Seleeke Flingai

Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants

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.

A synthetic consensus anti–spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

A consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge. Emerging vaccines Public outcry drives vaccine research during outbreaks of emerging infectious disease, but public support for vaccine development dries up when the outbreaks are resolved, frequently leaving promising vaccine candidates sitting on the shelf. DNA vaccines, with their potential for rapid large-scale production, may help overcome this hurdle. Muthumani et al. report the development of a synthetic DNA vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) that induces neutralizing antibodies in mice, macaques, and camels—natural hosts of MERS-CoV. Indeed, macaques vaccinated with this DNA vaccine were protected from viral challenge. These promising results support further development of DNA vaccines for emerging infections. 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.

Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

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.

Protection against dengue disease by synthetic nucleic acid antibody prophylaxis/immunotherapy

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.

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

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.

Rapid and Long-Term Immunity Elicited by DNA-Encoded Antibody Prophylaxis and DNA Vaccination Against Chikungunya Virus

Abstract 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.

Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

401. In Vivo Expression of Plasmid Encoded IgG for PD-1 or LAG3 by Synthetic DNA as a New Tool for Cancer Immunotherapy

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.

428. Generation of DNA Plasmid-Encoded Neutralizing Monoclonal Antibodies In Vivo

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.

433. DNA Monoclonal Antibodies Target Influenza Virus In Vivo

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.