Engineered DNA-Mediated Antibody Gene Transfer for Prophylaxis Against Infectious Diseases

Monoclonal antibodies (mAbs) have become important therapeutic and prophylactic agents for a number of indications, including infectious diseases. However, due to many issues, particularly the high cost of antibody production, mAb therapies are limited to the world’s richest populations. Furthermore, lengthy product development programs mean only a small number of mAb products can be produced at any one time. Engineering novel, low-cost, and simple methods of developing and delivering mAbs would be highly advantageous, potentially expanding the utility of antibody approaches into a wider array of applications. Here, we describe an approach to deliver human IgG 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 developed antibody-encoding DNA plasmids that could reproducibly deliver human mAbs to mouse serum. We show that these plasmid-encoded antibodies have similar binding capacity and functionality to in vitro-produced purified antibodies. Then, we use a mouse model to show that intramuscular delivery of pDVSF-3 LALA, which encodes a human anti-Dengue virus (DENV) IgG1 neutralizing antibody modified with a mutation that abrogates Fcγ receptor binding, produces anti-DENV antisera capable of binding to 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. To build upon these initial findings, we evaluated targeted genetic approaches and alternative delivery regimens in order to increase DMAb expression in vivo. Using DMAbs encoding human IgG1 antibodies against Borrelia burgdorferi (the causative agent of Lyme disease) as a model, we show that specific amino acid modifications to the framework regions of antibody variable domains confer increased in vitro and in vivo DMAb expression levels compared to the original DMAb sequences. Of note, these modifications were found to have no detrimental effect on the antibody’s borreliacidal activity. Lastly, we observed that pre-treatment of the DMAb injection site with hyaluronidase resulted in a 2.4 to 6.4-fold increase in human IgG concentration levels in vivo compared to mice receiving EP-mediated DMAb delivery only. Taken together, these data establish DNA plasmid-based antibody gene transfer as a safe, effective means of delivering tailored, protective monoclonal antibodies to hosts

An Analysis of Racial Disparities in Police Traffic Stops in Suffolk County, Massachusetts, from 2010 to 2019

The murder of George Floyd in May 2020 spurred a national reckoning around how Black people are viewed and treated by law enforcement and the criminal legal system. Some elected officials, prosecutors, and police have acknowledged their moral responsibility to pursue racial justice by examining racial disparities and inequities. This report addresses one such practice—non-traffic-safety stops. These occur when police stop and detain people for minor traffic violations that pose no identifiable risk of harm to people outside of the vehicle. Vera partnered with the Suffolk County (Massachusetts) District Attorney's Office from July 2020 to March 2022 to study racial disparities in the criminal legal system. Vera's analysis revealed that non-traffic-safety stops in Suffolk County are worsening racial disparities in traffic enforcement. This report shares findings from Vera's analysis, along with proposed solutions that prohibit or deter such stops

Pre-exposure Immunoprophylaxis by Genetically Encoded DMAb anti-OspA Human Monoclonal Antibody to Prevent Lyme Disease

Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease. Animal studies have demonstrated that transmission of Borrelia from tick vector to the mammalian host can be blocked by antibodies against outer surface protein A (OspA). We have recently developed borreliacidal human IgG1 monoclonal antibodies (HuMabs) directed against OspA. HuMab 319-44 was borreliacidal against B. burgdorferi (IC50Borreliatransmission after a single dose of 2 mg/kg administered on the day of tick challenge. Since passively administered IgG1 antibodies do not have a sufficient half-life to provide protection for the 6-7 month peak risk period, we investigated a novel approach of vector-mediated gene transfer of HuMabs that could potentially provide protection against Lyme disease during the seasonal risk period. A modified HuMab, 319-44 mod, expressed by a synthetic DNA plasmid (DMAb) was optimized and characterized in in vitro OspA binding and bactericidal assays. To assess in vivo protection, mice were administered a single DMAb injection into the quadriceps followed by electroporation. The mice were then challenged by B. burgdorferi-infected nymphs. Tissue samples were monitored by dark-field microscopy for spirochete growth. Serum samples were analyzed by ELISA to determine antibody concentrations. The modified 319-44 DMAb maintained in vitro biological activity comparable to the un-modified wild type antibody, and formulation-based delivery of DMAb resulted in long-term expression. This led to effective pre-exposure prophylaxis preventing transmission of spirochetes in 80% of mice in the murine model of tick-transmitted Lyme disease. These studies represent the first demonstration of employing DNA transfer as a rapid, novel delivery system for biologically relevant functional full-length HuMAbs in an in vivo animal model and provide support for such an approach for pre-exposure immunoprophylaxis to prevent Lyme disease

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

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

Anti-OspA DNA-Encoded Monoclonal Antibody Prevents Transmission of Spirochetes in Tick Challenge Providing Sterilizing Immunity in Mice

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 \u3e 6 mug/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

Design of animal groups for DNA prime-protein boost immunization study in BALB/c.

<p>Design of animal groups for DNA prime-protein boost immunization study in BALB/c.</p

Serum IgG ELISA responses in guinea pig immunized with HIV-1 envelope.

<p>(A) Timeline for the DNA prime-protein boost immunization study in guinea pigs. Serum samples from the immunized and control guinea pigs were obtained as indicated. (B&C) Anti-gp120 antibody-binding titers were determined by ELISA two weeks after the first protein boost (<i>n = 5</i>). Data are presented as the mean endpoint titers ± D.</p

Characterization of antisera directed against HIV-1 Env.

<p>Binding of mouse antisera from DNA prime-protein boosts with subtypes A, B C and D envelope DNA and subtype B proteins. ELISA plates were coated with recombinant gp120 (subtype B) envelope glycoproteins. (A&B) End-point anti-gp120 IgG titers obtained from mice (<i>n = 4</i>) immunized with different Env immunogens as indicated, data shown titers at day 35, one week after the second protein boost. (C) Correlation between the binding antibody titers and SFU obtained by T-cell ELISpot assay.</p