Vaccine-associated enhanced respiratory pathology in COVID-19 hamsters after TH2-biased immunization.

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in coronavirus disease 2019 (COVID-19), we evaluate two vaccine leads utilizing a severe hamster infection model: a T helper type 1 (TH1)-biased measles vaccine-derived candidate and a TH2-biased alum-adjuvanted, non-stabilized spike protein. The measles virus (MeV)-derived vaccine protects the animals, but the protein lead induces VAERD, which can be alleviated by dexamethasone treatment. Bulk transcriptomic analysis reveals that our protein vaccine prepares enhanced host gene dysregulation in the lung, exclusively up-regulating mRNAs encoding the eosinophil attractant CCL-11, TH2-driving interleukin (IL)-19, or TH2 cytokines IL-4, IL-5, and IL-13. Single-cell RNA sequencing (scRNA-seq) identifies lung macrophages or lymphoid cells as sources, respectively. Our findings imply that VAERD is caused by the concerted action of hyperstimulated macrophages and TH2 cytokine-secreting lymphoid cells and potentially links VAERD to antibody-dependent enhancement (ADE). In summary, we identify the cytokine drivers and cellular contributors that mediate VAERD after TH2-biased vaccination.

Small-molecule polymerase inhibitor protects non-human primates from measles and reduces shedding.

Measles virus (MeV) is a highly contagious pathogen that enters the human host via the respiratory route. Besides acute pathologies including fever, cough and the characteristic measles rash, the infection of lymphocytes leads to substantial immunosuppression that can exacerbate the outcome of infections with additional pathogens. Despite the availability of effective vaccine prophylaxis, measles outbreaks continue to occur worldwide. We demonstrate that prophylactic and post-exposure therapeutic treatment with an orally bioavailable small-molecule polymerase inhibitor, ERDRP-0519, prevents measles disease in squirrel monkeys (Saimiri sciureus). Treatment initiation at the onset of clinical signs reduced virus shedding, which may support outbreak control. Results show that this clinical candidate has the potential to alleviate clinical measles and augment measles virus eradication.

Spontaneous (Hashimoto-like) chronic lymphocytic thyroiditis in a rhesus macaque (Macaca mulatta).

A case of a female, 10-year-old rhesus macaque (Macaca mulatta) with spontaneous chronic lymphocytic thyroiditis is presented. At necropsy, the thyroid gland was slightly enlarged, with up to 2 mm large, round, confluent, beige foci on the surface of both lobes. Histopathologic features resembled human Hashimoto's thyroiditis: multifocally, the interstitium was infiltrated by lymphocytes and variably sized lymphoid follicles. In the pituitary gland, there were increased numbers of large, basophilic cells throughout the adenohypophysis. Using a human electrochemiluminescence immunoassay (ECLIA), no autoantibodies against thyroglobulin, thyroid peroxidase, or thyroid-stimulating hormone receptor were detected.

Unintended importation of tropical jumping spiders (Salticidae) into a laboratory monkey colony via banana supply.

This report describes a case of unintended importation of tropical baby jumping spiders to a laboratory monkey colony. The spiders were detected in a cocoon attached to a banana for monkey consumption. In identifying the family of spiders as jumping spiders (Salticidae), it turned out that these spiders would not have been venomous to humans and they most likely would not have had the potential to establish a new spider colony in the facility.

Reactive mesothelial hyperplasia mimicking mesothelioma in an African green monkey (Chlorocebus aethiops).

A spontaneous reactive mesothelial hyperplasia occurred in a female, 15.7-year-old African green monkey (grivet; Chlorocebus aethiops). At necropsy, massive effusions were found in the abdomen, the thorax, and the pericardium. Additionally, multiple small, beige-gray nodules were detected on the serosal surfaces of the abdominal organs. Histopathologically, the mesothelial cells resembled the epithelioid subtype of a mesothelioma, but no infiltrative or invasive growth could be demonstrated. The mesothelial cells on the thoracis, liver, and intestinal serosa were accompanied by chronic serositis. Mesothelial cells expressed cytokeratin, vimentin, calretinin, desmin, Wilms Tumor 1 (WT-1) protein, and epithelial membrane antigen (EMA). Cells were negative for carcinoembryonic antigen (CEA), cluster of differentiation 15 (CD15), and podoplanin. Ultrastructurally, cells revealed a moderate amount of microvilli of medium length, perinuclear tonofilament bundles, and long desmosomes. In fluorescence in situ hybridization (FISH) for the detection of characteristic gene loss (p16; CDKN2A), NF2, and MTAP, no deletions were detected. No asbestos fibers and no presence of Simian virus 40 antigen (SV40) could be demonstrated.

Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia.

Influenza viruses constitute a major health threat and economic burden globally, frequently exacerbated by preexisting or rapidly emerging resistance to antiviral therapeutics. To address the unmet need of improved influenza therapy, we have created EIDD-2801, an isopropylester prodrug of the ribonucleoside analog N 4-hydroxycytidine (NHC, EIDD-1931) that has shown broad anti-influenza virus activity in cultured cells and mice. Pharmacokinetic profiling demonstrated that EIDD-2801 was orally bioavailable in ferrets and nonhuman primates. Therapeutic oral dosing of influenza virus-infected ferrets reduced group pandemic 1 and group 2 seasonal influenza A shed virus load by multiple orders of magnitude and alleviated fever, airway epithelium histopathology, and inflammation, whereas postexposure prophylactic dosing was sterilizing. Deep sequencing highlighted lethal viral mutagenesis as the underlying mechanism of activity and revealed a prohibitive barrier to the development of viral resistance. Inhibitory concentrations were low nanomolar against influenza A and B viruses in disease-relevant well-differentiated human air-liquid interface airway epithelia. Correlating antiviral efficacy and cytotoxicity thresholds with pharmacokinetic profiles in human airway epithelium models revealed a therapeutic window >1713 and established dosing parameters required for efficacious human therapy. These data recommend EIDD-2801 as a clinical candidate with high potential for monotherapy of seasonal and pandemic influenza virus infections. Our results inform EIDD-2801 clinical trial design and drug exposure targets.

Spontaneous meningioma in a pig-tailed macaque (Macaca nemestrina).

We present a case of spontaneous meningioma in a female pig-tailed macaque (Macaca nemestrina) more than 24 years old. Clinically, the monkey displayed slow, weak, and insecure movements and poor vision. A tumorous mass was present at the floor of the cranial vault extending from the optic chiasm towards the foramen magnum. It compressed adjacent parts of the brain, infiltrated the sphenoidal and occipital bone, and showed transcranial expansion into the pharyngeal area. Histologically, the tumor was consistent with a meningioma displaying mostly meningothelial and some microcystic components. Since only six cases of meningiomas in nonhuman primates have been reported so far and only two of these meningiomas have been described in detail, the findings of each case should be reported to expand the knowledge base of this type of tumor. In addition, this is the first description of a meningioma in pig-tailed macaques.

A case of gallstones in an African green monkey (Chlorocebus aethiops).

Spontaneous cholelithiasis was found in a male African green monkey (Chlorocebus aethiops) at necropsy. Choleliths varied in size, shape and colour. Gallstones were analysed using accepted analytical methods. Results showed that the gallstones were composed of cholesterol and protein in varying proportions. Histologically, the gallbladder showed diffuse mild to moderate lymphocytic infiltration. The etiology of the cholelithiasis in the examined individual remains unknown.

Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge.

Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection.

Dental disease in slender lorises (Loris tardigradus).

Necropsies were performed in 25 slender lorises (Loris tardigradus) from a colony at Bochum University. In seven adult individuals, teeth were missing, loose, or severely affected by dental calculus. In one of these cases, a severe periodontal disease was seen without abscess formation. In another four lorises purulent/gangrenous inflammation was found next to the teeth either in the maxillary or the mandibular region.

Intrahepatic application of suicide gene-armed measles virotherapeutics: a safety study in transgenic mice and rhesus macaques.

Abstract Oncolytic viruses such as measles virus (MV) represent a new class of therapeutic agents that might help to overcome current limitations in cancer therapy. Although MV-based virotherapeutics already have entered clinical testing for various tumor entities, the preclinical safety of MV virotherapeutics so far has not been elucidated for particular regimens with high medical need, such as (1) direct injection into hepatic tumor sites, (2) employing high doses ibidem, and (3) concurrent usage of arming with cytotoxic genes required to further enhance oncolytic efficiency. Here, we assessed the safety and pharmacokinetics of suicide gene-armed vector MV-SCD when administered intrahepatically in two animal models, IFNAR(tm)-CD46(Ge) (interferon-α receptor deficient and CD46 MV receptor knock-in) transgenic mice and rhesus macaques (Macaca mulatta). Clinically, singular direct intrahepatic applications of MV-SCD were found to be well tolerated. Quantitative RT-PCR demonstrated the transient presence of viral RNA in various organs, whereas no shedding of infectious virus particles was observed at any time point. Histological analyses of organs did not exhibit adverse effects attributable to the test article. Blood parameters including liver enzymes revealed no deviations from normal. In both species an antiviral humoral immune response was mounted shortly after virus administration. Surprisingly, daily repeated systemic applications of MV-SCD under concomitant prodrug administration resulted in side effects in IFNAR(tm)-CD46(Ge) mice, but were less pronounced than in a 5-fluorouracil standard therapy control cohort. Taken together, these data indicate that "single shot" direct intrahepatic injections of MV-SCD in conjunction with systemic prodrug administration are safe and could be used in future virotherapeutic treatments of liver cancers.

Gallstones in slender lorises (Loris tardigradus).

A total of 32 slender lorises (Loris tardigradus) kept in captivity were investigated postmortem. In five adult lorises (4 females, 1 male), the gallbladder contained either one gallstone or multiple gallstones. Except for one 5-yr-old female, the affected individuals were between 11 and 13 yr of age. All of the gallstones consisted of 100% cholesterol. Besides other predisposing factors such as species, sex, increasing age, and diabetes mellitus, nutrition may play a major role in the occurrence of these gallstones.

Expression of porcine endogenous retroviruses (PERV) in different organs of a pig.

Porcine endogenous retroviruses (PERVs) represent a particular risk for xenotransplantation using pig cells, tissues or organs. PERVs are integrated in the genome of all pig strains and can be released as particles that infect human cells. We performed for the first time a systematic analysis of PERV expression in different organs of a miniature pig using in parallel quantitative real-time RT-PCR, Western blot analysis, and immunohistochemistry. All three types of PERV, PERV-A, PERV-B and PERV-C were present in the germ line of the animal. In addition, recombinant PERV-A/C were detected in some tissues, but not in the germ line. Expression of the viral full-length and spliced mRNA and proteins was found in many organs, but at different levels. A high expression was found in lymphoid organs.

Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease.

BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

Airborne Mycobacterium avium infection in a group of red-shanked douc langurs (Pygathrix nemaeus nemaeus).

BACKGROUND: This report describes an airborne Mycobacterium avium (MA)-infection in two red-shanked douc langurs (Pygathrix nemaeus nemaeus) from Cologne zoo. METHODS: The two individuals and their tissues were investigated clinically (including x-rays), in pathology, in pathohistology, in classical bacteriology and by polymerase chain reaction (PCR). RESULTS: Clinically, one individual displayed emaciation and a positive reaction in an intrapalpebral testing for M. bovis/MA. The other individual was without any symptoms and did not show any reaction in the intrapalpebral test. In x-ray photos of the lungs, calcified nodules were detected. In pathology, calcified and necrotic nodules were observed within the lungs and the bronchial lymph nodes. In pathohistology, both classical tuberculous granulomas, and few acid fast rods were seen in Ziehl-Neelsen-stain. However, classical bacteriology could not demonstrate mycobacteria. In PCR, MA-infection could be confirmed in one individual using the bronchial lymph nodes. CONCLUSIONS: It was an airborne infection; however, the definite source of infection in these cases remained unclear. Animals in contact to the langurs (house sparrows and mice) as well as water used in the building are the most promising candidates. The risk for a zoonotic transmission in these cases has been calculated to be low.

No in vivo infection of triple immunosuppressed non-human primates after inoculation with high titers of porcine endogenous retroviruses.

UNLABELLED: Porcine endogenous retroviruses (PERVs) released from pig tissue can infect selected human cells in vitro and therefore represent a safety risk for xenotransplantation using pig cells, tissues, or organs. Although PERVs infect cells of numerous species in vitro, attempts to establish reliable animal models failed until now. Absence of PERV transmission has been shown in first experimental and clinical xenotransplantations; however, these trials suffered from the absence of long-term exposure (transplant survival) and profound immunosuppression. METHODS: We conducted infectivity studies in rhesus monkeys, pig-tailed monkeys, and baboons under chronic immunosuppression with cyclosporine A, methylprednisolone, and the rapamycin derivative. These species were selected because they are close to the human species and PERVs can be transmitted in vitro to cells of these species. In addition, the animals received twice, a C1 esterase inhibitor to block complement activation before inoculation of PERV. In order to overcome the complications of microchimerism, animals were inoculated with high titers of cell-free PERV. In addition, to enable transmission via cell-cell contact, some animals also received virus-producing cells. For inoculation the primate cell-adapted strain PERV/5 degrees was used which is characterized by a high infectious titer. Produced on human cells, this virus does not express alpha 1,3 Gal epitopes, does not contain porcine antigens on the viral surface and is therefore less immunogenic in non-human primates compared with pig cell-derived virus. Finally, we present evidence that PERV/5 degrees productively infects cells from baboons and rhesus monkeys. RESULTS: In a follow-up period of 11 months, no antibody production against PERV and no integration of proviral DNA in blood cells was observed. Furthermore, no PERV sequences were detected in the DNA of different organs taken after necropsy. CONCLUSION: These results indicate that in a primate model, in the presence of chronic immunosuppression, neither the inoculation of cell-free nor cell-associated PERV using a virus already adapted to primate cells results in an infection; this is despite the fact that peripheral blood mononuclear cells of the same animals are infectible in vitro.

Polycystic nephropathy in slender lorises (Loris lydekkerianus).

In a colony of slender lorises, 20 deaths that occurred over a period of 11 years were investigated postmortem. Juvenile/adult polycystic nephropathy was observed in one newborn and 13 adult slender lorises. Although polycystic kidney disease (PKD) in humans and other animals is known to be inherited, it is not clear whether kidney alterations in slender lorises are genetically transmitted, stress related, or induced by microbiological influences.

Cataracts in a laboratory colony of African green monkeys (Chlorocebus aethiops).

Three cases of spontaneous cataracts were investigated in a colony of African green monkeys (Chlorocebus aethiops). Clinical, pathological and microbiological investigations were conducted in two cases of juvenile cataracts and in one case of a mature cataract. These investigations revealed no indication of an infection as the cause of cataract development. A genetic correlation existed between the affected individuals. Clinical chemistry gave a hint that calcium might be a "key factor" in the development of these cataracts: in both cases of the juvenile cataracts, the calcium content in the serum and in the aqueous humor was clearly decreased in the affected babies. The calcium uptake in the affected baby itself was not altered but the calcium content in the mother's milk was low.

[: A comparison of commercially available adjuvants in BALB/c-mice immunised with a weekly immunogenic peptide]

The antibody-stimulating activities and side effects of commercially available adjuvants were compared in BALB/c mice immunised with the immunosuppressive (ISU) peptide of HIV I. Clinical and pathological-histological parameters as well as behavioural changes, were used to assess the distress and pain caused to the animals. Complete Freund's Adjuvant (FAk) was used as the positive control and PBS as the negative control. The oil-based adjuvants Montanide ISA 51(R) (M 51), Specol(R), and Hunter's TiterMax Gold(R) (HTMG) were used with the ISU-peptide conjugated to KLH. The water-soluble Gerbu Adjuvant(R) was administered together with the antigen KLH conjugate and also with the ISU-peptide conjugated to cholera toxin B subunit (ChTxB). The Dutch "Code of Practice" was used as a guideline for all immunisations. No changes in animal activity or behaviour was observed in any of the groups. All the oil-based adjuvants gave rise to swellings and encapsulations, which were most pronounced in the HTMG-group. Although body weight increased throughout the study, no increase was seen in any adjuvant group for a short period after each booster immunisation, nor after the first immunisation in the HTMG group. FAk induced a light fever after all immunisations. FAk, Specol and M 51 as well as Gerbu-ChTxB induced antibody titres which were detectable in the ELISA, but no detectable antibody the water-soluble adjuvants or following administration of HTMG applied with the ISU-peptide-KLH conjugate.