Selective blockade of CD28-mediated T cell costimulation protects rhesus monkeys against acute fatal experimental autoimmune encephalomyelitis.

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the ß-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

Antagonizing the α4ß1 integrin, but not α4ß7, inhibits leukocytic infiltration of the central nervous system in rhesus monkey experimental autoimmune encephalomyelitis.

The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the α(4) integrins expressed by T lymphocytes. The α(4)ß(1) integrin mediates migration of memory T lymphocytes into the CNS, whereas the α(4)ß(7) integrin mediates migration preferentially into gastrointestinal tissue. This paradigm was established primarily from investigations in rodents; thus, the objective of this investigation was to determine if blocking the α(4)ß(7) integrin exclusively would affect migration of T lymphocytes into the CNS of primates. The effects of the dual α(4)ß(1) and α(4)ß(7) antagonist natalizumab were compared with those of the α(4)ß(7) antagonist vedolizumab on experimental autoimmune encephalomyelitis in the rhesus monkey. Animals received an initial i.v. bolus of placebo, natalizumab (30 mg/kg), or vedolizumab (30 mg/kg) before intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein and then Ab once weekly thereafter. Natalizumab prevented CNS inflammation and demyelination significantly (p < 0.05), compared with time-matched placebo control animals, whereas vedolizumab did not inhibit these effects, despite saturating the α(4)ß(7) integrin in each animal for the duration of the investigation. These results demonstrate that blocking α(4)ß(7) exclusively does not inhibit immune surveillance of the CNS in primates.

Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection.

To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis-derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies.

Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination.

BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces.

Deficits in the IgG+ memory B-cell recovery after anthracycline treatment is confined to the spleen of rhesus macaques.

OBJECTIVES: Loss of vaccine-induced antibodies (Abs) after chemotherapy against paediatric acute lymphoblastic leukaemia (ALL) is common and often necessitates re-immunisation after cessation of treatment. Even so, some ALL survivors fail to mount or to maintain protective Abs. Germinal centres (GCs) are clusters of proliferating B cells in follicles of secondary lymphoid tissues (SLTs) formed during adaptive immune responses and the origins of long-lived memory B and plasma cells that are the source of Abs. Furthermore, productive GC reactions depend on T follicular helper (TFH) cells. To understand why chemotherapy induces deficits in Ab responses, we examined how SLTs were affected by chemotherapy. METHODS: Rhesus macaques were infused with either three cycles of the anthracycline doxorubicin or saline, followed by immunisation with a de novo and booster antigen. Spleen and lymph nodes were removed, and memory B, bulk T and TFH cells were examined. RESULTS: Despite adequate GC morphology, a diminished memory and IgG+ B-cell population along with diminished total and booster vaccine-specific IgG-producing memory B cells were noted in the spleens of macaques with past doxorubicin exposure compared to the saline-treated controls (P < 0.05). Intact bulk T and TFH cells were found in the SLTs of treated macaques, which displayed higher CD40L upregulation capacity by their splenic CXCR5+ helper T cells (P < 0.01). In contrast to the spleen, the immune cell populations studied were comparable between the lymph nodes of both saline- and doxorubicin-treated macaques. CONCLUSION: Our findings suggest that the splenic memory B-cell subset, compared to its lymph node counterpart, is more severely altered by anthracycline treatment.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

Disparate Tuberculosis Disease Development in Macaque Species Is Associated With Innate Immunity.

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques.

Tuberculosis (TB) remains the deadliest infectious disease1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (TH17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB.

Characterization of naturally occurring CD4+CD25+ regulatory T cells in rhesus monkeys.

BACKGROUND: Translational research in a relevant preclinical model is recommended before Treg-inducing protocols can be implemented in humans. We have characterized rhesus monkey CD25 cells phenotypically and functionally. METHODS: The phenotype of CD4(+)CD25(high) cells was determined by FACS, focusing on established markers of mouse and human Treg cells. Percentages of cells positive for CD45RA, CD62L, and intracellular CTLA-4 and FOXP3 were compared between CD4(+)CD25(high) and CD4(+)CD25(-) cells. CD25 cells stimulated with anti-CD3, ConA, and/or allogeneic peripheral blood mononuclear cells were mixed with freshly isolated CD25 cells. The suppressive activity of the CD25 cells in vitro was assessed using several experimental conditions. RESULTS: Rhesus monkey CD4(+)CD25(high) cells expressed high intracellular levels of CTLA-4 and FOXP3, whereas expression was negligible in CD4(+)CD25(-) cells. The CD25(high) population was mostly CD45RA(-), indicative of a memory phenotype. The CD25(+) cells were anergic, because they showed low proliferative responses, no interleukin-2 production, and some interferon-gamma and interleukin-10 production. Proliferation of CD4(+)CD25(-) cells stimulated by anti-CD3 or allogeneic cells was decreased when CD4(+)CD25(-) cells were added at a 1:1 ratio. In addition, we found that CD25 cells inhibited the interleukin-2 and interferon-gamma production by anti-CD3-stimulated CD25 cells in a dose-dependent fashion, through a cell-cell contact-dependent mechanism. CONCLUSIONS: Rhesus monkey CD4(+)CD25(+) cells have similar phenotypic and functional characteristics as natural Tregs in humans. These findings allow testing of Treg expansion and induction protocols in a relevant preclinical model, the rhesus monkey.

Hinge-deleted IgG4 blocker therapy for acetylcholine receptor myasthenia gravis in rhesus monkeys.

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

No synergy between ATG induction and costimulation blockade induced kidney allograft survival in rhesus monkeys.

BACKGROUND: Costimulation blockade with antibodies directed against human CD40 and CD86 leads to prolonged kidney allograft survival in rhesus monkeys, but fails to induce permanent graft acceptance. We have tested whether costimulation blockade is more effective after peripheral T-cell ablation with antithymocyte globulin (ATG), with the aim to remove already primed autoreactive cells present in the normal repertoire. METHODS: Rhesus monkeys were transplanted with a mismatched kidney allograft. ATG was given around the time of transplantation (day -1 and 0). Costimulation blockade with anti-CD40+anti-CD86 was given at tapering dosages from day -1 to 56. Cyclosporin A (CsA) was given from day 42 onwards and first rejections occurring after day 42 were treated with prednisone. RESULTS: We observed accelerated rejection in ATG-treated monkeys, compared to animals receiving only costimulation blockade. The accelerated rejection of the kidney allograft occurred despite the application of rejection therapy with steroids and CsA. Three of the five ATG-treated animals were found seropositive for donor-specific alloantibodies. Early biopsies (day 21) from animals treated with ATG and anti-CD40+anti-CD86 show substantially reduced expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and forkhead box P3 (FOXP3) in focal infiltrates as compared to animals treated with only costimulation blockade. Furthermore, we observed the rapid reappearance of CD8 T-cells with a memory phenotype (disappearance of naive CD95/CD11a T-cells) in peripheral blood. CONCLUSION: We conclude that (subtotal) T-cell depletion using ATG does not add to costimulation blockade induced kidney allograft survival.

An Evaluation of 20 Years of EU Framework Programme-Funded Immune-Mediated Inflammatory Translational Research in Non-Human Primates.

Aging western societies are facing an increasing prevalence of chronic inflammatory and degenerative diseases for which often no effective treatments exist, resulting in increasing health-care expenditure. Despite high investments in drug development, the number of promising new drug candidates decreases. We propose that preclinical research in non-human primates can help to bridge the gap between drug discovery and drug prescription. Translational research covers various stages of drug development of which preclinical efficacy tests in valid animal models is usually the last stage. Preclinical research in non-human primates may be essential in the evaluation of new drugs or therapies when a relevant rodent model is not available. Non-human primate models for life-threatening or severely debilitating diseases in humans are available at the Biomedical Primate Research Centre (BPRC). These have been instrumental in translational research for several decades. In order to stimulate European health research and innovation from bench to bedside, the European Commission has invested heavily in access to non-human primate research for more than 20 years. BPRC has hosted European users in a series of transnational access programs covering a wide range of research areas with the common theme being immune-mediated inflammatory disorders. We present an overview of the results and give an account of the studies performed as part of European Union Framework Programme (EU FP)-funded translational non-human primate research performed at the BPRC. These data illustrate the value of translational non-human primate research for the development of new therapies and emphasize the importance of EU FP funding in drug development.

Costimulation blockade followed by a 12-week period of cyclosporine A facilitates prolonged drug-free survival of rhesus monkey kidney allografts.

Costimulation blockade as a single immunosuppressive treatment modality is not sufficient to prevent graft rejection. Here, we report an induction therapy using antagonistic antibodies against CD40 and CD86, given twice weekly from day -1 until day 56, followed by a delayed 12-week course of low-dose cyclosporine A (CsA) treatment in the rhesus monkey kidney-allograft model. Low-dose CsA treatment was initiated on day 42 and tapered until total cessation of all treatment on day 126. Treatment with anti-CD40/86 alone resulted in graft survival of 61, 71, 75, 78, and 116 days. Costimulation blockade followed by CsA resulted in more than 3-year drug-free survival in two of four animals. None of the animals developed donor-specific alloantibodies. Transforming growth factor-beta producing cells are present in early as well as in late kidney-graft biopsies and could play a role in the observed long-term drug-free graft survival.

Lymphoid-Like Structures with Distinct B Cell Areas in Kidney Allografts are not Predictive for Graft Rejection. A Non-human Primate Study.

Kidney allograft biopsies were analyzed for the presence of B cell clusters/aggregates using CD20 staining. Few B cells were found in the diffuse interstitial infiltrates, but clusters of B cells were found in nodular infiltrates. These nodular infiltrates were smaller shortly after transplantation, and their size increased over time. At the time of clinical rejection, the nodules often presented as tertiary lymphoid structures (TLS) with lymphoid-like follicles. The presence of small B cell clusters during the first 2 months after transplantation was not associated with early rejection. Even in animals that did not reject their allograft, TLS-like structures were present and could disappear over time. Although TLS were more often found in samples with interstitial fibrosis and tubular atrophy (IFTA), TLS were also present in samples without IFTA. The presence and density of clusters resembling tertiary lymphoid structures most likely reflect an ongoing immune response inside the graft and do not necessarily signify a poor graft outcome or IFTA.

The translational value of non-human primates in preclinical research on infection and immunopathology.

The immune system plays a central role in the defense against environmental threats - such as infection with viruses, parasites or bacteria - but can also be a cause of disease, such as in the case of allergic or autoimmune disorders. In the past decades the impressive development of biotechnology has provided scientists with biological tools for the development of highly selective treatments for the different types of disorders. However, despite some clear successes the translation of scientific discoveries into effective treatments has remained challenging. The often-disappointing predictive validity of the preclinical animal models that are used in the selection of the most promising vaccine or drug candidates is the Achilles heel in the therapy development process. This publication summarizes the relevance and usage of non-human primates as pre-clinical model in infectious and autoimmune diseases, in particular for biologicals, which due to their high species-specificity are inactive in lower species.

Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates.

BACKGROUND: Costimulation blockade has been proposed to induce allograft tolerance. We combined an antagonist anti-CD40 monoclonal antibody (mAb) with an antagonist anti-CD86 mAb in a rhesus monkey kidney allograft model. We chose this combination because it leaves CD80-CD152 signaling unimpaired, allowing for the down-regulatory effect of CD152 signaling to take place through this pathway. METHODS: Rhesus monkeys underwent transplantation with a major histocompatibility complex-mismatched kidney. One group of animals received anti-CD40 alone, and a second group received the combination of anti-CD40 and anti-CD86, twice weekly for 56 days. RESULTS: Three animals with low levels of anti-CD40 rejected the transplanted kidney while still receiving treatment. Three animals with high levels of anti-CD40 rejected at days 91, 134, and 217 with signs of chronic rejection. Animals treated with the combination of anti-CD40 and anti-CD86 mAbs rejected their kidneys at days 61, 75, and 78, shortly after cessation of treatment. Two animals were killed on days 71 and 116 with a blocked ureter. These animals developed virtually no signs of tubulitis or infiltration during treatment and no donor-specific alloantibodies. CONCLUSIONS: Both treatment protocols prevented rejection for the duration of the treatment in most animals. Blocking costimulation by anti-CD40 or by anti-CD40 plus anti-CD86 may be an effective method to prevent graft rejection and may obviate the need for other immunosuppressive drugs, especially in the immediate posttransplantation period.

T-cell-specific immunosuppression results in more than 53 days survival of porcine islets of langerhans in the monkey.

BACKGROUND: Transplantation of islets of Langerhans can restore insulin production in diabetic patients. Because of the shortage of human donor organs, transplantation of porcine islets may be an alternative solution. The present study was aimed at the characterization of rejection mechanisms of porcine islets transplanted into eight nondiabetic monkeys under the kidney capsule. METHODS: Cultured adult pig islets were used, which showed no expression of the galactose(alpha1,3)galactose epitope, major histocompatibility complex class II, or CD45, and no binding of antibodies or complement after exposure to monkey serum. Immunosuppression consisted of cyclophosphamide, cyclosporine A (CsA), and steroids (group 1); or antithymocyte globulin, anti-interleukin-2 receptor antibody, CsA, and steroids (group 2). In three animals of group 2, islets were also transplanted in the portal vein. RESULTS: Although all monkeys had preformed anti-pig antibodies, no correlation was found between antibody titers and rejection and no deposition of antibodies or complement was observed in the grafts. Group 1 showed islets up to day 11, followed by T-cell infiltration and rejection at approximately day 14. In group 2, two monkeys showed infiltrates consisting predominantly of T cells starting at approximately day 29, whereas two monkeys showed well-preserved islets without infiltration up to day 53. In the livers of the three monkeys that also received islets intraportally and were resectioned on days 21, 33, and 49, no islets could be detected. CONCLUSIONS: This study demonstrates that cultured adult pig islets can survive in the monkey for more than 53 days without signs of rejection under standard immunosuppression.

Blockade of CD40-CD154 at the time of donor-specific blood transfusion does not lead to prolonged kidney allograft survival in nonhuman primates.

BACKGROUND: In rodents it has been demonstrated that blockade of the CD40-CD154 (CD40L) pathway at the time of donor-specific blood transfusion (DST) can result in indefinite graft survival. Because it has been reported in the past that DST in monkeys can have a favorable effect on graft outcome and that blockade of the CD40-CD154 pathway can lead to prolonged kidney graft survival in monkeys, we have combined anti-CD154 treatment with DST in a monkey kidney graft model. The aim of this study was to investigate the immunosuppressive potential of blocking the CD40-CD154 interaction at the time of a DST in rhesus monkeys. METHODS: One donor-derived blood transfusion was given on day -15 after the first anti-CD154 injection. The anti-CD154 antibody was given on days -15, -13, -11, -9, and -7. The kidney was transplanted on day 0. Cyclosporine was given after kidney trans-plantation. RESULTS: No major difference in graft survival was observed between the groups. The animals died due to grade II acute rejection. At the time of transplantation, no antibody response could be detected directed against donor antigens. After transplantation, all animals surviving for more than 3 weeks had antidonor antibodies. There were no differences in the intragraft events analyzed by real time reverse transcriptase-polymerase chain reaction. CONCLUSIONS: DST under the cover of relatively high levels of anti-CD154 failed to result in prolonged graft survival or prevent the formation of antidonor antibodies, when cyclosporine was given after transplantation.

Improvement of preclinical animal models for autoimmune-mediated disorders via reverse translation of failed therapies.

The poor translational validity of autoimmune-mediated inflammatory disease (AIMID) models in inbred and specific pathogen-free (SPF) rodents underlies the high attrition of new treatments for the corresponding human disease. Experimental autoimmune encephalomyelitis (EAE) is a frequently used preclinical AIMID model. We discuss here how crucial information needed for the innovation of current preclinical models can be obtained from postclinical analysis of the nonhuman primate EAE model, highlighting the mechanistic reasons why some therapies fail and others succeed. These new insights can also help identify new targets for treatment.