key: cord-0013338-j3assowx
authors: Guibinga, Ghiabe H.; Sahay, Bikash; Brown, Heather; Cooch, Neil; Chen, Jing; Yan, Jian; Reed, Charles; Mishra, Meerambika; Yung, Bryan; Pugh, Holly; Schultheis, Katherine; Esquivel, Rianne N.; Weiner, David B.; Humeau, Laurent H.; Broderick, Kate E.; Smith, Trevor R.F.
title: Protection against Borreliella burgdorferi infection mediated by a synthetically engineered DNA vaccine
date: 2020-08-12
journal: nan
DOI: 10.1080/21645515.2020.1789408
sha: 2d08b9cf3756ee71d1ac026ba8df23ab02f8300c
doc_id: 13338
cord_uid: j3assowx

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.

Lyme disease is highly prevalent in Europe and North America. 1 The etiological agents are spirochetes of the Borreliella burgdorferi group and related species. 2 These bacteria are transmitted by ticks, with small mammals serving as reservoirs. In the United States, Borreliella burgdorferi is the most prevalent Lyme diseaseassociated species, and the number of reported cases have doubled over the past 15 years. 3 Thirty-thousand cases of Lyme disease are reported to the CDC in the US each year, and predicts the number of clinician-diagnosed cases exceeds 300,000 a year. 3, 4 The majority of patients respond adequately to antibiotic treatment. However, a significant number of patients develop persistent symptoms with local and systemic manifestations that vary in severity and duration that can last for months to years, 5 termed post-treatment Lyme disease syndrome (PTLDS). The armamentarium available to the medical community to tackle the increasing incidence of Lyme disease, and to effectively prevent or treat PTLDS is limited.

To develop effective countermeasures against Lyme disease several targets and mechanisms have been investigated. Multiple studies have demonstrated that the transmission of Borreliella Burgdorferi from tick to mammalian host can be interrupted with antibodies targeting the OspA antigen. [6] [7] [8] [9] OspA mediates the attachment of the spirochetes to the tick midgut, and its expression is downregulated during the blood-feeding process when the bacteria migrate from the midgut to salivary glands; consequently, anti-OspA antibodies harbored in the host can block bacterial transmission during hematophagy. Vaccines targeting OspA have been developed. The recombinant OspA protein vaccine, LYMErix demonstrated efficacy, and was briefly available on the market. 10 The efficacy of this recombinant protein OspA vaccine was modest, 49% after 2 immunizations, and yearly boosts were required. Furthermore, there were concerns surrounding reports of immune cross-reactivity between an epitope present in OspA and the human LFA-1 antigen, and the postulation that vaccination may initiate an autoreactive immune mechanism leading to Lyme-associated arthritis. 11 Additionally, poor representation of the vaccine in the media and lack of public enthusiasm resulted in poor sales. The vaccine was ultimately pulled from the market in 2002. Post hoc assessment revealed the vaccine to be safe with no difference in arthritis cases between vaccinated and controls, and the postulation that the OspA vaccine-elicited cross-reactive anti-LFA-1 responses that could mediate arthritis was ultimately withdrawn. 11, 12 Currently, there is no Lyme disease vaccine available for human use.

Newer engineered synthetic nucleic acid vaccines delivered by advanced CELLECTRA® adaptive electroporation have proven to be highly effective in eliciting robust and long-lasting immunity in multiple clinical studies against infectious disease targets, including Zika, Ebola and MERS. [13] [14] [15] In these studies, rapid seroconversion at durable high titers, neutralizing antibodies, passive transfer protection to animal challenges from human sera, and induction of CD4 and CD8 T cells has been observed in most subjects. [13] [14] [15] Furthermore, in other therapeutic studies involving HPV-related cervical infections, strong T cell responses and T cell invasion into the infected tissues as well as viral clearance have been observed. 16, 17 Based on efficacy in clearing HPV infection in the cervix, a phase III study is in progress. Collectively these studies have established a strong safety database in over 1600 individuals, and demonstrated that this advanced platform can reproducibly drive humoral and cellular immunity in humans with relevance to the prevention of infections or clearance of infectious disease. Potentially the induction of T cell responses by this vaccine platform may afford improved B cell help to generate durable humoral immunity. 18 The current manuscript describes the development of a synthetic consensus (SynCon®) OspA DNA vaccine, pLD1 in combination with CELLECTRA® in vivo delivery technology. The data were benchmarked against an OspA and OspC chimeric recombinant protein vaccine, VANGUARDcrLyme, an approved veterinary vaccine, henceforth referred to as crLyme. The data show that pDL1 DNA vaccine elicits robust humoral and cellular immune responses in mice against the OspA antigen that confers protection against Borreliella burgdorferi spirochete tissue colonization following a live bacterial challenge. Also, intradermal immunization of guinea pigs with pLD1 leads to a durable and robust antibody response which persists for at least a year. Moreover, antibodies from the immunized guinea pigs effectively compete for OspA binding with a borreliacidal human mAb 319-44. 19, 20 

Human embryonic kidney 293 T cells were cultured in Dulbecco modified eagle's medium (DMEM) and supplemented with 10% heat-inactivated fetal bovine serum (FBS), along with 100 U/ml of penicillin and streptomycin each. Cells were incubated at 37°C and 5% CO 2 .

For Immunofluorescence analysis, 1.5 × 10 6 cells were seeded per well into a 6-well cell culture plate, and transfected with either GFP (transfection control), pVAX or pLD1 using the Lipofectamine 3000 transfection kit (ThermoFisher, Waltham, MA) following the manufacturer's instructions. Fortyeight hours later transfected cells were washed thrice with PBS, and incubated overnight at 4°C with a mouse anti-OspA monoclonal antibody (Clone 0551 LSBio, LS-C103210). Cells were washed with PBS and incubated with Alexa fluor® 555 donkey anti-mouse secondary antibody (Life Technologies, A-31570). The nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min. Images were captured using ImageXpress Pico automated cell imaging system (Molecular Devices).

A proprietary synthetic consensus sequence was generated to target OspA serotype 1 (ST1). (pLD1). The sequences were codon and RNA optimized. 21, 22 The resulting nucleotide sequences were synthesized, digested with BamHI and XhoI, and cloned into the expression vector pVAX under the control of the human cytomegalovirus immediate-early promoter and a bovine growth hormone polyadenylation signal (Genscript, Piscataway, New Jersey).

Female Hartley Guinea pigs (8-10 weeks) weighing 500-600 g and C3 H/HeN female mice (6-8 weeks) were grouped and housed with ad libitum access to food and water. Animals were housed at Acculab (San Diego, CA 92121) or the University of Florida (Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608). All housing, handling and treatment protocols were approved and handled according to the standards of the Institutional Animal Care and Use Committee.

30 µl DNA vaccine pLD1 or pVAX formulations were administered by 25 G needle intramuscular to the tibialis anterior muscle and this step was followed by CELLECTRA® in vivo electroporation using the 3P array. The CELLECTRA® EP delivery consists of two sets of pulses with 0.2 Amp constant current. Second pulse set is delayed 3 s. Within each set, there are two 52 ms pulses with a 198 ms delay between the pulses. The vaccine dose was 25 µg of plasmid DNA per injection. Each group was dosed on week 0, 2 and 4 for a total of three immunizations.

Hartley guinea pigs were immunized either by intradermal injection (Mantoux technique) of pLD1 on the abdominal left flank followed by CELLECTRA®-EP with the 3p array (as described above) or subcutaneous (SC) injection of a recombinant protein vaccine. Treatments consisted of a total of three immunizations each at a dose of 0.1 mg in 0.1 mL volume of pLD1 administrated at weeks 0, 3 and 6. The crLyme vaccine group of animals was immunized SC with 250 µl of vaccine, commercially available for Lyme disease prevention in dogs (VANGUARDcrLYME by Zoetis).

Briefly, spleens from mice were collected individually in 5 mL of RPMI1640 media supplemented with 10% FBS (R10), processed into single-cell suspensions with a gentle MACS Dissociator (Miltenyi Biotec, Auburn, CA), then centrifuged at 1500 rpm for 5 min. Cell pellets were resuspended in 5 mL of ACK lysis buffer (Life Technologies, Carlsbad, CA) for 5 min at room temperature, and 10 ml PBS was then added to dilute the lysis buffer. The samples were again centrifuged at 1500 rpm for 5 min, cell pellets resuspended in R10, and then passed through a 45 um nylon filter before use in ELISpot assay.

To assess cellular IFNγ responses, mouse ELISpot assays were performed using commercial Mabtech pre-coated IFNy ELISpot kits (Mabtech 3321-4APW-10, Sweden). The 96-well ELISpot plates were washed 3 times with sterile 1X PBS and blocked with R10 medium for 1 h. The R10 was removed and 200,000 mouse splenocytes in R10 media were added to each well and incubated at 37°C in 5% CO 2 the presence of peptide pool 1, 2 or 3, each consisting of 15-mers overlapping by 9 amino acids, together these pools spanned the length of OspA protein, DMSO (negative control), ConA (positive control for mouse). After 18-20 h, plates were washed and developed according to the manufacturer's protocols, and IFN-γ positive spots were counted by an automated ELISpot reader (CTL, Shaker Heights, OH). OspA peptide-reactive responses were calculated by subtracting the number of spots in DMSO from peptide wells. Results (Cellular responses or Interferon-gamma ELISpot responses) are shown as individual animal spot-forming units (SFU)/10 6 PBMCs.

ELISAs were performed to determine sera IgG antigenbinding titers. Nunc ELISA plates were coated with 1 µg/ml recombinant OspA protein (Immune Technology, New York, NY) in DPBS overnight at 4°C. Plates were washed three times then blocked with 3% BSA DPBS with 0.05% Tween 20 for 2 h at 37°C. Plates were then washed and incubated with serial dilutions of mouse or guinea pig sera and incubated for 2 h at 37°C. Plates were again washed and then incubated with HRP conjugated-species specific secondary antibodies (1 in 10,000 dilution) and incubated for 1 h at 37°C. After the final wash, plates were developed using SureBlue TMB Component peroxidase substrate and the reaction stopped with TMB stop reagent (KPL). Plates are then read at 450 nm within 30 min using a SpectraMax Plus384 Microplate Reader (Molecular Devices).

ciELISAs were performed to determine the presence of antibodies in the serum after OspA immunization in guinea pigs competed with 319-44 human monoclonal antibody known to bind the OspA epitope targeted by the LA-2 antibody. 19, 20 ELISA plates were coated with 0.5 µg/ml recombinant OspA protein (Immune Technology, New York, NY) in DPBS overnight at 4°C. Plates were washed three times then blocked with 3% BSA DPBS with 0.05% Tween 20 for 2 h at 37°C. Plates were then washed and incubated with serial dilutions (1 in 3) of guinea pig sera and incubated for 2 h at 37°C. Plates were washed and then incubated with 0.75 µg/ml of 319-14 human antibody for 1 h at room temperature. HRP conjugated-anti-human kappa light chain (Bethyl Laboratories) was added for 1 h at room temperature. After the final wash, plates were developed using SureBlue TMB component peroxidase substrate and the reaction stopped with TMB stop reagent (KPL). Plates were then read at 450 nm within 30 min using a SpectraMax Plus384 Microplate Reader (Molecular Devices).

Low-passage B. burgdorferi strain 297 was maintained at 23°C in Barbour-Stoenner-Kelley medium containing 6% normal rabbit serum (BSK Media, Complete; Sigma Aldrich, St. Louis, MO; Cat # B8291) and then temperature-shifted to 37°C. Increased expression of OspC as a virulence factor and presence of OspA as a target for the vaccine was confirmed by Coomassie staining of whole borrelial lysates separated by SDS-PAGE.

Mice were challenged with 1 × 10 5 bacteria injected subcutaneously at the sternum as described previously. 23 Bacteria were confirmed OspA+ by PCR. All the mice were bled 2 days before the challenge from a facial vein using. Four weeks postinoculation, mice were anesthetized for intracardiac bleeding, and euthanized for tissue collection that included joints, heart, ear and bladder, respectively.

DNA was isolated from the excised tissues using a Quick-DNA miniprep kit (Zymo Research; Cat # D3024). Bacterial burden in tissues was determined using genomic DNA as previously described. 24 Briefly, 80 ng of extracted genomic DNA was along with B. burgdorferi-specific flaB primers (200 nM) and probe (320 nM) or primers (400 nM) and probe (320 nM) directed against the mouse actin gene, as described elsewhere. 25 Standards were developed to quantify the number of B. burgdorferi flaB and mouse actin copies across a range of 10°-10 6 copies. Amplification data were acquired and analyzed using the MIC Real-time PCR system, and quantification of target DNA was accomplished as previously described. 26 

Freshly grown temperature-shifted B. burgdorferi strain 297 was counted, and 1 × 10 5 bacteria incubated with 200 µl two-fold serially diluted serum from the study animals for 18 h in 200ul tubes. At this stage, we included naïve serum from C3 H/HeN mice as a negative control as well as a heat-inactivated counterpart. 50 µl of the aliquots (in triplicate) were inoculated in 1.5 ml microfuge tubes with 1.2 ml of BSK complete media and sealed with parafilm to create microaerobiosis. These tubes were kept at 37°C for 2 weeks. The cultures were monitored daily for change in pH, and if pH drops down, the bacteria were visualized. The presence of bacteria visualized in all the tubes under a dark-field microscope. The highest dilution of the serum that prevented the growth of B. burgdorferi was recorded as a bactericidal titer of the serum.

Data were presented as mean ± SEM for all data points. The statistical difference between individual groups was assessed using Mann Whitney test with statistical significance set at p < .05.

Data that support the findings of this study are available from the corresponding author upon reasonable request.

A synthetic consensus sequence was generated to target OspA serotype 1 (ST1), the vaccine is intended for North American use. Thirteen full-length protein sequences from 13 orthologous open reading frames of Borreliella burgdorferi were collected. A consensus was generated by alignment of the OspA protein sequences using ClustalW, sharing 98.5%-100% identity with each individual OspA sequence. Three residues of the consensus were replaced with the corresponding residues of an OspA protein sequence from B. afzelii in order to eliminate an epitope homology with human leucocyte function antigen 1 (LFA-1) (Y165 F, V166 T, T170 K). 27 The pLD1 construct was generated using this modified OspA consensus sequence (Figure 1(a) ). SynCon® OspA ST1 (Design A, pLD1), a potentially exposed N-terminal Cys17 was deleted from the full length modified OspA consensus to avoid possible posttranslational modifications and covalent conjugations and the endogenous N-terminal leader sequence was replaced with an immunoglobulin E (IgE) leader sequence to enhance the translation of the construct which has been demonstrated to increase the immunogenicity of the translated product. 28 The final sequence was codon and RNA optimized and cloned into the pVAX plasmid (see Figure 1 (b)). Expression of the OspA was confirmed in vitro using immuno-fluorescence of 293 T cells transfected with pLD1 (Figure 1(c) ).

We evaluated the immunogenicity of the OspA antigen expressed after PLD1 vaccination in C3 H/HeN mice. Immunogenicity analyses revealed a robust immune response mediated by the pLD1 vaccine after a single dose, and the response was boosted after subsequent doses (Figure 2(a) ). On a different set of animals, we examined the cellular immune response to pLD1 vaccination against three pools of overlapping peptides, which together spanned the length of the OspA antigen. Here, groups of 8 mice received three doses of either pLD1, pVAX or crLyme, recombinant protein LD vaccine. All animals were female, we did not address the sex of the animals as a biological variable. Two weeks after the final immunization cellular IFN-γ response was evaluated to OspA peptide recall. As illustrated on Figure 2 (c), pLD1 elicited a significantly stronger cellular response than the control (pVAX1) and crLyme groups, with the epitopes in pool 3 being where the cellular response predominantly targeted.

We evaluated the efficacy of pLD1 in a live bacterial challenge. Schematic of the challenge study is illustrated in Figure 3 (a). C3 H/HeN mice are susceptible to Borreliella burgdorferi infection, and the model recapitulates some pathophysiology features of human Lyme disease, such as sub-acute arthritis, which is a consequence of the bacterial spread and colonization in the animal's organs and tissues. 23, 29, 30 Three weeks after the final immunization mice were inoculated with 100,000 of Borreliella burgdorferi strain 297, and 4 weeks later bacterial burden was evaluated by PCR in a double-blinded manner. As shown in Figure 3(b) , only the pVAX control group presented with detectable bacterial burden in joints, heart and bladder as well as ear tissues. In groups of mice that received one, two or three dose of the pLD1 vaccine or three doses of crLyme vaccine, a bacterial presence in the tissues tested was not detected. We proceeded to determine the presence of functional anti-B. burgdorferi antibodies in the animals' serum employing a bacterial killing assay. This assay measures the dilution of serum at which the bacterial growth is enabled. As illustrated in Figure 3 (c) bacterial growth was enabled at a serum dilution of approximately 256 for pLD1 and crLyme immunized mice indicating robust bactericidal activity. These results from both bacterial tissue burden and bacterial growth inhibition assay demonstrate the efficacy of pLD1 and crLyme vaccines against Borreliella burgdorferi infection in the C3 H/HeN mouse needle challenge model. 

We assessed the immunogenicity of pLD1 in guinea pigs, an established model for intradermal vaccine delivery. 31, 32 We measured the OspA antigen-binding titers of IgG in the serum up to 52 weeks after initial vaccination. Animals in Group one received 0.1 mg of pLD1 vaccine via ID-CELLECTRA®EP delivery on weeks 0, 3 and 6. Group two received 250 µl (1/4 dose recommended for dogs) of crLyme vaccine administered subcutaneously at weeks 0, 3 and 6. Humoral immune responses against OspA antigen were measured up to Week 52. A robust humoral response against OspA was detected across both groups. We proceeded to evaluate the quality of the anti-OspA humoral immune response by assessing the generation of antibodies targeting the LA-2 epitope. To accomplish this we used the LA-2 equivalent mAb 319-44 in a competition assay with the serum from immunized guinea pigs. 20 Similar assays have previously been shown to be a pre-clinical correlate of protection. 33 The humoral response elicited by pLD1 and crLyme vaccines demonstrated the ability to compete and inhibit the binding of human mAb 319-44 ( Figure 5 ). These results underscore the potential of the pLD1 vaccine to provide durable antibody titers to OspA epitopes associated with correlates of protection.

Previous studies employing DNA vaccines encoding OspA have shown promise in small animal studies, 34-36 however they have yet to advance to clinical testing. Here we have employed advanced delivery technology of in vivo EP to enhance the immunogenicity of the Lyme disease vaccine pLD1 after administration to the C3 H/HeN mouse muscle or Hartley guinea pig skin. Such technology increases in vivo gene expression up to 1000 fold, and the subsequent cellular and humoral immune responses elicited are substantively enhanced. 37 Here, the described study is the first to employ this vaccine delivery technology as a development concept for Lyme disease prevention. We show that a synthetically engineered vaccine pLD1 elicits robust and durable anti-OspA IgG levels, and confers protection against Borreliella burgdorferi infection in a C3 H/HeN mouse needle challenge model.

The pLD1 vaccine induced a robust anti-OspA immune response in C3 H/HeN mice ( Figure 2 ). The ability to induce strong T cell immunity is likely due to the intracellular antigen expression pathway associated with DNA vaccines, assuring antigen processing in the MHC class I and II pathways, driving broad T cell responses. Furthermore, the activation of Toll-like receptors by the un-methylated CpG motifs contained within synthetic nucleotides, and the activation of the intracellular STING pathway may provide innate signals that lead to enhanced cellular responses. 38, 39 Activated T cells can provide help to B cells leading to increased antibody levels and memory cell development, enhancing the humoral immunity. In reference to Lyme disease immune responses, an OspA T cell epitope that promotes OspA antibody response has been described, 18 supporting the importance of anti-OspA T cell responses in promoting antibody production. 40 Data here show one dose of pLD1 generates high antibody titers (see Figure 2(a,b) ), enough to provide protection against bacterial challenge (Figure 3(b) ).

We recently reported intradermal delivery of an Ebola GP Synthetic DNA vaccine resulted in 100% seroconversion and induction of T cell responses in 70% of subjects within weeks, by simple dose sparing ID formulations. 13 In the current study, we evaluated the immunogenicity of the pLD1 DNA vaccine in guinea pigs employing intradermal delivery. Guinea pigs represent an excellent ID immunization model, because unlike mice they possess a well-defined epidermis. An important aspect of targeting vaccine delivery to the skin is that it is rich in professional antigen-presenting cells that are capable of mediating host immune responses. ID delivery of pLD1 elicited a robust humoral immune response (Figure 4 ). One limitation in this current study was that it did not consider the role of OspC-targeted immune responses of the comparator vaccine. crLyme is a chimeric vaccine containing OspA ST1 and a chimeric protein of multiple OspC antigens. Unlike OspA, OspC is not downregulated and is present on spirochetes entering the host, and vaccines containing OspC antigens may have the ability to elicit responses to spirochetes which have entered the host. 41 crLyme has a USDA-approved 15-month duration of immunity on its label. In contrast to OspA, over 30 different OspC genotypes have been defined, with many circulating in the USA. 42 The inclusion of OspC antigens may be considered in formulations.

The ability of serum from OspA-immunized guinea pigs to competitively inhibit the binding of OspA specific-319-44 mAb was also evaluated ( Figure 5 ). This type of competition assay can serve as a correlate of protective anti-OspA response as previously shown for the murine LA-2 antibody. 27, 33 The LA-2 antibody has been identified as protective against Borreliella burgdorferi infection in passive transfer and active immunization studies targeting OspA protein. 33, 43, 44 319-44 mAb has demonstrated the equivalent capability of mediating protection against Borreliella burgdorferi infection in antibody transfer experiments as the LA-2 mAb. 19, 20 In the context of the present study, antibodies from sera of pLD1 immunized guinea pigs provided inhibition of 319-44 mAb OspA binding. These data indicate the immune response elicited by pLD1 can generate antibodies which can bind to the same OspA epitopes as mAbs which have been shown to prevent transmission of Lyme disease spirochetes.

While there is an obvious unmet need for a Lyme disease vaccine, it is important to consider the hurdles faced by previous vaccine programs which were ultimately not successful. The efficacy of this recombinant protein OspA vaccine was only 49% after 2 immunizations and required yearly boosts to maintain protective antibody titers. Furthermore, there was speculation about side effects due to the vaccine containing an epitope with homology to a human epitope in the LFA-1 protein. With pLD1 we have aimed to enhance the durability of the antibody titers which would potentially result a reduction in the frequency of boosts required to maintain protective serum titers. Additionally, we replaced the epitope with homology to human LFA-1 (Figure 1(a) ). Preliminary surveys are ongoing to assess the current market for a Lyme disease vaccine. With the increasing incidence and impact of this disease, we are optimistic about the demand and the level of utilization of such a medical countermeasure. Future studies are planned to address the limitations of the scope of work presented here. These will focus on efficacy testing in Borreliella burgdorferi infected tick challenges, which will reflect a more natural exposure route than the bacterial needle challenge model reported here. Studies will be designed to test vaccine efficacy after tick challenge in the dog, a large animal model which exhibits similarities in LD pathophysiology with humans.

We thank Maria Yang and team for the manufacturer of plasmid DNA vaccines. 

GHG, HB, NC, JC, JY, CH, BY, HP, LH, KEB and TRFS are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. DBW has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board services. Remuneration received by DBW includes direct payments or stock options, and in the interest of disclosure he notes potential conflicts associated with this work with Inovio and possibly others. In addition, he has a patent DNA vaccine delivery pending to Inovio. All other authors report there are no competing interests.

This work was supported financially by INOVIO PHARMACEUTICALS, Inc.

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