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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.

We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease. Plasmid DNA-encoded anti-OspA HuMAbs inoculated in mice achieved a serum antibody concentration of >6 μg/mL. Among mice injected with DNA-encoded monoclonal antibodies, 75%–77% were protected against an acute challenge by Borrelia-infected ticks. Our results represent the first demonstration of employing DNA transfer as a delivery system for antibodies that block transmission of Borrelia in animal models.

The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens., DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.

Influenza virus remains a significant public health threat despite innovative vaccines and antiviral drugs. A major limitation to current vaccinations and therapies against influenza virus is pathogenic diversity generated by shift and drift. A simple, cost-effective passive immunization strategy via in vivo production of cross-protective antibody molecules may augment existing vaccines and antiviral drugs in seasonal and pandemic outbreaks. We engineered synthetic plasmid DNA to encode two novel and broadly cross-protective monoclonal antibodies targeting influenza A and B. We utilized enhanced in vivo delivery of these plasmid DNA-encoded monoclonal antibody (DMAb) constructs and show that this strategy induces robust levels of functional antibodies directed against influenza A and B viruses in mouse sera. Mice receiving a single inoculation with anti-influenza A DMAb survive lethal Group 1 H1 and Group 2 H3 influenza A challenges, while inoculation with anti-influenza B DMAb yields protection against lethal Victoria and Yamagata lineage influenza B morbidity and mortality. Furthermore, these two DMAbs can be delivered coordinately resulting in exceptionally broad protection against both influenza A and B. We demonstrate this protection is similar to that achieved by conventional protein antibody delivery. DMAbs warrant further investigation as a novel immune therapy platform with distinct advantages for sustained immunoprophylaxis against influenza., Nucleic acid delivery: Instant, wide-ranging protection against influenza A and B A novel innoculation technique involving the injection of antibody-producing plasmid DNA has shown to be effective against influenza in mice. The flu is responsible for up to half a million deaths each year and up to five million cases of severe disease, while also posing a substantial pandemic threat, even with our current repertoire of vaccines. A team of researchers led by Sarah Elliott and David Weiner of The Wistar Institute of Anatomy and Biology, Philadelphia, developed potent plasmid-based constructs that, once injected, entered hosts’ cells and utilized cellular machinery to encode antibodies protective against a range of influenza A and B subtypes. DNA inoculation conferred acute protection from disease, with treated individuals also being immune to subsequent exposure. This approach warrants further investigation as an alternative technology for practical delivery of monoclonal antibody therapeutics.

Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.

Dengue virus (DENV) is the most important mosquito-borne viral infection in humans. In recent years, the number of cases and outbreaks has dramatically increased worldwide. While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes. Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes. Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs. Engineering novel methods of delivering monoclonal antibodies could tip the scale in the fight against DENV. Here we demonstrate that simple intramuscular delivery by electroporation of synthetic DNA plasmids engineered to express modified human nAbs against multiple DENV serotypes confers protection against DENV disease and prevents antibody-dependent enhancement (ADE) of disease in mice. This synthetic nucleic acid antibody prophylaxis/immunotherapy approach may have important applications in the fight against infectious disease.

Dengue virus (DENV) is the most important mosquito-borne viral infection in humans, with nearly 400 million infections occurring each year and a continually expanding geographic reach. While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes. Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes. Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs. Engineering nucleic acid-based strategies for delivering monoclonal antibodies could tip the scale in the fight against DENV, as well as other emerging infectious diseases.Here, we describe an approach to delivering cross-reactive neutralizing antibodies against DENV into the host circulation using DNA plasmid-mediated antibody gene transfer. This approach, which we term DNA mAb (DMAB) delivery, generates biologically relevant levels of mAbs after a single intramuscular injection of antibody-encoding DNA followed by in vivo electroporation (EP). Since this approach allows for genetic tailoring of the exact features of the desired antibody, we incorporated Fc region modifications to a naturally occurring human anti-DENV neutralizing antibody to enhance antibody function in vivo. We demonstrate that intramuscular delivery in mice of pDVSF-3 LALA, which encodes a human anti-DENV1-3 IgG1 neutralizing antibody modified with a mutation that abrogates FcgR binding, produces anti-DENV antisera capable of binding and neutralizing DENV1-3. Importantly, mice receiving pDVSF-3 LALA, but not the unmodified pDVSF-3 WT, were protected from both virus-only disease and antibody-enhanced lethal disease. These data establish this novel platform as a safe, effective means of delivering protective monoclonal antibodies to a host.This work was supported by grants funded to DBW through the National Institutes of Health, the DARPA-PROTECT award, and Inovio Pharmaceuticals Inc.

Despite promising innovations, influenza vaccines and antiviral drugs fail to provide full protection from seasonal infection, and provide little defense against novel and potentially pandemic viral strains. Broadly cross-protective monoclonal antibodies have been developed with the aim of providing protection against highly divergent influenza viruses. However, the utility of delivering purified protein antibody as therapy or prophylaxis against influenza is limited, especially in pandemic settings. Use of gene therapy to generate monoclonal antibodies in vivo provides a simplified, flexible, and relatively inexpensive alternative to protein antibody treatment.In this study, we used intramuscular electroporation of plasmid DNA encoding immunoglobulin to express DNA monoclonal antibodies (DMAb) against influenza hemagglutinin (HA) surface protein in mice. Multiple aspects of plasmid construction, antibody design, and delivery were optimized to enhance expression of DMAb from muscle cells in vivo. We investigated multiple antibody clones, including the broadly-neutralizing anti-influenza-H1 antibody 5J8. The 5J8 DMAb was expressed at µg/mL levels in serum of both nude and immune-competent mice. Serum DMAb produced from muscle in vivo were functional in vitro - with the ability to bind influenza HA, block hemagglutination of red blood cells, and neutralize influenza virus. Serum DMAb expression levels approximate those required for protection. Influenza challenge studies of mice treated with 5J8 DMAb are underway.DMAb provide an important new approach to immune therapy. DNA has an excellent safety profile and averts challenges of pre-existing serology associated with many viral vectors. Here, we demonstrate that DNA can be used to deliver consistently high levels of potent monoclonal antibodies for protection against a viral pathogen.

The development of vaccines against arthropod-borne infectious diseases has been wrought with difficulties. Recent advances in human antibody isolation have uncovered neutralizing monoclonal antibodies (mAbs) that are capable of providing protection against pathogen challenge in various animal models. Yet generating and delivering biologically-relevant levels of such antibodies using conventional monoclonal antibody methodology is impractical, often requiring huge expenses and repeated administrations for clinical benefit. Creating new methods of delivering monoclonal antibodies could drastically tip the scale in the fight against a number of devastating pathogens.Here, we describe an approach to delivering neutralizing mAbs in vivo using DNA plasmid-mediated antibody gene transfer. This approach, which we term DNA mAb (DMAb) delivery, generates biologically relevant levels of mAbs after a single intramuscular injection of antibody-encoding DNA followed by in vivo electroporation (EP). First, we demonstrate the ability of DMAb technology to deliver cross-reactive neutralizing antibodies against DENV into the host circulation. Since this approach allows for genetic tailoring of the exact features of the desired antibody, we incorporated Fc region modifications to a naturally occurring human anti-DENV neutralizing antibody to enhance antibody function in vivo. We show that intramuscular delivery in mice of pDVSF-3 LALA, which encodes a human anti-DENV1-3 IgG1 neutralizing antibody modified with a mutation that abrogates FcγR binding, produces anti-DENV antisera capable of binding and neutralizing DENV1-3. Importantly, mice receiving pDVSF-3 LALA, but not the unmodified pDVSF-3 WT, were protected from both virus-only disease and antibody-enhanced lethal disease.Using a similar, targeted genetic approach to antibody modifications, we also show that DMAbs encoding antibodies against Borrelia burgdorferi (the causative agent of Lyme disease) can undergo extensive amino acid modifications that substantially increase in vivo mAb production levels compared to wild-type DMAb sequences. These data illustrate a subset of the functional optimizations made possible with the DMAb platform.This work was supported by grants funded to DBW through the National Institutes of Health, the DARPA-PROTECT award, and Inovio Pharmaceuticals Inc.

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.

In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.

Therapeutic monoclonal antibodies (mAb) are approved for treatment of several diseases including primary immunodeficiencies, cancer, asthma, and graft transplantation. Yet, only 2 mAbs are approved for administration against infectious diseases: palivizumab (respiratory syncytial virus) and raxibacumab (inhalational anthrax). Numerous protective mAbs targeting recurring and emerging bacterial and viral pathogens have been isolated, however, the high dosage (mg/kg) and associated cost of mAb manufacturing are significant hurdles for routine therapeutic delivery. Recently we have described the development of DNA vector-encoded monoclonal antibodies (DMAbs), as a possible alternative technology. This delivery targets skeletal muscle for invivo transfection to transiently produce and secrete mAb. By optimizing gene design protective levels of antibody are produced in vivo by this technology. We show that expression can last a period of weeks. These designed DMAb formulations encoding the mAb heavy and light chain genes are delivered in vivo by intramuscular injection followed by electroporation (IM-EP). Several DMAbs were developed targeting antimicrobial resistant bacteria, a serious global health concern. Additionally, we also designed DMAbs against a range of viral infections including frequent, emerging, and neglected tropical diseases. Through a series of sequence and formulation optimizations, we are able to achieve serum levels >5ug/mL and as high as 100ug/mL, depending on the DMAb. DMAb serum levels match the protective trough level range of their purified mAb counterparts and perform on par in functional assays (e.g. killing assays, neutralization). DMAb candidates are protective against bacterial and viral challenges in mice, illustrating functionality in vivo. Data from the challenge studies will be presented. Ongoing studies are investigating DMAb gene delivery in larger animal models including rabbits, guinea pigs, and non-human primates. DNA-delivered mAbs is a flexible platform that transforms mAb delivery, allowing for repeat administration, significantly lower production costs, and expands the utility of DNA vector technology for therapeutic gene therapy. This approach may have benefit for routine DMAb gene delivery to prevent nosocomial and community-acquired infections and can be rapidly deployed during an infectious disease outbreak.

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.

First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.