Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Development of a Well-Characterized Cynomolgus Macaque Model of Sudan Virus Disease for Support of Product Development.

The primary objective of this study was to characterize the disease course in cynomolgus macaques exposed to Sudan virus (SUDV), to determine if infection in this species is an appropriate model for the evaluation of filovirus countermeasures under the FDA Animal Rule. Sudan virus causes Sudan virus disease (SVD), with an average case fatality rate of approximately 50%, and while research is ongoing, presently there are no approved SUDV vaccines or therapies. Well characterized animal models are crucial for further developing and evaluating countermeasures for SUDV. Twenty (20) cynomolgus macaques were exposed intramuscularly to either SUDV or sterile phosphate-buffered saline; 10 SUDV-exposed animals were euthanized on schedule to characterize pathology at defined durations post-exposure and 8 SUDV-exposed animals were not part of the scheduled euthanasia cohort. Survival was assessed, along with clinical observations, body weights, body temperatures, hematology, clinical chemistry, coagulation, viral load (serum and tissues), macroscopic observations, and histopathology. There were statistically significant differences between SUDV-exposed animals and mock-exposed animals for 26 parameters, including telemetry body temperature, clinical chemistry parameters, hematology parameters, activated partial thromboplastin time, serum viremia, and biomarkers that characterize the disease course of SUDV in cynomolgus macaques.

Development of a Well-Characterized Cynomolgus Macaque Model of Marburg Virus Disease for Support of Vaccine and Therapy Development.

Marburg virus (MARV) is a filovirus that can infect humans and nonhuman primates (NHPs), causing severe disease and death. Of the filoviruses, Ebola virus (EBOV) has been the primary target for vaccine and therapeutic development. However, MARV has an average case fatality rate of approximately 50%, the infectious dose is low, and there are currently no approved vaccines or therapies targeted at infection with MARV. The purpose of this study was to characterize disease course in cynomolgus macaques intramuscularly exposed to MARV Angola variant. There were several biomarkers that reliably correlated with MARV-induced disease, including: viral load; elevated total clinical scores; temperature changes; elevated ALT, ALP, BA, TBIL, CRP and decreased ALB values; decreased lymphocytes and platelets; and prolonged PTT. A scheduled euthanasia component also provided the opportunity to study the earliest stages of the disease. This study provides evidence for the application of this model to evaluate potential vaccines and therapies against MARV and will be valuable in improving existing models.

SARS-CoV-2 targets glial cells in human cortical organoids.

Coronavirus disease 2019 (COVID-19) patients have manifested a variety of neurological complications, and there is still much to reveal regarding the neurotropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human stem cell-derived brain organoids offer a valuable in vitro approach to study the cellular effects of SARS-CoV-2 on the brain. Here we used human embryonic stem cell-derived cortical organoids to investigate whether SARS-CoV-2 could infect brain tissue in vitro and found that cortical organoids could be infected at low viral titers and within 6 h. Importantly, we show that glial cells and cells of the choroid plexus were preferentially targeted in our model, but not neurons. Interestingly, we also found expression of angiotensin-converting enzyme 2 in SARS-CoV-2 infected cells; however, viral replication and cell death involving DNA fragmentation does not occur. We believe that our model is a tractable platform to study the cellular effects of SARS-CoV-2 infection in brain tissue.

Development of a Well-Characterized Rhesus Macaque Model of Ebola Virus Disease for Support of Product Development.

Ebola virus (EBOV) is a negative-sense RNA virus that can infect humans and nonhuman primates with severe health consequences. Development of countermeasures requires a thorough understanding of the interaction between host and pathogen, and the course of disease. The goal of this study was to further characterize EBOV disease in a uniformly lethal rhesus macaque model, in order to support development of a well-characterized model following rigorous quality standards. Rhesus macaques were intramuscularly exposed to EBOV and one group was euthanized at predetermined time points to characterize progression of disease. A second group was not scheduled for euthanasia in order to analyze survival, changes in physiology, clinical pathology, terminal pathology, and telemetry kinetics. On day 3, sporadic viremia was observed and pathological evidence was noted in lymph nodes. By day 5, viremia was detected in all EBOV exposed animals and pathological evidence was noted in the liver, spleen, and gastrointestinal tissues. These data support the notion that EBOV infection in rhesus macaques is a rapid systemic disease similar to infection in humans, under a compressed time scale. Biomarkers that correlated with disease progression at the earliest stages of infection were observed thereby identifying potential "trigger-to-treat" for use in therapeutic studies.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters.

An urgent global quest for effective therapies to prevent and treat coronavirus disease 2019 (COVID-19) is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987 and REGN10933) that targets nonoverlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques, which may model mild disease, and golden hamsters, which may model more severe disease. We demonstrate that REGN-COV-2 can greatly reduce virus load in the lower and upper airways and decrease virus-induced pathological sequelae when administered prophylactically or therapeutically in rhesus macaques. Similarly, administration in hamsters limits weight loss and decreases lung titers and evidence of pneumonia in the lungs. Our results provide evidence of the therapeutic potential of this antibody cocktail.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.

Infection of Amblyomma ovale by Rickettsia sp. strain Atlantic rainforest, Colombia.

Our goal was to understand rickettsial spotted fevers' circulation in areas of previous outbreaks reported from 2006 to 2008 in Colombia. We herein present molecular identification and isolation of Rickettsia sp. Atlantic rainforest strain from Amblyomma ovale ticks, a strain shown to be pathogenic to humans. Infected ticks were found on dogs and a rodent in Antioquia and Córdoba Provinces. This is the first report of this rickettsia outside Brazil, which expands its known range considerably.

Phenotype of the anti-Rickettsia CD8(+) T cell response suggests cellular correlates of protection for the assessment of novel antigens.

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8(+) T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8(+) T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8(+) T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8(+) T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3(+)CD8(+)CD44(high) cells, (2) production of Granzyme B by CD27(low)CD43(low) antigen-experienced CD8(+) T cells, (3) generation of memory-type CD8(+) T cells [Memory Precursor Effector Cells (MPECs), as well as CD127(high)CD43(low), and CD27(high)CD43(low) CD8(+) T cells], and (4) generation of effector-like memory CD8(+) T cells (CD27(low)CD43(low)). We propose that these correlates could be useful for the general assessment of the quality of the CD8(+) T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.

Discovery of novel cross-protective Rickettsia prowazekii T-cell antigens using a combined reverse vaccinology and in vivo screening approach.

Rickettsial agents are some of the most lethal pathogens known to man. Among them, Rickettsia prowazekii is a select agent with potential use for bioterrorism; yet, there is no anti-Rickettsia vaccine commercially available. Owing to the obligate intracellular lifestyle of rickettsiae, CD8(+) T cells are indispensable for protective cellular immunity. Furthermore, T cells can mediate cross-protective immunity between different pathogenic Rickettsia, a finding consistent with the remarkable similarity among rickettsial genomes. However, Rickettsia T cell antigens remain unidentified. In the present study, we report an algorithm that allowed us to identify and validate four novel R. prowazekii vaccine antigen candidates recognized by CD8(+) T cells from a set of twelve in silico-defined protein targets. Our results highlight the importance of combining proteasome-processing as well as MHC class-I-binding predictions. The novel rickettsial vaccine candidate antigens, RP778, RP739, RP598, and RP403, protected mice against a lethal challenge with Rickettsia typhi, which is indicative of cross-protective immunity within the typhus group rickettsiae. Together, our findings validate a reverse vaccinology approach as a viable strategy to identify protective rickettsial antigens and highlight the feasibility of a subunit vaccine that triggers T-cell-mediated cross-protection among diverse rickettsiae.

Discovery of a protective Rickettsia prowazekii antigen recognized by CD8+ T cells, RP884, using an in vivo screening platform.

Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8(+) T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.

A neurodegenerative disease affecting synaptic connections in Drosophila mutant for the tumor suppressor morphogen Patched.

The tumor suppressor morphogen, Patched (Ptc), has an extensive homology to the Niemann-Pick-C 1 (NPC1) protein. The NPC disease is a paediatric, progressive and fatal neurodegenerative disorder thought to be due to an abnormal accumulation of cholesterol in neurons. Here, we report that patched mutant adults develop a progressive neurodegenerative disease and their brain contains membranous and lamellar inclusions. There is also a significant reduction in the number of synaptic terminals in the brain of the mutant adults. Interestingly, feeding cholesterol to wild type flies generates inclusions in the brain, but does not cause the disease. However, feeding cholesterol to mutant flies increases synaptic connections and suppresses the disease. Our results suggest that sequestration of cholesterol in the mutant brain in the form of membranous material and inclusions affects available pool of cholesterol for cellular functions. This, in turn, negatively affects the synaptic number and contributes to the disease-state. Consistent with this, in ptc mutants there is a reduction in the pool of cholesterol esters, and cholesterol-mediated suppression of the disease accompanies an increase in cholesterol esters. We further show that Ptc does not function directly in this process since gain of function for Hedgehog also induces the same disease with a reduction in the level of cholesterol esters. We believe that loss of function for ptc causes neurodegeneration via two distinct ways: de-repression of genes that interfere with lipid trafficking, and de-repression of genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections.

Genetic analysis of the ADGF multigene family by homologous recombination and gene conversion in Drosophila.

Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.

The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases.

The Drosophila melanogaster Fork head and Bombyx mori SGF1/Fork head proteins are key regulators of tissue specific gene expression in the modified larval labial glands. Here we use the competitive electrophoretic mobility shift assay to create a detailed Fork head binding matrix and we investigate some unusual features of the Fork head interaction with DNA. We found that the Fork head-DNA interaction is context dependent--the binding specificity of the protein is partly determined by specific combinations of neighbouring bases. Although the total number of the sub-optimal dinucleotide steps is not high, the negative cooperation of neighbouring bases significantly contributes to the overall binding site specificity. Our results allow efficient recognition of insect Fork head binding sites and we show that the putative Fork head cognate elements preferentially accumulate in the near upstream region of genes abundantly expressed in the labial gland.

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine.

We describe a protein family in Drosophila containing six adenosine deaminase-related growth factors (ADGFs), which are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine.