Free PEG Suppresses Anaphylaxis to PEGylated Nanomedicine in Swine.

Covalent conjugation of poly(ethylene glycol) (PEG) is frequently employed to enhance the pharmacokinetics and biodistribution of various protein and nanoparticle therapeutics. Unfortunately, some PEGylated drugs can induce elevated levels of antibodies that can bind PEG, i.e., anti-PEG antibodies (APA), in some patients. APA in turn can reduce the efficacy and increase the risks of allergic reactions, including anaphylaxis. There is currently no intervention available in the clinic that specifically mitigates allergic reactions to PEGylated drugs without the use of broad immunosuppression. We previously showed that infusion of high molecular weight free PEG could safely and effectively suppress the induction of APA in mice and restore prolonged circulation of various PEGylated therapeutics. Here, we explored the effectiveness of free PEG as a prophylaxis against anaphylaxis induced by PEG-specific allergic reactions in swine. Injection of PEG-liposomes (PL) resulted in anaphylactoid shock (pseudoanaphylaxis) within 1-3 min in both naïve and PL-sensitized swine. In contrast, repeated injection of free PEG alone did not result in allergic reactions, and injection of free PEG effectively suppressed allergic reactions to PL, including in previously PL-sensitized swine. These results strongly support the further investigation of free PEG for reducing APA and allergic responses to PEGylated therapeutics.

Biodistribution of Biomimetic Drug Carriers, Mononuclear Cells, and Extracellular Vesicles, in Nonhuman Primates.

Discovery of novel drug delivery systems to the brain remains a key task for successful treatment of neurodegenerative disorders. Herein, the biodistribution of immunocyte-based carriers, peripheral blood mononuclear cells (PBMCs), and monocyte-derived EVs are investigated in adult rhesus macaques using longitudinal PET/MRI imaging. 64 Cu-labeled drug carriers are introduced via different routes of administration: intraperitoneal (IP), intravenous (IV), or intrathecal (IT) injection. Whole body PET/MRI (or PET/CT) images are acquired at 1, 24, and 48 h post injection of 64 Cu-labeled drug carriers, and standardized uptake values (SUVmean and SUVmax ) in the main organs are estimated. The brain retention for both types of carriers increases based on route of administration: IP < IV < IT. Importantly, a single IT injection of PBMCs produces higher brain retention compared to IT injection of EVs. In contrast, EVs show superior brain accumulation compared to the cells when administered via IP and IV routes, respectively. Finally, a comprehensive chemistry panel of blood samples demonstrates no cytotoxic effects of either carrier. Overall, living cells and EVs have a great potential to be used for drug delivery to the brain. When identifying the ideal drug carrier, the route of administration could make big differences in CNS drug delivery.

Improved Tolerability of a Salmonella enterica Serovar Typhimurium Live-Attenuated Vaccine Strain Achieved by Balancing Inflammatory Potential with Immunogenicity.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA We determined that S Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (109 CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD50 (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

Mycobacterium kansasii Isolated from Tuberculinpositive Rhesus Macaques (Macaca mulatta) in the Absence of Disease.

Mycobacterial infections are of primary health concern in NHP colonies in biomedical research. NHP are constantly monitored and screened for Mycobacterium spp. We report 6 Chinese-origin rhesus macaques infected with Mycobacterium kansasii that exhibited positive tuberculin skin tests in the absence of disease. Two of these macaques were being used for research purposes; the remaining 4 macaques were residing at the contract quarantine company. Histopathology and acid-fast staining of fixed tissues from all macaques showed that all were free of disease. Thoracic radiographs were negative for any signs of disease or infection. Samples from bronchial lavage and tissues including lung, spleen, hilar and mesenteric lymph nodes tested negative by PCR assay for Mycobacterium spp. One of the research macaques tested culture-positive for M. kansasii and a poorly characterized M. avium complex organism. One macaque from the contract quarantine facility tested culture positive for M. kansasii. Genomic testing and target gene RNA expression analysis of the 2 M. kansasii isolates were performed to evaluate possible kinship and affected genes that might contribute to susceptibility to mycobacterial infection. Genotyping of the 2 isolates revealed 2 genetically distinct strains (strains 1 and 4). The presence of positive tuberculin skin tests in the absence of disease raises serious concerns regarding diagnostic methods used for infected NHP.

Differential response of the cynomolgus macaque gut microbiota to Shigella infection.

Little is known about the role of gut microbiota in response to live oral vaccines against enteric pathogens. We examined the effect of immunization with an oral live-attenuated Shigella dysenteriae 1 vaccine and challenge with wild-type S. dysenteriae 1 on the fecal microbiota of cynomolgus macaques using 16 S rRNA analysis of fecal samples. Multi-dimensional cluster analysis identified different bacterial community types within macaques from geographically distinct locations. The fecal microbiota of Mauritian macaques, observed to be genetically distinct, harbored a high-diversity community and responded differently to Shigella immunization, as well as challenge compared to the microbiota in non-Mauritian macaques. While both macaque populations exhibited anti-Shigella antibody responses, clinical shigellosis was observed only among non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and consideration of host genetic backgrounds in conducting vaccine studies.

Histopathology of chronic rejection in a nonhuman primate model of vascularized composite allotransplantation.

BACKGROUND: Chronic rejection of vascularized composite allografts (VCA) is an emerging phenomenon that may decrease long-term allograft survival and impair allograft function. Although intimal hyperplasia has been reported in human hand transplants, chronic changes in VCAs remain poorly described. METHODS: We developed a nonhuman primate model of face transplantation to evaluate the effect of various immunosuppressive regimens on allograft survival that we have previously reported. Nineteen grafts were successfully transplanted and serially biopsied to assess for rejection. Five VCA grafts with long-term survival (>200 days) were weaned off immunosuppression. We performed additional histologic and immunohistochemical studies on previously collected samples. RESULTS: All five grafts developed features consistent with chronic rejection, including neointimal proliferation, transplant vasculopathy, vessel wall fibrosis, progressive luminal occlusion, and tertiary lymphoid follicles. Review of 186 serial allograft skin and subcutaneous tissue biopsies revealed that tertiary follicles and vascular changes developed in the absence of acute skin rejection. No relationship was found between alloantibody production and these changes. CONCLUSIONS: Recognition of these characteristics of VCA chronic rejection is important for diagnosis in clinical hand and face transplantation. Studies directed towards minimizing VCA chronic rejection responses may be required to improve long-term outcomes.

Infrared fluorescence imaging of lymphatic regeneration in nonhuman primate facial vascularized composite allografts.

BACKGROUND: Clinical vascularized composite allografts (VCA), although performed with good success, have been characterized by rejection episodes and postoperative graft edema. We investigated lymphatic donor-recipient reconstitution and lymphatic regeneration in a nonhuman primate facial VCA model. METHODS: Heterotopic partial face (n = 9) VCAs were performed in cynomolgus macaques. Grafts were monitored for rejection episodes and response to immunosuppressive therapies as previously described. Donor and recipient lymphatic channels were evaluated using a near-infrared handheld dual-channel light-emitting diode camera system capable of detecting fluorescence from indocyanine green injections. Graft lymphatic channels were serially evaluated from postoperative day 0 to 364. RESULTS: Preoperative imaging demonstrated superficial lymphatic anatomy similar to human anatomy. Initial resolution of facial allograft swelling coincided with superficial donor-recipient lymphatic channel reconstitution. Reconstitution occurred despite early acute rejection episodes in 2 animals. However, lymphatic channels demonstrated persistent functional and anatomic pathology, and graft edema never fully resolved. No differences in lymphatic channels were noted between grafts that developed transplant vasculopathy (n = 3) and those that did not (n = 6). Dynamic changes in patterns of lymphatic drainage were noted in 4 animals following withdrawal of immunosuppression. CONCLUSIONS: Donor-recipient lymphatic channel regeneration following VCA did not result in resolution of edema. Technical causes of graft edema may be overcome with alternative surgical techniques, allowing for direct investigation of the immunologic relationship between VCA graft edema and rejection responses. Mechanisms and timing of dynamic donor-recipient lymphatic channel relationships can be evaluated using fluorescent imaging systems to better define the immunologic role of lymphatic channels in VCA engraftment and rejection responses, which may have direct clinical implications.

Nonhuman primate model of fibula vascularized composite tissue allotransplantation demonstrates donor-recipient bony union.

BACKGROUND: Vascularized composite tissue allotransplantation has demonstrated clinical success with standard immunosuppression in hand and upper extremity transplantation. The authors developed a fibular vascularized composite tissue allotransplantation model in nonhuman primates to investigate healing and rejection patterns of bone and associated tissues. METHODS: Five fibular vascularized composite tissue allotransplantations were performed between mismatched cynomolgus macaques (Macaca fascicularis). Vascularized fibular segments with associated muscle and skin were transplanted to recipient forearm radius defects. Recipients were treated with either tacrolimus monotherapy or tacrolimus plus co-stimulatory blockade with a novel anti-CD28 antibody. Animals were followed for 6 months with serial radiographs, blood sample collection, and biopsies. At the study endpoint, angiographic, biomechanical, histologic, and immunologic assays were performed. RESULTS: All animals survived to the experimental endpoint of 180 days. Rapid or immediate skin loss was evident secondary to vascular compromise (n = 3) or rejection (n = 1) in four animals. Despite loss of nonbony segments and the development of transplant arteriopathy consistent with chronic rejection in two animals, serial radiologic imaging and histology demonstrated bone healing and donor-recipient bony union by 10 weeks in all animals. Histology confirmed the presence of viable cortical and marrow elements. Biomechanical analysis supported donor-recipient bony union. Short-tandem repeated genotypic analysis revealed that donor marrow had been completely replaced by recipient marrow. CONCLUSIONS: In contrast to successes in extremity vascularized composite tissue allotransplantation, the authors' nonhuman primate fibular vascularized composite tissue allotransplantation model showed early skin loss, replacement of donor bone marrow, and chronic rejection. Donor-recipient bone union did occur and supports the potential for reconstruction of bony continuity defects using isolated vascularized bone allotransplants.

A challenge model for Shigella dysenteriae 1 in cynomolgus monkeys (Macaca fascicularis).

Shigella dysenteriae type 1 can cause devastating pandemics with high case fatality rates; a vaccine for Shigella is unavailable currently. Because of the risks associated with performing challenge studies with wild-type S. dysenteriae 1 in human clinical trials to advance vaccine development, an improved nonhuman primate model is needed urgently. In the present study, cynomolgus macaques (Macaca fascicularis) were challenged with various doses of S. dysenteriae 1 strain 1617 to establish a dose that would produce shigellosis. Further, different routes of delivery of S. dysenteriae 1 were compared to establish the most appropriate route for infection. Animals receiving 10(11) cfu S. dysenteriae 1 intragastrically consistently developed signs of shigellosis characterized by the onset of diarrhea and dysentery within 2 to 3 d. Administration of as many as 10(9) cfu S. dysenteriae 1 intraduodenally did not elicit signs characteristic of infection in macaques despite fecal shedding of bacteria for as long as 10 d. S. dysenteriae 1 administered intraduodenally at 10(9) cfu or intragastrically at 10(11) cfu elicited robust IgG and IgA antibody responses to LPS. We have developed a reliable challenge model of infection with wild-type S. dysenteriae 1 in cynomolgus macaques that reproducibly induces disease and elicits robust immune responses. We believe that this animal model may provide unique insights into the immunologic mechanisms of protection to S. dysenteriae 1 infection and in advancing development of a vaccine against shigellosis.

Prolonged survival of composite facial allografts in non-human primates associated with posttransplant lymphoproliferative disorder.

BACKGROUND: Composite tissue allotransplantation may have different immunosuppressive requirements and manifest different complications compared with solid organ transplantation. We developed a non-human primate facial composite tissue allotransplantation model to investigate strategies to achieve prolonged graft survival and immunologic responses unique to these allografts. METHODS: Composite facial subunits consisting of skin, muscle, and bone were heterotopically transplanted to mixed lymphocyte reaction-mismatched Cynomolgus macaques. Tacrolimus monotherapy was administered via continuous intravenous infusion for 28 days then tapered to daily intramuscular doses. RESULTS: Five of the six animals treated with tacrolimus monotherapy demonstrated rejection-free graft survival up to 177 days (mean, 113 days). All animals with prolonged graft survival developed posttransplant lymphoproliferative disorders (PTLD). Three animals converted to rapamycin after 28 days of rejection of their allografts, but did not develop PTLD. Genotypic analysis of PTLD tumors demonstrated donor origin in three of the five analyzed by short-tandem repeats. Sustained alloantibodies were detected in rejecting grafts and absent in nonrejecting grafts. CONCLUSIONS: Tacrolimus monotherapy provided prolonged rejection-free survival of composite facial allografts in a non-human primate model but was associated with the development of a high frequency of donor-derived PTLD tumors. The transplantation of a large volume of vascularized bone marrow in composite tissue allografts may be a risk factor for PTLD development.

Methicillin-resistant Staphylococcus non-aureus infection in an irradiated rhesus macaque (Macaca mulatta).

We describe a case of methicillin-resistant Staphylococcus non-aureus infection in a rhesus macaque (Macaca mulatta). The nonhuman primate described was part of a research project that involved whole-body gamma irradiation and subsequently developed acute generalized dermatitis with skin dryness, peeling, and erythema around the eyes. After initial evaluation, which included microbiologic culture and 6 d of medical treatment, the animal was euthanized due to concern regarding a possible outbreak of infectious or zoonotic disease. On the basis of skin culture, diagnosis of methicillin-resistant Staphylococcus non-aureus was confirmed. This report underscores the importance of the occupational risk of methicillin-resistant Staphylococcus non-aureus to research and animal care staff in a research animal facility setting.

Diagnosis and prevention of dissemination of tuberculosis in a recently imported rhesus macaque (Macaca mulatta).

Thirty-four (34) days after arrival at our facility, a recently imported rhesus macaque demonstrated a grade 4/5 reaction to intradermal testing with mammalian old tuberculin and a strong positive response in a serum interferon gamma (IFN-gamma) stimulation assay (Primagam). The affected animal was euthanized to prevent further exposure of the other rhesus in the quarantine room. Necropsy revealed enlarged, caseating mediastinal lymph nodes. Further analysis confirmed the presence of Mycobacterium tuberculosis complex organisms. Strict isolation measures were initiated and intensive testing of all animals in the affected room began immediately. After 13 weeks of additional testing, none of the animals in the room showed any positive response and all were released from quarantine. This case illustrates the importance of prolonged quarantine of non-human primates (NHP) and illustrates the usefulness of serology-based diagnostics as an adjunct to intradermal testing (molecular-based diagnostics typically refers to polymerase chain reaction, whereas this assay is really serology based, even though it is an in vitro IFN-gamma stimulation assay). It also demonstrates that with proper isolation procedures, the spread of tuberculosis can be prevented in NHP facilities.

A heterotopic primate model for facial composite tissue transplantation.

The purpose of this study was to develop a nonhuman primate model for heterotopic composite tissue facial transplantation in which to study the natural history of facial transplantation and evaluate immunosuppressive regimens.A composite oromandibular facial segment transplant based on the common carotid artery was evaluated. Flaps from 7 cynomolgus monkeys were transplanted to the groins of 7 recipients at the superficial femoral artery and vein. The immunosuppressive regimen consisted of thymoglobulin, rapamycin, and tacrolimus. Allograft survival ranged from 6 to 129 days. Histology performed in the long-term survivor at the time of necropsy revealed extensive inflammation and necrosis of the allograft skin; however, muscle and bone elements were viable, with minimal inflammation. This heterotopic facial transplantation model avoids the potential morbidity of mandibular resection and orthotopic facial transplantation. Our work also concurs with the work of other groups who found that the skin component is the most antigenic.

Disseminated simian varicella virus infection in an irradiated rhesus macaque (Macaca mulatta).

We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.