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Cytolytic T cells (CTL) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). Electroporation was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by electroporation, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8+ T-cell–dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs are important.

Supplementary figures 1-4 and Supplementary Table 1

Supplementary Figure 1 shows the anti-mouse CTLA-4 DMAb design. Supplementary Figure 2 shows the efficacy of the recombinant 9D9 antibody in the Sa1N tumor model. Supplementary Figure 3 shows that the mouse anti-mouse CTLA-4 DMAb induces immune memory and protection from tumor re-challenge. Supplementary Figure 4 shows the efficacy of the mouse anti-mouse CTLA-4 DMAb delivered at an earlier time point. Supplementary Figure 5 shows that the anti-mouse CTLA-4 DMAb induces T cell infiltration into tumors. Supplementary Figure 6 shows the efficiency of CD4 and CD8 depletion antibodies.

Supplementary Figure 1: Construct design and expression. Supplementary Figure 2: Comparison of Native and SynCon FAP vaccines in C57Bl/6 mice. Supplementary Figure 3: Comparison of Native and SynCon FAP vaccines in Balb/c mice. Supplementary Figure 4: Efficacy of FAP vaccine in therapeutic prostate and breast tumor models. Supplementary Figure 5: Alteration of the tumor microenvironment using hyaluronidase is less effective at synergizing with DNA vaccines. Supplementary Figure 6: Immune responses from mice receiving combination mTERT + FAP vaccination. Supplementary Figure 7: Impact of SynCon FAP vaccine on immune cell subsets by flow cytometry. Supplementary Figure 8: FAP expression and immune cell infiltration in patients.

Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti–CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.Significance: DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies. Cancer Res; 78(22); 6363–70. ©2018 AACR.

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen–specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration.Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190–201. ©2018 AACR.

PDF file - 50KB, Protective efficacy maintained by mtrIL-33- and proIL-33-adjuvanted ConE6E7. In a prophylactic study, groups of B6 (10 mice/group) were immunized (three times at three week intervals) with ConE6E7 with or without 7μg of mtrIL-33 or proIL-33 and challenged with 5x104 TC-1 cells one week after last immunization to assess the anti-tumor effects. All vaccination groups prevented tumor growth upon TC-1 implantation.

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PDF file - 58KB, IL-33 expands the frequency of the Ag-specific CD8+ T cells. P14 chimera mice (n=5) were vaccinated twice (on day 0 and day 41) containing GP33 with or without proIL-33. Displayed are representative fluorescent intensity plots of GP33/Ly5.1-specific CD8+ T cells responses in the blood of vaccinated mice at days 14, 21 and 31 after first vaccination and day 48 (day 7 after second immunization). Numbers indicate the percentage of Ag-specific CD8+ T Cells within the total CD8+ T cell populations. Experiments were performed independently at least two times and data are representative of the results.

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Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)–associated model for cancer immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-γ responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4+ and CD8+ T-cell responses, with a large proportion of CD8+ T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8+ T-cell responses and elicit effector–memory CD8+ T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy. Cancer Res; 74(6); 1789–800. ©2014 AACR.

Summary Background Middle East respiratory syndrome (MERS) coronavirus causes a highly fatal lower-respiratory tract infection. There are as yet no licensed MERS vaccines or therapeutics. This study (WRAIR-2274) assessed the safety, tolerability, and immunogenicity of the GLS-5300 MERS coronavirus DNA vaccine in healthy adults. Methods This study was a phase 1, open-label, single-arm, dose-escalation study of GLS-5300 done at the Walter Reed Army Institute for Research Clinical Trials Center (Silver Spring, MD, USA). We enrolled healthy adults aged 18–50 years; exclusion criteria included previous infection or treatment of MERS. Eligible participants were enrolled sequentially using a dose-escalation protocol to receive 0·67 mg, 2 mg, or 6 mg GLS-5300 administered by trained clinical site staff via a single intramuscular 1 mL injection at each vaccination at baseline, week 4, and week 12 followed immediately by co-localised intramuscular electroporation. Enrolment into the higher dose groups occurred after a safety monitoring committee reviewed the data following vaccination of the first five participants at the previous lower dose in each group. The primary outcome of the study was safety, assessed in all participants who received at least one study treatment and for whom post-dose study data were available, during the vaccination period with follow-up through to 48 weeks after dose 3. Safety was measured by the incidence of adverse events; administration site reactions and pain; and changes in safety laboratory parameters. The secondary outcome was immunogenicity. This trial is registered at ClinicalTrials.gov (number NCT02670187) and is completed. Findings Between Feb 17 and July 22, 2016, we enrolled 75 individuals and allocated 25 each to 0·67 mg, 2 mg, or 6 mg GLS-5300. No vaccine-associated serious adverse events were reported. The most common adverse events were injection-site reactions, reported in 70 participants (93%) of 75. Overall, 73 participants (97%) of 75 reported at least one solicited adverse event; the most common systemic symptoms were headache (five [20%] with 0·67 mg, 11 [44%] with 2 mg, and seven [28%] with 6 mg), and malaise or fatigue (five [20%] with 0·67 mg, seven [28%] with 2 mg, and two [8%] with 6 mg). The most common local solicited symptoms were administration site pain (23 [92%] with all three doses) and tenderness (21 [84%] with all three doses). Most solicited symptoms were reported as mild (19 [76%] with 0·67 mg, 20 [80%] with 2 mg, and 17 [68%] with 6 mg) and were self-limiting. Unsolicited symptoms were reported for 56 participants (75%) of 75 and were deemed treatment-related for 26 (35%). The most common unsolicited adverse events were infections, occurring in 27 participants (36%); six (8%) were deemed possibly related to study treatment. There were no laboratory abnormalities of grade 3 or higher that were related to study treatment; laboratory abnormalities were uncommon, except for 15 increases in creatine phosphokinase in 14 participants (three participants in the 0·67 mg group, three in the 2 mg group, and seven in the 6 mg group). Of these 15 increases, five (33%) were deemed possibly related to study treatment (one in the 2 mg group and four in the 6 mg group). Seroconversion measured by S1-ELISA occurred in 59 (86%) of 69 participants and 61 (94%) of 65 participants after two and three vaccinations, respectively. Neutralising antibodies were detected in 34 (50%) of 68 participants. T-cell responses were detected in 47 (71%) of 66 participants after two vaccinations and in 44 (76%) of 58 participants after three vaccinations. There were no differences in immune responses between dose groups after 6 weeks. At week 60, vaccine-induced humoral and cellular responses were detected in 51 (77%) of 66 participants and 42 (64%) of 66, respectively. Interpretation The GLS-5300 MERS coronavirus vaccine was well tolerated with no vaccine-associated serious adverse events. Immune responses were dose-independent, detected in more than 85% of participants after two vaccinations, and durable through 1 year of follow-up. The data support further development of the GLS-5300 vaccine, including additional studies to test the efficacy of GLS-5300 in a region endemic for MERS coronavirus. Funding US Department of the Army and GeneOne Life Science.

Semen is the vehicle for virion dissemination in the female reproductive tract (FRT) in male-to-female HIV transmission. Recent data suggests that higher frequency semen exposure is associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, we use a non-human primate (NHP) model to show that repeated vaginal exposure to semen significantly reduces subsequent infection by repeated low-dose vaginal SIVmac251 challenge. Repeated semen exposures result in lower CCR5 expression in circulating CD4+ T-cells, as well as higher expression of Mx1 (in correlation with IFNε expression) and FoxP3 in the cervicovaginal mucosa, and increased infiltration of CD4+ T-cells. Establishing in vivo evidence of competing effects of semen on transmission impacts our basic understanding of what factors may determine HIV infectivity in humans. Our results clearly indicate that repeated semen exposure can profoundly modulate the FRT microenvironment, paradoxically promoting host resistance against HIV acquisition., High frequency semen exposure has been associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, Abdulhaqq et al. show that repeated vaginal exposure to semen reduces vaginal infection by SIV in non-human primates, and is associated with lower CCR5 expression in CD4 T-cells and a local type-I interferon response.

Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti–CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.Significance: DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies. Cancer Res; 78(22); 6363–70. ©2018 AACR.

SUMMARY Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings., Graphical Abstract, In Brief Monoclonal antibodies are an important approach for emerging infectious disease prevention. Patel et al. demonstrate engineering and in vivo delivery of DNA-encoded monoclonal antibodies (DMAbs) targeting the Zaire ebolavirus (EBOV) glycoprotein. DMAbs protect against lethal mouse-adapted EBOV and are useful for rapid evaluation of fully human mAbs in live animal models.

Despite extensive efforts, an efficacious HIV vaccine remains elusive. HIV Envelope (Env) is the target of all known neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. However, mice are the most cost effective and widely used vaccine model and mouse immunizations with NLTs have generally failed to induce tier-2 nAbs. Here, we explored if DNA-encoded NLTs would fold properly in vivo and induce an informed humoral response. We observed DNA encoding a BG505 NLT induces autologous tier-2 nAbs in mice, identified murine monoclonal neutralizing antibodies, mapped the responses to a new murine neutralizing Env C3/V5 epitope and determined the structures of Env-nAbs complexes. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

Background: Several techniques are under investigation to improve the immunogenicity of HIV-1 DNA vaccine candidates. DNA vaccines are advantageous due to their ease of design, expression of multiple antigens, and safety. Methods: The HVTN 098 trial assessed the PENNVAX®-GP DNA vaccine (encoding HIV env, gag, pol) administered with or without plasmid IL-12 at 0-, 1-, 3-, and 6-month timepoints via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, adult participants. We report on safety, tolerability, and acceptability. Results: HVTN 098 enrolled 94 participants: 85 received PENNVAX®-GP and nine received placebo. Visual analog scale (VAS) pain scores immediately after each vaccination were lower in the ID/EP than in the IM/EP group (medians 4.1–4.6 vs. 6–6.5, p &lt, 0.01). IM/EP participants reported greater pain and/or tenderness at the injection site. Most ID/EP participants had skin lesions such as scabs/eschars, scars, and pigmentation changes, which resolved within 6 months in 51% of participants (24/55). Eighty-two percent of IM/EP and 92% of ID/EP participant survey responses showed acceptable levels of discomfort. Conclusions: ID/EP and IM/EP are distinct experiences, however, HIV-1 DNA vaccination by either route was safe, tolerable and acceptable by most study participants.

In article number https://doi.org/10.1002/advs.201902802, David B. Weiner, Daniel W. Kulp, and co‐workers demonstrate that synthetic DNA delivery and adaptive electroporation can launch de novo assembly of different nanoparticle vaccines in the hosts to elicit significantly improved responses while reducing required doses. Next‐generation designer vaccines can now be rapidly evaluated in vivo to facilitate clinical translation for the betterment of human and animal health.

BACKGROUND: HVTN 098, a randomized, double-blind, placebo-controlled trial, evaluated the safety, tolerability, and immunogenicity of PENNVAX-GP HIV DNA vaccine, administered with or without plasmid IL-12 (pIL-12), via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, HIV-uninfected adults. The study tested whether PENNVAX-GP delivered via ID/EP at one-fifth the dose could elicit equivalent immune responses to delivery via IM/EP and whether inclusion of pIL-12 provided additional benefit. METHODS: Participants received DNA encoding HIV-1 env/gag/pol in 3 groups: 1.6 mg ID (ID no IL-12 group, n = 20), 1.6 mg ID + 0.4 mg pIL-12 (ID + IL-12 group, n = 30), 8 mg IM + 1 mg pIL-12 (IM + IL-12 group, n = 30), or placebo (n = 9) via EP at 0, 1, 3, and 6 months. Results of cellular and humoral immunogenicity assessments are reported. RESULTS: Following vaccination, the frequency of responders (response rate) to any HIV protein based on CD4(+) T cells expressing IFN-γ or IL-2 was 96% for both the ID + IL-12 and IM + IL-12 groups; CD8(+) T cell response rates were 64% and 44%, respectively. For ID delivery, the inclusion of pIL-12 increased CD4(+) T cell response rate from 56% to 96%. The frequency of responders was similar (≥90%) for IgG binding antibody to gp140 consensus Env across all groups, but the magnitude was higher in the ID + IL-12 group compared with the IM + IL-12 group. CONCLUSION: PENNVAX-GP DNA induced robust cellular and humoral immune responses, demonstrating that immunogenicity of DNA vaccines can be enhanced by EP route and inclusion of pIL-12. ID/EP was dose sparing, inducing equivalent, or in some aspects superior, immune responses compared with IM/EP. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431767. FUNDING: This work was supported by National Institute of Allergy and Infectious Diseases (NIAID), U.S. Public Health Service grants, an HIV Vaccine Design and Development Team contract, Integrated Preclinical/Clinical AIDS Vaccine Development Program, and an NIH award.

Cytolytic T cells (CTLs) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation (EP)-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). EP was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by EP, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8(+) T cell–dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs is important.

Background There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

Prostate-specific membrane antigen (PSMA) is expressed at high levels on malignant prostate cells and is likely an important therapeutic target for the treatment of prostate carcinoma. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines as well as passive administration of PSMA-specific monoclonal antibodies (mAb). Conventional mAb immunotherapy has numerous logistical and practical limitations, including high production costs and a requirement for frequent dosing due to short mAb serum half-life. In this report, we describe a novel strategy of antibody-based immunotherapy against prostate carcinoma that utilizes synthetic DNA plasmids that encode a therapeutic human mAb that target PSMA. Electroporation-enhanced intramuscular injection of the DNA-encoded mAb (DMAb) plasmid into mice led to the production of functional and durable levels of the anti-PSMA antibody. The anti-PSMA produced in vivo controlled tumor growth and prolonged survival in a mouse model. This is likely mediated by antibody-dependent cellular cytotoxicity (ADCC) effect with the aid of NK cells. Further study of this novel approach for treatment of human prostate disease and other malignant conditions is warranted.

Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 μg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.

Vaccination remains one of the greatest medical breakthroughs in human history and has resulted in the near eradication of many formerly lethal diseases in many countries, including the complete eradication of smallpox. However, there remain a number of diseases for which there are no or only partially effective vaccines. There are numerous hurdles in vaccine development, of which knowing the appropriate immune response to target is one of them. Recently, tissue-resident T cells have been shown to mediate high levels of protection for several infections, although the best way to induce these cells is still unclear. Here we compare the ability to generate skin-resident T cells in sites distant from the immunization site following intramuscular and intradermal injection using optimized synthetic DNA vaccines. We found that mice immunized intradermally with a synthetic consensus DNA HIV envelope vaccine by electroporation (EP) are better able to maintain durable antigen-specific cellular responses in the skin than mice immunized by the intramuscular route. We extended these studies by delivering a synthetic DNA vaccine encoding Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK) by EP and again found that the intradermal route was superior to the intramuscular route for generating skin-resident PEPCK-specific T cells. We observed that when challenged with Leishmania major parasites, mice immunized intradermally exhibited significant protection, while mice immunized intramuscularly did not. The protection seen in intradermally vaccinated mice supports the viability of this platform not only to generate skin-resident T cells but also to promote durable protective immune responses at relevant tissue sites.

Identification of novel molecular adjuvants which can boost and enhance vaccine-mediated immunity and provide dose-sparing potential against complex infectious diseases and for immunotherapy in cancer is likely to play a critical role in the next generation of vaccines. Given the number of challenging targets for which no or only partial vaccine options exist, adjuvants that can address some of these concerns are in high demand. Here, we report that a designed truncated Interleukin-36 gamma (IL-36 gamma) encoded plasmid can act as a potent adjuvant for several DNA-encoded vaccine targets including human immunodeficiency virus (HIV), influenza, and Zika in immunization models. We further show that the truncated IL-36 gamma (opt-36&gamma, t) plasmid provides improved dose sparing as it boosts immunity to a suboptimal dose of a Zika DNA vaccine, resulting in potent protection against a lethal Zika challenge.

Vaccination remains one of the greatest medical breakthroughs in human history and has resulted in the near eradication of many formerly lethal diseases in many countries, including the complete eradication of smallpox. However, there remain a number of diseases for which there are no or only partially effective vaccines. There are numerous hurdles in vaccine development, of which knowing the appropriate immune response to target is one of them. Recently, tissue-resident T cells have been shown to mediate high levels of protection for several infections, although the best way to induce these cells is still unclear. Here we compare the ability to generate skin-resident T cells in sites distant from the immunization site following intramuscular and intradermal injection using optimized synthetic DNA vaccines. We found that mice immunized intradermally with a synthetic consensus DNA HIV envelope vaccine by electroporation (EP) are better able to maintain durable antigen-specific cellular responses in the skin than mice immunized by the intramuscular route. We extended these studies by delivering a synthetic DNA vaccine encoding Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK) by EP and again found that the intradermal route was superior to the intramuscular route for generating skin-resident PEPCK-specific T cells. We observed that when challenged with Leishmania major parasites, mice immunized intradermally exhibited significant protection, while mice immunized intramuscularly did not. The protection seen in intradermally vaccinated mice supports the viability of this platform not only to generate skin-resident T cells but also to promote durable protective immune responses at relevant tissue sites.

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.

Specific antibody therapy, including mAbs and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production, thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery, and in vivo production would be an important advance. Here we describe the development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2dMAb) and delivered it into animals through adaptive electroporation. HER2dMAb was efficiently expressed in vitro and in vivo, reaching levels of 50 μg/ml in mouse sera. Mechanistically, HER2dMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2dMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2dMAb single-chain variable fragment portion to engineer a DNA-encoded BiTE (DBiTE). This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate an approach to generate DBiTEs in vivo, which represent promising immunotherapies for HER2(+) tumors, including ovarian and potentially other cancers.

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.

There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG responses in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P

Background Despite vigorous and ongoing efforts, active immunizations have yet to induce broadly neutralizing antibodies (bNAbs) against HIV-1. An alternative approach is to achieve prophylaxis with long-term expression of potent biologic HIV-1 inhibitors with Adeno-associated Virus (AAV), which could however be limited by hosts' humoral and cellular responses. An approach that facilitates in vivo production of these complex molecules independent of viral-vectored delivery will be a major advantage. Methods We used synthetic DNA and electroporation (DNA/EP) to deliver an anti-HIV-1 immunoadhesin eCD4-Ig in vivo. In addition, we engineered a TPST2 enzyme variant (IgE-TPST2), characterized its intracellular trafficking patterns and determined its ability to post-translationally sulfate eCD4-Ig in vivo. Findings With a single round of DNA injection, a peak expression level of 80-100μg/mL was observed in mice 14 days post injection (d.p.i). The engineered IgE-TPST2 enzyme trafficked efficiently to the Trans-Golgi Network (TGN). Co-administrating low dose of plasmid IgE-TPST2 with plasmid eCD4-Ig enhanced the potency of eCD4-Ig by three-fold in the ex vivo neutralization assay against the global panel of HIV-1 pseudoviruses. Interpretation This work provides a proof-of-concept for delivering anti-HIV-1 immunoadhesins by advanced nucleic acid technology and modulating protein functions in vivo with targeted enzyme-mediated post-translational modifications. Funding This work is supported by NIH IPCAVD Grant U19 Al109646-04, Martin Delaney Collaboration for HIV Cure Research and W.W. Smith Charitable Trust., Graphical abstract Unlabelled Image, Highlights • Use of DNA/electroporation technology to promote in vivo expression of anti-HIV-1 immunoadhesin eCD4-Ig for 6 months • Engineered a Tyrosylprotein Sulfotransferase 2 variant (IgE-TPST2) that efficiently sulfate eCD4-Ig in vivo at low dose • IgE-TPST2 mediated sulfation enhanced potency of eCD4-Ig by 3-fold in the neutralizaiton of HIV global panel isolates

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

ISG15 is an ubiquitin-like protein induced by type I interferon associated with antiviral activity. ISG15 is also secreted and known to function as an immunomodulatory molecule. However, ISG15's role in influencing the adaptive CD8 T-cell responses has not been studied. Here, we demonstrate the efficacy of ISG15 as a vaccine adjuvant, inducing human papilloma virus (HPV) E7-specific IFNγ responses as well as the percentage of polyfunctional, cytolytic, and effector CD8 T-cell responses. Vaccination with ISG15 conferred remarkable control and/or regression of established HPV-associated tumor-bearing mice. T-cell depletion coupled with adoptive transfer experiments revealed that ISG15 protective efficacy was CD8 T-cell mediated. Importantly, we demonstrate that ISG15 vaccine-induced responses could be generated independent of ISGylation, suggesting that responses were mostly influenced by free ISG15. Our results provide more insight into the immunomodulatory properties of ISG15 and its potential to serve as an effective immune adjuvant in a therapeutic tumor or infectious disease setting.

Identifying new molecular adjuvants that elicit effective vaccine-induced CD8(+) T cell immunity may be critical for the elimination of many challenging diseases including Tuberculosis, HIV and cancer. Here, we report that co-administration of molecular adjuvant IL-33 during vaccination enhanced the magnitude and function of antigen (Ag)-specific CD8(+) T cells against a model Ag, LCMV NP target protein. These enhanced responses were characterized by higher frequencies of Ag-specific, polyfunctional CD8(+) T cells exhibiting cytotoxic characteristics. Importantly, these cells were capable of robust expansion upon Ag-specific restimulation in vivo and conferred remarkable protection against a high dose lethal LCMV challenge. In addition, we demonstrate the ability of IL-33 to amplifying the frequency of Ag-specific KLRG1(+) effector CD8(+) T cells. These data show that IL-33 is a promising immunoadjuvant at improving T cell immunity in a vaccine setting and suggest further development and understanding of this molecular adjuvant for strategies against many obstinate infectious diseases and cancer.

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.

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines. Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data. Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen–specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration. Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190–201. ©2018 AACR.

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.

Interleukin 33 (IL-33) has emerged as a cytokine that can exhibit pleiotropic properties. Here we examine IL-33 for its immunoadjuvant effects in an HPV-associated cancer immune therapy model in which cell-mediated immunity is critical for protection. It is known that two biologically active forms of IL-33 exist: full-length IL-33 and mature IL-33. The potential ability of both isoforms to act as vaccine adjuvants to influence the CD4 Th1 and CD8 T cell immune responses has not been well defined. We show that both isoforms of IL-33 are capable of enhancing potent antigen (Ag)-specific effector and memory T cell immunity in vivo in a DNA vaccine setting. We also show that while both forms of IL-33 drove robust IFN-γ responses, neither form drove high secretion of IL-4 or any elevation of IgE levels. Moreover, both isoforms augmented vaccine-induced Ag-specific polyfunctional CD4+ and CD8+ T cell responses, with a large proportion of CD8+ T cells undergoing cytolytic plurifunctional degranulation. Therapeutic studies indicated that established TC-1-bearing mice undergo rapid and complete regression after therapeutic vaccination with both IL-33 adjuvant isoforms used in conjunction with an HPV DNA vaccine. Furthermore, using the P14 transgenic mouse model, we show that IL-33 can significantly expand the magnitude of Ag-specific CD8+ T cell responses and elicit bonafide effector-memory CD8+ T cells. Overall, the data suggests the potential use of these two IL-33 isoforms as immunoadjuvant candidates in future vaccination against other pathogens and in the context of anti-tumor immune-based therapy.

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.

The search for an efficacious human immunodeficiency virus type 1 (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1-specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small-animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4 + and CD8 + T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA-only or protein-only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding and neutralizing and antibody-dependent cellular cytotoxicity (ADCC) activity after immunization with the DNA prime-protein boost regimen, with the multiple-Env formulation inducing a more robust and broader response than the single-Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce, which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses. IMPORTANCE Even with effective antiretroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine regimen due to the resource-limited setting in which the HIV pandemic is most rampant. DNA vaccination lends itself well to increasing the amount of diversity included in a vaccine due to the ease of manufacturing multiple plasmids and formulating them as a single immunization. By increasing the number of Envs within a formulation, we were able to show an increased breadth of responses as well as improved functionality induced in a nonhuman primate model. This increased breadth could be built upon, leading to better coverage against circulating strains with broader vaccine-induced protection.

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.