No abstract

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates complete discrimination of these two groups. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Among the potential nonviral vectors for human gene therapy are DNA-liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA-liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA-liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

A stable single vial lipoplex formulation has been of a T-connector. Homogenous cationic liposome prepdeveloped that can be stored frozen without losing either arations were prepared by extrusion in two different size biological activity or physical stability. This formulation was ranges of either 400 or 100 nm. Extruded liposomes proidentified by systematically controlling several formulation duced more monodisperse and physically stable lipoplex variables and without introducing either stabilizers or surformulations than unextruded liposomes, but the formufactants. Analytical assays were used to unambiguously lations prepared with 100 nm liposomes were less active characterize the formulations. The critical formulation parain in vitro transfection assays than either the 400 nm or meters were: (1) the size of the cationic liposomes; (2) the unextruded liposomes. Low ionic strength and 5% sorbitol rate and method of DNA and cationic liposome mixing; and were required for the lipoplex formulations to survive freez-(3) the ionic strength of the suspending vehicle. The mixing ing and thawing. A frozen lipoplex formulation stored for conditions were precisely controlled by using a novel, spemore than a year maintained its biological activity. These cially designed continuous flow pumping system in which results have broad implications for the pharmaceutical the DNA and liposome solutions were mixed at the junction development of lipoplex formulations for gene delivery.

c Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1-or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1-and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli. Herpes simplex virus 1 (HSV-1) and HSV-2 cause significant human morbidity. HSV-2 is the causative agent of most recurrent genital herpes lesions and is sexually transmitted. Infections are often asymptomatic, and most infected individuals are unaware of the infection, yet HSV-2 is associated with an increased risk of HIV acquisition (33) and an increased risk during pregnancy of spontaneous abortion, premature birth, and perinatal herpes (12, 13). Unawareness of HSV-2 infection is also a major contributing factor to transmission to uninfected partners (64,65). In contrast, HSV-1 is usually transmitted during childhood and is found to be associated predominantly with orolabial infections (cold sores). Also, HSV-1 infection of the eye (ocular herpes) is the most common cause of infectious corneal blindness in industrialized countries. Both HSV-1 and HSV-2 establish lifelong latent infections within the dorsal root and trigeminal ganglia and are characterized by periodic reactivation and virus shedding from mucocutaneous epithelium. Owing to the different natural histories and outcomes of HSV-1 and -2 infections, accurate diagnosis of the HSV type is important for patient management and prognosis and controlling potential transmission. For example, knowing the specific HSV type can help the patient take appropriate precautions to prevent transmission of the disease to others. In particular, the identification of unrecognized HSV-2 infection can be used to carefully monitor virus shedding during pregnancy and minimize the risk of perinatal infection.Laboratory tests for HSV infection include virus culture, virus neutralization, PCR, and serological tests. Virus culture is considered the "gold standard" in the early stages of a primary infection. However, it is less sensitive during the healing stage of infection or during recurrent infections. Culture testi...

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization—a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo . The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-β, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-β). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward T H 2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.