In the past 25 years, the world has witnessed outbreaks of illnesses in humans from three different coronaviruses. Both the SARS-CoV outbreak of 2003 and the MERS-CoV outbreak of 2013 resulted in overall low fatalities in part due to inefficient human-to-human spread of each virus. In contrast, SARS-CoV-2, which emerged in 2019, was highly efficient at human-to-human spread and caused a global pandemic resulting in millions of casualties. Zoonotic transmission of viruses, including the three coronaviruses, poses an ongoing threat that cannot be ignored. In this review, we have focused on the diagnostics and therapeutics fronts using SARS-CoV-2 as a model. Specifically, we have selected proteins associated with the virus particles as targets and discussed various platform technologies. These insights hold the potential to inform the development of more effective therapeutics and vaccines not only for SARS-CoV-2 but also for future viral pandemics, thus contributing to global health on a broader scale.

Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These mAbs are developed from naturally occurring antibodies and target specific epitopes of single molecules, minimizing off-target effects. Antibodies can also be designed to target particular pathogens or modulate immune function by activating or suppressing certain pathways. Despite their benefit for patients, the production and administration of monoclonal antibody therapeutics are laborious, costly, and time-consuming. Administration often requires inpatient stays and repeated dosing to maintain therapeutic levels, limiting their use in underserved populations and developing countries. Researchers are developing alternate methods to deliver monoclonal antibodies, including synthetic nucleic acid-based delivery, to overcome these limitations. These methods allow for in vivo production of monoclonal antibodies, which would significantly reduce costs and simplify administration logistics. This review explores new methods for monoclonal antibody delivery, including synthetic nucleic acids, and their potential to increase the accessibility and utility of life-saving treatments for several diseases.

Cancers employ various strategies to escape immune surveillance including the exploitation of immune checkpoint inhibitors. Checkpoint inhibitors are receptors found on immune and stromal cells whose function can impact the duration or potency of an immune response. Tumor cells often upregulate ligands for these receptors to protect themselves from the host immune response. Monoclonal antibody (MAb) therapeutics which block checkpoint inhibitor-ligand interactions restore T cell destruction of cancer cells in vivo. MAbs that target the inhibitory T cell signaling mediated by CTLA-4 and/or PD-1 checkpoint inhibitors have recently gained regulatory approval for the treatment of some cancers based on remarkable clinical outcomes. Here we have focused on a new method to improve MAb delivery through direct engineering of MAb in the form of synthetic DNA plasmids. This technology would improve many aspects of such a therapy by lowering cost, increasing in vivo expression times and allowing for simple combination formulations in the absence of a host anti-vector immune response, possibly extending use of these groundbreaking therapies to disadvantaged patient populations. We report that "enhanced and optimized" DNA plasmid technology can be used to direct in vivo production of immunoglobulin heavy and light chains of established monoclonal antibodies which can target the immune checkpoint inhibitors LAG3 and PD-1 as determined in Flow cytometry, ELISA and Western blot assays. Both antibodies are produced at physiologically relevant levels in blood and other tissues of mice using electroporation-enhanced delivery of DNA plasmids encoding genes for each antibody. We report that serum antibodies from inoculated animals retain the ability to bind to their targets and are bioactive in vivo and exhibit immune stimulatory effects for host T cells. These studies have significant implications for prophylactic and therapeutic strategies for cancer and other important diseases and warrants further attention.

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR −/− ) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR −/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans.

To date, there have been several million infections by the Chikungunya virus (CHIKV), a mosquito-transmitted emerging pathogen that is considered to be taxonomically an Old World RNA virus. Although original CHIKV outbreaks were restricted to India, East Asian countries, Northern Italy, and France, a recent sharp rise had been identified in 41 countries or territories in the Caribbean, Central America, South America, and North America. A total of 1,012,347 suspected and 22,579 laboratory-confirmed CHIKV cases have been reported from these areas, which signals an increasing risk to the US mainland. Unlike past epidemics that were usually associated with Ae. aegypti transmission, the Caribbean outbreak was associated with Ae. albopictus transmission as the principal mosquito vector. In addition, the substantial increase in the number of deaths during this epidemic, as well as incidence of neurologic disease, suggests that CHIKV may have become more virulent. Currently, there are no licensed vaccines or therapeutics available for CHIKV or its associated disease pathologies. Therefore, development of new vaccines and therapies that could confer immunity and/or treat clinical symptoms of CHIKV is greatly desired. This chapter describes the use of entirely cutting edge technologies/methodologies developed by our group for the development and evaluation of novel DNA vaccines against CHIKV.

Background. Vaccination and passive antibody therapies are critical for controlling infectious diseases. Passive antibody administration has limitations, including the necessity for purification and multiple injections for efficacy. Vaccination is associated with a lag phase before generation of immunity. Novel approaches reported here utilize the benefits of both methods for the rapid generation of effective immunity.Methods. A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti-chikungunya virus (CHIKV) envelope monoclonal antibody (dMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy.Results. One intramuscular injection of dMAb produced antibodies in vivo more rapidly than active vaccination with an anti-CHIKV DNA vaccine. This dMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of dMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection.Conclusions. A DNA-based dMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short-and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

Background: Previous studies describe a relationship between pannus mass and panniculectomy-related complication rates. Patient management may be improved by elucidating the key factors influencing pannus formation. Methods: A retrospective review was conducted of 135 patients who had undergone laparoscopic Roux-en-Y gastric bypass from 1996 to 2010 and subsequent panniculectomy. Outcome measures included age, sex, body mass index, time of surgery, resected pannus mass, comorbidities, and panniculectomy-related complications. Nonparametric continuous and nominal variables were assessed using Spearman rank-correlation and Mann-Whitney U tests, respectively. Results: One hundred thirty-five patients (123 women and 12 men; mean age, 44.7 years) were included in analysis. All patients had body contouring surgery more than 1 year after bariatric surgery (median time interval, 2.1 years). Median body mass index at the time of bypass, 1 year after bypass, and at the time of body contouring surgery was 48.7, 30.0, and 29.4 kg/m2, respectively. Median pannus mass was 2.2 kg. Larger pannus mass was associated with greater age at gastric bypass surgery (p = 0.034), higher pre–gastric bypass body mass index (p = 0.031), higher prepanniculectomy body mass index (p < 0.001), and longer time interval between gastric bypass and panniculectomy (p = 0.046). Female patients requiring blood transfusions had a significantly larger pannus mass than those who did not (p = 0.048). Conclusion: Performing bariatric surgery on patients at a younger age or having patients reduce body mass index as much as possible before bariatric surgery may be useful for minimizing symptomatic pannus formation and in turn may decrease rates of panniculectomy-related complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Abstract Introduction: Breast surgery often results in physical deformities that significantly diminish patient's quality of life. A natural and aesthetically pleasing breast reconstruction can be achieved through autologous fat grafting and the success of this technique has largely been attributed to the regenerative properties of adipose-derived stem cells (ASCs) within fat graft. However, in the setting of resected breast cancer, the growth stimulating and angiogenic effects of ASCs pose a risk of increasing local recurrence rates. Although the safety of autologous fat grafting in the breast cancer population has yet to be determined, potential recurrence risk may be minimized through incorporating tumor-suppressing elements in the graft. This study aimed to determine if doxorubicin and paclitaxel could be used to inhibit breast cancer cells while maintaining the viability and functionality of ASCs in vitro. Materials and methods: Human ASCs were isolated from non-diabetic female patients between 35 and 60 years of age (n = 3). BT-474 and MDA-MB-231 were obtained from ATCC. ASCs, ASCs undergoing adipogenic differentiation, and breast cancer cells were each exposed to a range of doxorubicin-HCl (0, 10, 30, 100, 300, 1000, 3000, or 10000nM) or paclitaxel (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300nM) concentrations. Proliferation, viability, and differentiation capacity were assessed with commercially available CyQuant, MTT, and AdipoRed assay kits, respectively. Results: Dose-dependent inhibition was observed for doxorubicin in ASCs and both breast cancer cell lines. The IC50 of doxorubicin on ASCs, BT-474, and MDA-MB-231 were 901.3, 656.5, and 333 nM, respectively. Dose-dependent inhibition for paclitaxel was only observed in BT-474 and MDA-MB-231 cells, with IC50 values of 1.809 and 3.477 nM, respectively. ASCs maintained greater than 80% viability over the range of paclitaxel concentrations tested and consequently and IC50 value could not be determined. ASCs differentiation into mature adipocytes was not inhibited by doxorubicin exposure. Discussion: In vitro cytotoxicity studies demonstrated greater doxorubicin and paclitaxel sensitivity in BT-474 and MDA-MB-231 than in ASCs. Furthermore, the presence of doxorubicin did not inhibit ASC differentiation into mature adipocytes. These findings suggest that incorporating chemotherapeutic drugs in fat grafts for breast reconstruction following primary breast cancer surgery may be a viable option for decreasing the risk of cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-03.

Vascularization is crucial for implantation of engineered tissues in reconstructive surgery. Polypeptides encapsulated in microspheres can be efficiently transported to their site of action and released in a sustained dosage. We evaluated the effect of delivering vascular endothelial growth factor (VEGF)-encapsulated microspheres in a lipoaspirate scaffold on vascularization and tissue survival. The VEGF-loaded (n = 6) and empty (n = 6) poly(lactic-co-glycolic acid) microspheres in human lipoaspirate and the human lipoaspirate alone (n = 6) were injected subcutaneously into the flanks of athymic nude mice. Three mice from each group were killed, and grafts were explanted at weeks 3 and 6. Increases in mass and volume of VEGF samples, as well as decreases in empty and lipoaspirate-only samples, were observed at 3 and 6 weeks, reaching statistical significance at 6 weeks. Hematoxylin and eosin and CD31(+) imaging demonstrated significantly greater vascularization in VEGF samples than in both the empty and lipoaspirate-only groups at both 3 and 6 weeks.

SummaryDNA vaccination is a novel immunization strategy that has great potential for the development of vaccines and immune therapeutics. This strategy has been highly effective in mice, but is less immunogenic in non‐human primates and in humans. Enhancing DNA vaccine potency remains a challenge. It is likely that antigen‐presenting cells (APCs), and especially dendritic cells (DCs), play a significant role in the presentation of the vaccine antigen to the immune system. A new study reports the synergistic recruitment, expansion and activation of DCs in vivo by high‐mobility group box 1 (HMGB1) protein. Such combinational strategies for delivering vaccine in a single, simple platform will hypothetically bolster the cellular immunity in vivo. Here, we combined plasmid encoding human immunodeficiency virus‐1 (HIV‐1) Gag and Env with an HMGB1 plasmid as a DNA adjuvant in BALB/c mice (by intramuscular immunization via electroporation), and humoral and cellular responses were measured. Co‐administration of this potent immunostimulatory adjuvant strongly enhanced the cellular interferon‐γ (IFN‐γ) and humoral immune response compared with that obtained in mice immunized with vaccine only. Our results show that co‐immunization with HMGB1 can have a strong adjuvant activity, driving strong cellular and humoral immunity that may be an effective immunological adjuvant in DNA vaccination against HIV‐1.

acknowledge and thank VGX Pharmaceuticals Inc, Blue Bell, PA for support and J. Faust for technical assistance with the flow cytometric analysis. In addition, we also acknowledge the AIDS Research and Reference Reagent Program of the National Institutes of Health for providing peptides and other reagents as well as the University of Pennsylvania Center for AIDS Research (CFAR) for additional support. Vance M. Lambert is a student in the University of South Florida Honors College. Drs. Kim and Weiner declare their affiliation with VGX Pharmaceuticals, Inc. which supported a portion of the presented studies.

Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses. Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4(+) T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known. Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.

Chikungunya virus (CHIKV) is an emerging arbovirus and is an important human pathogen. Infection of humans by CHIKV can cause a syndrome characterized by fever, headache, rash, nausea, vomiting, myalgia, arthralgia and occasionally neurological manifestations such as acute limb weakness. It is also associated with a fatal haemorrhagic condition. CHIKV is geographically distributed from Africa through Southeast Asia and South America, and its transmission to humans is mainly through the Aedes aegypti species mosquitoes. The frequency of recent epidemics in the Indian Ocean and La Reunion islands suggests that a new vector perhaps is carrying the virus, as A. aegypti are not found there. In fact, a relative the Asian tiger mosquito, Aedes albopictus, may be the culprit which has raised concerns in the world health community regarding the potential for a CHIK virus pandemic. Accordingly steps should be taken to develop methods for the control of CHIKV. Unfortunately, currently there is no specific treatment for Chikungunya virus and there is no vaccine currently available. Here we present data of a novel consensus-based approach to vaccine design for CHIKV, employing a DNA vaccine strategy. The vaccine cassette was designed based on CHIKV capsid- and envelope-specific consensus sequences with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The expression of capsid, envelope E1 and E1 was evaluated using T7-coupled transcription/translation and immunoblot analysis. A recently developed, adaptive constant-current electroporation technique was used to immunize C57BL/6 mice with an intramuscular injection of plasmid coding for the CHIK-Capsid, E1 and E2. Analysis of cellular immune responses, including epitope mapping, demonstrates that electroporation of these constructs induces both potent and broad cellular immunity. In addition, antibody ELISAs demonstrate that these synthetic immunogens are capable of inducing high titer antibodies capable of recognizing native antigen. Taken together, these data support further study of the use of consensus CHIK antigens in a potential vaccine cocktail.

The mitochondrion is an organelle that regulates various cellular functions including the production of energy and programmed cell death. Aberrant mitochondrial function is often concomitant with various cytopathies and medical disorders. The mitochondrial membrane plays a key role in the induction of cellular apoptosis, and its destabilization, as triggered by both intracellular and extracellular stimuli, results in the release of proapoptotic factors into the cytosol. Not surprisingly, proteins from the human immunodeficiency virus type 1 (HIV) have been implicated in exploiting this organelle to promote the targeted depletion of key immune cells, which assists in viral evasion of the immune system and contributes to the characteristic global immunodeficiency observed during progression of disease. Here we review the mechanisms by which HIV affects the mitochondrion, and suggest that various viral-associated genes may directly regulate apoptotic cell death.