A Phase 1 Two-Arm, Randomized, Double-Blind, Active-Controlled Study of Live, Oral Plasmid-Derived Adenovirus Type 4 and Type 7 Vaccines in Seronegative Adults.

PXVX0047 is an investigational vaccine developed for active immunization to prevent febrile acute respiratory disease (ARD) caused by adenovirus serotypes 4 (Ad4) and 7 (Ad7). PXVX0047 consists of a modernized, plasmid-derived vaccine that was generated using a virus isolated from Wyeth Ad4 and Ad7 vaccine tablets. A phase 1 two-arm, randomized, double-blind, active-controlled study was conducted to evaluate the safety profile and immunogenicity of the investigational adenovirus vaccines. The two components of PXVX0047 were administered orally together in a single dose to 11 subjects. For comparison, three additional subjects received the Ad4/Ad7 vaccine that is currently in use by the US military. The results of this study show that the tolerability and immunogenicity of the PXVX0047 Ad7 component are comparable with that of the control Ad4/Ad7 vaccine; however, the immunogenicity of the PXVX0047 Ad4 component was lower than expected. Clinical trial number NCT03160339.

Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Clonogenic Cell Survival in Human Microvascular Endothelial Cells.

To better understand radiation-induced organ dysfunction at both high and low doses, it is critical to understand how endothelial cells (ECs) respond to radiation. The impact of irradiation (IR) on ECs varies depending on the dose administered. High doses can directly damage ECs, leading to EC impairment. In contrast, the effects of low doses on ECs are subtle but more complex. Low doses in this study refer to radiation exposure levels that are below those that cause immediate and necrotic damage. Mitochondria are the primary cellular components affected by IR, and this study explored their role in determining the effect of radiation on microvascular endothelial cells. Human dermal microvascular ECs (HMEC-1) were exposed to varying IR doses ranging from 0.1 Gy to 8 Gy (~0.4 Gy/min) in the AFRRI 60-Cobalt facility. Results indicated that high doses led to a dose-dependent reduction in cell survival, which can be attributed to factors such as DNA damage, oxidative stress, cell senescence, and mitochondrial dysfunction. However, low doses induced a small but significant increase in cell survival, and this was achieved without detectable DNA damage, oxidative stress, cell senescence, or mitochondrial dysfunction in HMEC-1. Moreover, the mitochondrial morphology was assessed, revealing that all doses increased the percentage of elongated mitochondria, with low doses (0.25 Gy and 0.5 Gy) having a greater effect than high doses. However, only high doses caused an increase in mitochondrial fragmentation/swelling. The study further revealed that low doses induced mitochondrial elongation, likely via an increase in mitochondrial fusion protein 1 (Mfn1), while high doses caused mitochondrial fragmentation via a decrease in optic atrophy protein 1 (Opa1). In conclusion, the study suggests, for the first time, that changes in mitochondrial morphology are likely involved in the mechanism for the radiation dose-dependent effect on the survival of microvascular endothelial cells. This research, by delineating the specific mechanisms through which radiation affects endothelial cells, offers invaluable insights into the potential impact of radiation exposure on cardiovascular health.

Discovery of Rickettsia spp. in mosquitoes collected in Georgia by metagenomics analysis and molecular characterization.

Arthropods have a broad and expanding worldwide presence and can transmit a variety of viral, bacterial, and parasite pathogens. A number of Rickettsia and Orientia species associated with ticks, fleas, lice, and mites have been detected in, or isolated from, patients with febrile illness and/or animal reservoirs throughout the world. Mosquitoes are not currently considered vectors for Rickettsia spp. pathogens to humans or to animals. In this study, we conducted a random metagenome next-generation sequencing (NGS) of 475 pools of Aedes, Culex, and Culiseta species of mosquitoes collected in Georgia from 2018 to 2019, identifying rickettsial gene sequences in 33 pools of mosquitoes. We further confirmed the findings of the Rickettsia by genus-specific quantitative PCR (qPCR) and multi-locus sequence typing (MLST). The NGS and MLST results indicate that Rickettsia spp. are closely related to Rickettsia bellii, which is not known to be pathogenic in humans. The results, together with other reports of Rickettsia spp. in mosquitoes and the susceptibility and transmissibility experiments, suggest that mosquitoes may play a role in the transmission cycle of Rickettsia spp.

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques.

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

Human Bocavirus as a Possible Contributor to Respiratory Disease in the Georgian Military Population.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated that new and devastating respiratory pathogens can emerge without warning. It is therefore imperative that Special Operations medical personnel be aware of the presence of emerging pathogens within their area of operation. Human bocavirus (HBoV) is a newly described member of a family of viruses known as the Parvovirinae that are often associated with acute respiratory illness. The presence of HBoV in the country of Georgia has not been previously reported. Nasal and throat swabs were collected from 95 symptomatic members of the Georgian military. HBoV was detected in 11 of them (12%). To our knowledge, this is the first report of HBoV infection in the country of Georgia. This finding may have a significant impact on members of the Special Operations community who train in Georgia as more data concerning the transmission, pathogenesis, and treatment of HBoV are accumulated and the role of HBoV in human disease is more clearly defined.

Live Oral Adenovirus Type 4 and Type 7 Vaccine Induces Durable Antibody Response.

Human adenoviruses (AdV) are mostly associated with minimal pathology. However, more severe respiratory tract infections and acute respiratory diseases, most often caused by AdV-4 and AdV-7, have been reported. The only licensed vaccine in the United States, live oral AdV-4 and AdV-7 vaccine, is indicated for use in the military, nearly exclusively in recruit populations. The excellent safety profile and prominent antibody response of the vaccine is well established by placebo-controlled clinical trials, while, long-term immunity of vaccination has not been studied. Serum samples collected over 6 years from subjects co-administered live oral AdV-4 and AdV-7 vaccine in 2011 were evaluated to determine the duration of the antibody response. Group geometric mean titers (GMT) at 6 years post vaccination compared to previous years evaluated were not significantly different for either AdV-4 or AdV-7 vaccine components. There were no subjects that demonstrated waning neutralization antibody (NAb) titers against AdV-4 and less than 5% of subjects against AdV-7. Interestingly, there were subjects that had a four-fold increase in NAb titers against either AdV-4 or AdV-7, at various time points post vaccination, suggesting either homotypic or heterotypic re-exposure. This investigation provided strong evidence that the live oral AdV-4 and AdV-7 vaccine induced long-term immunity to protect from AdV-4 and AdV-7 infections.

Targeted Sequencing of Respiratory Viruses in Clinical Specimens for Pathogen Identification and Genome-Wide Analysis.

A large number of viruses can individually and concurrently cause various respiratory illnesses. Metagenomic sequencing using next-generation sequencing (NGS) technology is capable of identifying a variety of pathogens. Here, we describe a method using a large panel of oligo probes to enrich sequence targets of 34 respiratory DNA and RNA viruses that reduces non-viral reads in NGS data and achieves high performance of sequencing-based pathogen identification. The approach can be applied to total nucleic acids purified from respiratory swabs stored in viral transport medium. Illumina TruSeq RNA Access Library procedure is used in targeted sequencing of respiratory viruses. The samples are subjected to RNA fragmentation, random reverse transcription, random PCR amplification, and ligation with barcoded library adaptors. The libraries are pooled and subjected to two rounds of enrichments by using a large panel of oligos designed to capture whole genomes of 34 respiratory viruses. The enriched libraries are amplified and sequenced using Illumina MiSeq sequencing system and reagents. This method can achieve viral detection sensitivity comparable with molecular assay and obtain partial to complete genome sequences for each virus to allow accurate genotyping and variant analysis.

Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

In vitro human bone marrow analog: clinical potential.

Bone marrow is the primary site of hematopoiesis in adult humans. Bone marrow can be cultured in vitro but few simple culture systems fully support hematopoiesis beyond a few months. Human bone marrow analogs are long-term in vitro cultures of marrow stromal and hematopoietic stem cells that can be used to produce cells and products normally harvested from human donors. Bone marrow analog systems should exhibit confluence of the stromal cell populations, persistence of hematopoietic progenitor cells, presence of active regions of hematopoiesis and capacity to produce mature cell types for extended periods of time. Although we are still years away from realizing clinical application of products formed by artificial bone marrow analogs, the process of transitioning this research tool from bench to bedside should be fairly straightforward. The most obvious application of artificial marrow would be for production of autologous hematopoietic CD34(+) stem cells as a stem cell therapy for individuals experiencing bone marrow failure due to disease or injury. Another logical application is for 'blood farming', a process for large-scale in vitro production of red blood cells, white blood cells or platelets, for transfusion or treatment. Other possibilities include production of nonhematopoietic stem cells such as osteogenic stromal cells, osteoblasts and rare pluripotent stem cells. Bone marrow analogs also have great potential as ex vivo human test systems and could play a critical role in drug discovery, drug development and toxicity testing in the future.

Tolerance by selective in vivo expansion of foreign major histocompatibility complex-transduced autologous bone marrow.

BACKGROUND: Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. METHODS: To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. RESULTS: After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. CONCLUSION: Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.

Competitive equality of donor cells expressing a disparate MHC antigen following stem cell-enriched bone marrow transplantation.

INTRODUCTION: Bone marrow cells expressing foreign MHC antigens survive poorly after transplantation. Stable mixed hematopoietic chimerism requires reconstitution with a relatively large number of foreign bone marrow cells and intensive depletion of host cells. In addition, when foreign MHC-transduced autologous bone marrow cells are transplanted, prolonged hematopoietic transgene expression requires extensive host conditioning. The competitive disadvantage associated with engraftment of donor cells expressing foreign MHC antigens is thought to result from a defect in engraftment secondary to donor-host incompatibility or immunologic resistance by the host. METHODS: We used a limiting-dilution competitive repopulation assay with cells from HLA-A2.1 transgenic mice to determine whether and to what extent foreign MHC antigen expression impairs engraftment in C57BL/6 hosts. Transplants were performed with Hoechst 33342 fluorescence-sorted side population (SP) cells, a subset of bone marrow enriched for stem cells. RESULTS.: Transplantation with 250 stem cell-enriched HLA-A2.1-transgenic side population cells successfully competed with nearly 5000 host C57BL/6 side population cells to produce stable long-term mixed chimerism. There was a direct relationship between the number of transplanted donor HLA-A2-expressing cells and the percentage of HLA-A2-expressing cells in the peripheral blood of reconstituted C57BL/6 mice (r2=0.1799, P=0.031). This correlation was maintained in secondary transplant recipients. CONCLUSIONS: HLA-A2-expressing hematopoietic cells do not have an engraftment defect when transplanted into C57BL/6 hosts and immunologic resistance did not limit chimerism following lethal irradiation. These results may have relevance to understanding long-term gene expression after hematopoietic stem cell based gene therapy.

Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells.

BACKGROUND: Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. METHODS: Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. RESULTS: After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. CONCLUSIONS: Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.