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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2, is a positive-sense single-stranded RNA virus with epithelial cell and respiratory system proclivity. Like its predecessor, SARS-CoV, COVID-19 can lead to life-threatening disease. Due to wide geographic impact affecting an extremely high proportion of the world population it was defined by the World Health Organization as a global public health pandemic. The infection is known to readily spread from person-to-person. This occurs through liquid droplets by cough, sneeze, hand-to-mouth-to-eye contact and through contaminated hard surfaces. Close human proximity accelerates SARS-CoV-2 spread. COVID-19 is a systemic disease that can move beyond the lungs by blood-based dissemination to affect multiple organs. These organs include the kidney, liver, muscles, nervous system, and spleen. The primary cause of SARS-CoV-2 mortality is acute respiratory distress syndrome initiated by epithelial infection and alveolar macrophage activation in the lungs. The early cell-based portal for viral entry is through the angiotensin-converting enzyme 2 receptor. Viral origins are zoonotic with genomic linkages to the bat coronaviruses but without an identifiable intermediate animal reservoir. There are currently few therapeutic options, and while many are being tested, although none are effective in curtailing the death rates. There is no available vaccine yet. Intense global efforts have targeted research into a better understanding of the epidemiology, molecular biology, pharmacology, and pathobiology of SARS-CoV-2. These fields of study will provide the insights directed to curtailing this disease outbreak with intense international impact. Graphical Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large. This Review highlights the progress that has been made in the development of diagnostic tools for the detection of SARS-CoV-2 in the fight against COVID-19.

Background Regulatory T cells (Tregs) maintain immune tolerance. While Treg-mediated neuroprotective activities are now well-accepted, the lack of defined antigen specificity limits their therapeutic potential. This is notable for neurodegenerative diseases where cell access to injured brain regions is required for disease-specific therapeutic targeting and improved outcomes. To address this need, amyloid-beta (Aβ) antigen specificity was conferred to Treg responses by engineering the T cell receptor (TCR) specific for Aβ (TCR A β ). The TCR Ab were developed from disease-specific T cell effector (Teff) clones. The ability of Tregs expressing a transgenic TCR Aβ (TCR Aβ -Tregs) to reduce Aβ burden, transform effector to regulatory cells, and reverse disease-associated neurotoxicity proved beneficial in an animal model of Alzheimer’s disease. Methods TCR A β -Tregs were generated by CRISPR-Cas9 knockout of endogenous TCR and consequent incorporation of the transgenic TCR Ab identified from Aβ reactive Teff monoclones. Antigen specificity was confirmed by MHC-Aβ-tetramer staining. Adoptive transfer of TCR Aβ -Tregs to mice expressing a chimeric mouse-human amyloid precursor protein and a mutant human presenilin-1 followed measured behavior, immune, and immunohistochemical outcomes. Results TCR Aβ -Tregs expressed an Aβ-specific TCR. Adoptive transfer of TCR Aβ -Tregs led to sustained immune suppression, reduced microglial reaction, and amyloid loads. 18 F-fluorodeoxyglucose radiolabeled TCR Aβ -Treg homed to the brain facilitating antigen specificity. Reduction in amyloid load was associated with improved cognitive functions. Conclusions TCR Aβ -Tregs reduced amyloid burden, restored brain homeostasis, and improved learning and memory, supporting the increased therapeutic benefit of antigen specific Treg immunotherapy for AD. Graphical Abstract

Antiretroviral therapy (ART) improves the quality of life for those living with the human immunodeficiency virus type one (HIV-1). However, poor compliance reduces ART effectiveness and leads to immune compromise, viral mutations, and disease co-morbidities. Here we develop a drug formulation in which a lipid-based nanoparticle (LBNP) carrying rilpivirine (RPV) is decorated with the C-C chemokine receptor type 5 (CCR5) targeting peptide. This facilitates extended drug persistence within myeloid cells. Particle delivery to viral reservoirs is tracked by positron emission tomography. The CCR5-mediated LBNP cell uptake and retention reduce HIV-1 replication in human monocyte-derived macrophages and infected humanized mice (hu mice). Focused ultrasound with microbubbles mediated blood brain barrier (BBB) disruption allows the CCR5-targeted LBNP to penetrate the BBB and reach brain myeloid cells. These findings offer a role for CCR5-targeted therapeutics in antiretroviral delivery to optimize HIV suppression. Here the authors made lipid-based CCR5-receptor targeted nanoparticles to facilitate cell-based delivery of the antiretroviral drug rilpivirine, improving HIV-1 suppression in cell and tissue reservoirs. Focused ultrasound facilitates penetrance of the nanoparticles across the blood-brain barrier where they enter myeloid cells in humanized mice.

Extracellular vesicles (EVs) are the common designation for ectosomes, microparticles and microvesicles serving dominant roles in intercellular communication. Both viable and dying cells release EVs to the extracellular environment for transfer of cell, immune and infectious materials. Defined morphologically as lipid bi-layered structures EVs show molecular, biochemical, distribution, and entry mechanisms similar to viruses within cells and tissues. In recent years their functional capacities have been harnessed to deliver biomolecules and drugs and immunological agents to specific cells and organs of interest or disease. Interest in EVs as putative vaccines or drug delivery vehicles are substantial. The vesicles have properties of receptors nanoassembly on their surface. EVs can interact with specific immunocytes that include antigen presenting cells (dendritic cells and other mononuclear phagocytes) to elicit immune responses or affect tissue and cellular homeostasis or disease. Due to potential advantages like biocompatibility, biodegradation and efficient immune activation, EVs have gained attraction for the development of treatment or a vaccine system against the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. In this review efforts to use EVs to contain SARS CoV-2 and affect the current viral pandemic are discussed. An emphasis is made on mesenchymal stem cell derived EVs' as a vaccine candidate delivery system.

The COVID-19 pandemic has affected more than 38 million people world-wide by person to person transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic and preventative strategies for SARS-CoV-2 remains a significant challenge. Within the past several months, effective treatment options have emerged and now include repurposed antivirals, corticosteroids and virus-specific antibodies. The latter has included convalescence plasma and monoclonal antibodies. Complete viral eradication will be achieved through an effective, safe and preventative vaccine. To now provide a comprehensive summary for each of the pharmacotherapeutics and preventative strategies being offered or soon to be developed for SARS-CoV-2. Graphical abstract

Regulatory T cells (Tregs) maintain immune tolerance. While Treg-mediated neuroprotective activities are now well-accepted, the lack of defined antigen specificity limits their therapeutic potential. This is notable for neurodegenerative diseases where cell access to injured brain regions is required for disease-specific therapeutic targeting and improved outcomes. To address this need, amyloid-beta (A[beta]) antigen specificity was conferred to Treg responses by engineering the T cell receptor (TCR) specific for A[beta] (TCR.sub.A.sub.[beta]). The TCR.sub.Ab were developed from disease-specific T cell effector (Teff) clones. The ability of Tregs expressing a transgenic TCR.sub.A[beta] (TCR.sub.A[beta] -Tregs) to reduce A[beta] burden, transform effector to regulatory cells, and reverse disease-associated neurotoxicity proved beneficial in an animal model of Alzheimer's disease. TCR.sub.A.sub.[beta] -Tregs were generated by CRISPR-Cas9 knockout of endogenous TCR and consequent incorporation of the transgenic TCR.sub.Ab identified from A[beta] reactive Teff monoclones. Antigen specificity was confirmed by MHC-A[beta]-tetramer staining. Adoptive transfer of TCR.sub.A[beta]-Tregs to mice expressing a chimeric mouse-human amyloid precursor protein and a mutant human presenilin-1 followed measured behavior, immune, and immunohistochemical outcomes. TCR.sub.A[beta]-Tregs expressed an A[beta]-specific TCR. Adoptive transfer of TCR.sub.A[beta]-Tregs led to sustained immune suppression, reduced microglial reaction, and amyloid loads. .sup.18F-fluorodeoxyglucose radiolabeled TCR.sub.A[beta]-Treg homed to the brain facilitating antigen specificity. Reduction in amyloid load was associated with improved cognitive functions. TCR.sub.A[beta]-Tregs reduced amyloid burden, restored brain homeostasis, and improved learning and memory, supporting the increased therapeutic benefit of antigen specific Treg immunotherapy for AD.

The purpose of this thesis was to study the pharmacokinetics of amoxicillin in skin following iontophoretic administration by using a stainless-steel patch. FDA labeled indication of amoxicillin includes treatment of skin infections. The pharmacokinetic behavior of amoxicillin (AMX) in skin is poorly studied. It has been observed that amoxicillin is used for anthrax and treats skin or soft tissue infection, cutaneous bacillus anthracis and bacterial Infection caused by susceptible Streptococcus (α- or β-hemolytic strains only), Staphylococcus, or Escherichia coli . Ciprofloxacin or doxycycline is the initial drug of choice for postexposure prophylaxis following a suspected or confirmed bioterrorism-related anthrax exposure whereas amoxicillin is an alternative for postexposure prophylaxis of anthrax following exposure to aerosolized bacillus anthracis spores (inhalational anthrax). Amoxicillin is also an alternative for treatment of cutaneous anthrax. Iontophoresis is a penetration enhancer technique that uses a mild electrical current to increase the penetration of charged ions through the skin. Since AMX is ionized at physiological pH it looks a suitable candidate for iontophoretic delivery. Skin concentrations were monitored via microdialysis sampling. Microdialysis is a semi-invasive separation technique that allows the sampling or the delivery of molecules in vivo, according to the concentration gradient between the solution perfusing the probe and the extracellular fluid surrounding it. Owing to selective access to the target site for most anti-infective drugs, microdialysis satisfies regulatory requirements for pharmacokinetic distribution studies and has become a reference technique for tissue distribution studies. The HPLC method selected and validated for the determination of amoxicillin in microdialysis samples consisted of a reversed phase C18 column, flow rate of 1 ml/min and a detection wavelength of 230 nm. Mobile phase used for microdialysis consisted of 90% 50mM phosphate buffer (pH=3) containing 0.1% triethylamine and 10% methanol. The retention time was 3.93 min. The calibration curves for microdialysis samples were linear in the range of 0.1 to 100 1.1.g/m1 with a correlation coefficient larger than 0.99. The lower limit of quantification (LLOQ) was 0.1 µg/ml. The kinetics of amoxicillin was investigated in 3 female pathogen-free New Zealand albino rabbits. Microdialysis probes were implanted into the upper dorsal shaved skin of a tranquilized rabbit and perfused with lactated ringer's solution. Patches donated by Dr. Phillip Friden (Transport Pharmaceuticals, Framingham, MA) were used to deliver amoxicillin for 1 hour at different current densities (100, 200, and 300/cm2) on different occasions in a randomized cross-over experimental design in same dose. Microdialysis samples were collected at selected time intervals. Retrodialysis was performed at the start of each experiment to assess probe recovery and correct the dialysate concentration to reflect the actual interstitial fluid concentration. The results show that measurable amoxicillin concentrations were reached immediately after the onset of the current in the skin. Though there was no considerable difference in Cmax and AUC across the different current densities, this study indicates the possibility of transdermal delivery of amoxicillin by iontophoresis.

Abstract A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high mutation rate of HIV-1 gives rise to numerous circulating strains with increased capacity for immune evasion and antiretroviral drug resistance. To facilitate viral elimination while accounting for this diversity, we propose genetic inactivation of proviral DNA with CRISPR-spCas9. We designed a library of “mosaic gRNAs” against a HIV-1 consensus sequence constructed from 4004 clinical strains, targeting the viral transcriptional regulator tat. Testing in 7 HIV-1 transmitted founder strains led, on average, to viral reductions of 82% with tandem TatD and TatE (TatDE) treatment. No off-target cleavages were recorded. Lentiviral transduction of TatDE attenuated latency reversal by 94% in HIV-infected, transcriptionally silent ACH2 T cells. In all, TatDE guide RNAs successfully disrupted 5 separate HIV-1 exons (tat1-2/rev1-2/gp41) providing a pathway for CRISPR-directed HIV-1 cure therapies. Significance Statement Over 38 million individuals worldwide are infected with HIV-1, which necessitates lifelong dependence on antiretroviral therapy (ART) to prevent viral replication that leads to AIDS. Efforts to rid hosts of HIV-1 are limited by the virus’ abilities to integrate proviral DNA in nuclei, mutate their genomes, and lay dormant for decades during ART treatment. We developed mosaic guide RNAs, TatD and TatE, for CRISPR-Cas9 that recognize the majority of known HIV-1 strains and inactivate 94% of proviral DNA in latently infected cells. Tandem TatDE-CRISPR inactivation of 5 viral exons (tat1-2, rev1-2, and gp41), which blocked HIV-1 replication for 28 days in CD4+ T cells without unwanted editing to the host genome, may serve as a viable strategy for HIV cure. Competing Interest Statement J.H., M.H., and H.E.G. are named inventors on provisional patents for the CRISPR therapy described in this report (62/985,392; 62/986,216). J.H., M.H., B.K., and H.E.G hold a patent on a virus-like particle-based delivery for HIV-1 CRISPR therapeutics (Docket No. 19040PCT; Serial No. PCT/US2020/016126; International Publication No. WO 2020/160418 A1). H.E.G is a member of the scientific advisory board at Longevity Biotech and a co-founder of Exavir Therapeutics, Inc. Footnotes * ↵1 Co-first authors * Competing Interest Statement: J.H., M.H., and H.E.G. are named inventors on provisional patents for the CRISPR therapy described in this report (62/985,392; 62/986,216). J.H., M.H., B.K., and H.E.G hold a patent on a virus-like particle-based delivery for HIV-1 CRISPR therapeutics (Docket No. 19040PCT; Serial No. PCT/US2020/016126; International Publication No. WO 2020/160418 A1). H.E.G is a member of the scientific advisory board at Longevity Biotech and a co-founder of Exavir Therapeutics, Inc. * doi:10.17632/phyy89w9c2.1

ABSTRACT Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to coronavirus disease 2019 (COVID-19). Virus-specific immunity controls infection, transmission and disease severity. With respect to disease severity, a spectrum of clinical outcomes occur associated with age, genetics, comorbidities and immune responses in an infected person. Dysfunctions in innate and adaptive immunity commonly follow viral infection. These are heralded by altered innate mononuclear phagocyte differentiation, activation, intracellular killing and adaptive memory, effector, and regulatory T cell responses. All of such affect viral clearance and the progression of end-organ disease. Failures to produce effective controlled antiviral immunity leads to life-threatening end-organ disease that is typified by the acute respiratory distress syndrome. The most effective means to contain SARS-CoV-2 infection is by vaccination. While an arsenal of immunomodulators were developed for control of viral infection and subsequent COVID-19 disease, further research is required to enable therapeutic implementation. Severe acute respiratory syndrome causing coronavirus 2 (SARS-CoV-2) has set off a pandemic with more than 3.8 million COVID-19 casualties. Although several emergency authorized vaccines are effective against the circulating strains, it is more important than ever to provide comprehensive information on the immunopathology of this agent.