononuclear cells, while amphotropic LNL6 marking levels were higher in PBMNCs than in granulocytes. The differential gene marking patterns suggest that RD114 and amphotropic vectors may target distinct progenitor or stem cell populations. There was no clear advantage for RD114-pseudotyped vectors in this predictive preclinical model in terms of overall long-term marking levels; however, optimization of transduction conditions by increasing m.o.i. or inducing the receptor could potentially improve results with this novel vector system.

Retroviral transduction and engraftment ability of primate hematopoietic progenitor and stem cells transduced under serum-free versus serum-containing conditions.

The ability to efficiently transduce hematopoietic stem and progenitor cells under serum-free conditions would be desirable for safety and standardization of clinical gene therapy protocols. Using rhesus macaques, we studied the transduction efficiency and engraftment ability of CD34-enriched SCF/G-CSF mobilized progenitor cells (PBSC) transduced with standard amphotropic marking vectors under serum-free and serum-containing conditions. Supernatants were collected from producer cells 16 hours after serum-free medium or medium containing 10% fetal calf serum was added. Vector titers were approximately two- to threefold higher when producer cells were cultured in serum-containing medium. However, retroviral transduction of rhesus CFU-GM was improved using serum-free vector-containing medium. For analysis of engraftment with transduced cells, three macaques had CD34+ peripheral blood stem cells split into two fractions for transduction. One fraction was transduced using serum-free vector-containing medium, and the other fraction was transduced using standard serum-containing medium. The two fractions were re-infused simultaneously following total body irradiation. In all three animals, there was equivalent marking from both vectors for 7-9 months post-transplantation. These data are encouraging regarding the removal of serum-containing medium from clinical hematopoietic cell transduction protocols, given the lack of a detrimental effect on transduction and engraftment with transduced cells.

Effect of chronic cytokine therapy on clonal dynamics in nonhuman primates.

Hematopoietic cytokines such as filgrastim are used extensively to stimulate granulocyte production or to mobilize hematopoietic progenitors into the circulation; however, their effect on more primitive hematopoietic progenitor and stem cells in vivo is unknown, particularly in large animals or humans. In particular, there is concern that chronic therapy with cytokines could result in stem cell exhaustion or clonal dominance; however, direct assessment of the dynamics of individual stem and progenitor cell clones in vivo has not been previously reported. A number of models can be proposed regarding the mechanisms by which the marrow responds to cytokine stimulation, including recruitment of previously quiescent clones, stimulation of proliferation of already active clones, or prevention of apoptosis of more mature progenitors from all clones. Using retroviral marking and comprehensive insertion site tracking of individual stem and progenitor cell clones in 2 rhesus macaques, we analyzed the effect of chronic administration of granulocyte colony-stimulating factor (G-CSF), or a combination of G-CSF plus stem cell factor (SCF). The overall number of contributing clones remained constant, and the relative output from each clone did not change significantly during or following cytokine treatments. These results suggest that individual transduced stem or progenitor cells can contribute to hematopoiesis for prolonged periods, with no evidence for an effect of G-CSF or G-CSF/SCF on the number, the lifespan, or the relative activity of individual stem or progenitor cell clones. These relevant large animal studies are reassuring regarding clinical applications of cytokines and provide new insights into their mechanisms of action.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors.

One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

Retrovirally transduced muscle-derived cells contribute to hematopoiesis at very low levels in the nonhuman primate model.

Recent studies have suggested a remarkable potential of adult stem cells from a variety of organs to give rise to cells of disparate organs, but evidence of such potential at a clonal level is lacking in most if not all studies to date. To assess directly the hematopoietic potential of muscle-derived cells in a relevant large animal, we initiated retroviral-tagging studies in the rhesus macaque to allow tracking at the clonal level by integration site analysis. Four rhesus macaques underwent transplantation with transduced muscle-derived cells after lethal irradiation followed by delayed infusion of an autologous hematopoietic graft. The first animal showed no evidence of hematopoietic recovery and, despite infusion of the backup hematopoietic graft, succumbed due to complications of prolonged cytopenias. In the remaining three animals, the overall contribution of retrovirally tagged muscle-derived cells toward hematopoiesis was exceedingly low. Retroviral integration site analysis among clonally derived muscle cells and bone marrow cells in vivo in one animal suggests a common source. These results demonstrate that harvesting disparate organs for cellular therapy is currently highly inefficient at best.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector.

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20-60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.