Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl-/- Mice

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

2021 Jun 02

Kinoshita, Y;Mohamed, FF;Amadeu de Oliveira, F;Narisawa, S;Miyake, K;Foster, BL;Millán, JL;
PMID: 34076297 | DOI: 10.1002/jbmr.4382

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl-/- ) mice, a murine model for severe infantile HPP. Alpl-/- mice received 3 × 1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi . Treatment extended life span of Alpl-/- mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl-/- mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl-/- mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl-/- mice versus controls. Treated Alpl-/- mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP.

Detailed analysis of the pathologic hallmarks of Nipah virus (Malaysia) disease in the African green monkey infected by the intratracheal route

PloS one

2022 Feb 10

Cline, C;Bell, TM;Facemire, P;Zeng, X;Briese, T;Lipkin, WI;Shamblin, JD;Esham, HL;Donnelly, GC;Johnson, JC;Hensley, LE;Honko, AN;Johnston, SC;
PMID: 35143571 | DOI: 10.1371/journal.pone.0263834

Disease associated with Nipah virus infection causes a devastating and often fatal spectrum of syndromes predominated by both respiratory and neurologic conditions. Additionally, neurologic sequelae may manifest months to years later after virus exposure or apparent recovery. In the two decades since this disease emerged, much work has been completed in an attempt to understand the pathogenesis and facilitate development of medical countermeasures. Here we provide detailed organ system-specific pathologic findings following exposure of four African green monkeys to 2.41×105 pfu of the Malaysian strain of Nipah virus. Our results further substantiate the African green monkey as a model of human Nipah virus disease, by demonstrating both the respiratory and neurologic components of disease. Additionally, we demonstrate that a chronic phase of disease exists in this model, that may provide an important opportunity to study the enigmatic late onset and relapse encephalitis as it is described in human disease.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Cell Stem Cell

2021 Dec 01

Jansen, J;Reimer, K;Nagai, J;Varghese, F;Overheul, G;de Beer, M;Roverts, R;Daviran, D;Fermin, L;Willemsen, B;Beukenboom, M;Djudjaj, S;von Stillfried, S;van Eijk, L;Mastik, M;Bulthuis, M;Dunnen, W;van Goor, H;Hillebrands, J;Triana, S;Alexandrov, T;Timm, M;Tideman van den Berge, B;van den Broek, M;Nlandu, Q;Heijnert, J;Bindels, E;Hoogenboezem, R;Mooren, F;Kuppe, C;Miesen, P;Grünberg, K;Ijzermans, T;Steenbergen, E;Czogalla, J;Schreuder, M;Sommerdijk, N;Akiva, A;Boor, P;Puelles, V;Floege, J;Huber, T;van Rij, R;Costa, I;Schneider, R;Smeets, B;Kramann, R;
| DOI: 10.1016/j.stem.2021.12.010

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor

Nature communications

2021 Sep 15

Yang, C;Lei, L;Collins, JWM;Briones, M;Ma, L;Sturdevant, GL;Su, H;Kashyap, AK;Dorward, D;Bock, KW;Moore, IN;Bonner, C;Chen, CY;Martens, CA;Ricklefs, S;Yamamoto, M;Takeda, K;Iwakura, Y;McClarty, G;Caldwell, HD;
PMID: 34526512 | DOI: 10.1038/s41467-021-25749-3

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens

Microbial pathogenesis

2021 Aug 25

Briggs, RE;Billing, SR;Boatwright, WD;Chriswell, BO;Casas, E;Dassanayake, RP;Palmer, MV;Register, KB;Tatum, FM;
PMID: 34454023 | DOI: 10.1016/j.micpath.2021.105159

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

Cell-mimicking nanodecoys neutralize SARS-CoV-2 and mitigate lung injury in a non-human primate model of COVID-19

Nature nanotechnology

2021 Jun 17

Li, Z;Wang, Z;Dinh, PC;Zhu, D;Popowski, KD;Lutz, H;Hu, S;Lewis, MG;Cook, A;Andersen, H;Greenhouse, J;Pessaint, L;Lobo, LJ;Cheng, K;
PMID: 34140674 | DOI: 10.1038/s41565-021-00923-2

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has grown into a global pandemic, and only a few antiviral treatments have been approved to date. Angiotensin-converting enzyme 2 (ACE2) plays a fundamental role in SARS-CoV-2 pathogenesis because it allows viral entry into host cells. Here we show that ACE2 nanodecoys derived from human lung spheroid cells (LSCs) can bind and neutralize SARS-CoV-2 and protect the host lung cells from infection. In mice, these LSC-nanodecoys were delivered via inhalation therapy and resided in the lungs for over 72 h post-delivery. Furthermore, inhalation of the LSC-nanodecoys accelerated clearance of SARS-CoV-2 mimics from the lungs, with no observed toxicity. In cynomolgus macaques challenged with live SARS-CoV-2, four doses of these nanodecoys delivered by inhalation promoted viral clearance and reduced lung injury. Our results suggest that LSC-nanodecoys can serve as a potential therapeutic agent for treating COVID-19.

COVID-19 during Pregnancy: Clinical and In Vitro Evidence against Placenta Infection at Term by SARS-CoV-2

The American journal of pathology

2021 Jun 07

Colson, A;Depoix, CL;Dessilly, G;Baldin, P;Danhaive, O;Hubinont, C;Sonveaux, P;Debiève, F;
PMID: 34111431 | DOI: 10.1016/j.ajpath.2021.05.009

Despite occasional reports of SARS-CoV-2 vertical transmission during pregnancy, the question of placental infection and its consequences for the newborn remain questionable. Here, we analyzed the placentas of 31 COVID-19-positive mothers by RT-PCR, immunohistochemistry and in situ hybridization. We only detected one case of placental infection, which was associated with intrauterine demise of the fetus. We then isolated and differentiated primary trophoblasts from non-pathological human placentas at term, and exposed them to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, we were not able to detect the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant four days after infection. As a mechanism of defense, we hypothesized that trophoblasts at term do not express ACE2 and TMPRSS, the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but the reported case raises concern about preterm infection.

One or two dose regimen of the SARS-CoV-2 synthetic DNA vaccine INO-4800 protects against respiratory tract disease burden in nonhuman primate challenge model

Vaccine

2021 Jun 01

Gooch, K;Smith, T;Salguero, F;Fotheringham, S;Watson, R;Dennis, M;Handley, A;Humphries, H;Longet, S;Tipton, T;Sarfas, C;Sibley, L;Slack, G;Rayner, E;Ryan, K;Schultheis, K;Ramos, S;White, A;Charlton, S;Sharpe, S;Gleeson, F;Humeau, L;Hall, Y;Broderick, K;Carroll, M;
| DOI: 10.1016/j.vaccine.2021.06.057

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we assessed the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induced both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 x 106 pfu) via the intranasal and intratracheal routes we observed significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 was associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provided no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.

Berbamine inhibits Japanese encephalitis virus (JEV) infection by compromising TPRMLs-mediated endolysosomal trafficking of low-density lipoprotein receptor (LDLR)

Emerging microbes & infections

2021 Dec 01

Huang, L;Li, H;Ye, Z;Xu, Q;Fu, Q;Sun, W;Qi, W;Yue, J;
PMID: 34102949 | DOI: 10.1080/22221751.2021.1941276

Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is an important pathogen that causes human and animal infectious diseases in Asia. So far, no effective antiviral agents are available to treat JEV infection. Here, we found that LDLR is a host factor required for JEV entry. Berbamine significantly decreases the level of LDLR at the plasma membrane by inducing the secretion of LDLR via extracellular vesicles (EVs), thereby inhibiting JEV infection. Mechanistically, berbamine blocks TRPMLs (Ca2+ permeable non-selective cation channels in endosomes and lysosomes) to compromise the endolysosomal trafficking of LDLR. This leads to the increased secretion of LDLR via EVs and the concomitant decrease in its level at the plasma membrane, thereby rendering cells resistant to JEV infection. Berbamine also protects mice from the lethal challenge of JEV. In summary, these results indicate that berbamine is an effective anti-JEV agent by preventing JEV entry.

Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection

Viruses

2021 Sep 14

Munnamalai, V;Sammudin, NH;Young, CA;Thawani, A;Kuhn, RJ;Fekete, DM;
PMID: 34578404 | DOI: 10.3390/v13091823

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

Intranasal Administration of a Monoclonal Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection

Viruses

2021 Jul 29

Halwe, S;Kupke, A;Vanshylla, K;Liberta, F;Gruell, H;Zehner, M;Rohde, C;Krähling, V;Gellhorn Serra, M;Kreer, C;Klüver, M;Sauerhering, L;Schmidt, J;Cai, Z;Han, F;Young, D;Yang, G;Widera, M;Koch, M;Werner, A;Kämper, L;Becker, N;Marlow, MS;Eickmann, M;Ciesek, S;Schiele, F;Klein, F;Becker, S;
PMID: 34452363 | DOI: 10.3390/v13081498

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Broad-Based Influenza-Specific CD8+ T Cell Response without the Typical Immunodominance Hierarchy and Its Potential Implication

Viruses

2021 Jun 05

Huang, M;Xu, R;Triffon, C;Mifsud, N;Chen, W;
PMID: 34198851 | DOI: 10.3390/v13061080

Syngeneic murine systems have pre-fixed MHC, making them an imperfect model for investigating the impact of MHC polymorphism on immunodominance in influenza A virus (IAV) infections. To date, there are few studies focusing on MHC allelic differences and its impact on immunodominance even though it is well documented that an individual's HLA plays a significant role in determining immunodominance hierarchy. Here, we describe a broad-based CD8+ T cell response in a healthy individual to IAV infection rather than a typical immunodominance hierarchy. We used a systematic antigen screen approach combined with epitope prediction to study such a broad CD8+ T cell response to IAV infection. We show CD8+ T cell responses to nine IAV proteins and identify their minimal epitope sequences. These epitopes are restricted to HLA-B*44:03, HLA-A*24:02 and HLA-A*33:03 and seven out of the nine epitopes are novel (NP319-330# (known and demonstrated minimal epitope positions are subscripted; otherwise, amino acid positions are shown as normal text (for example NP 319-330 or NP 313-330)), M1124-134, M27-15, NA337-346, PB239-49, HA445-453 and NS1195-203). Additionally, most of these novel epitopes are highly conserved among H1N1 and H3N2 strains that circulated in Australia and other parts of the world.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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