Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial.

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor ß-chain (TCRß) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV's mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .

A synDNA vaccine delivering neoAg collections controls heterogenous, multifocal murine lung and ovarian tumors via robust T cell generation.

Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings.

Intramuscular and Intradermal Electroporation of HIV-1 PENNVAX-GP® DNA Vaccine and IL-12 Is Safe, Tolerable, Acceptable in Healthy Adults.

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 < 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.

Robust antibody and cellular responses induced by DNA-only vaccination for HIV.

BACKGROUNDHVTN 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.METHODSParticipants 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.RESULTSFollowing 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.CONCLUSIONPENNVAX-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 REGISTRATIONClinicalTrials.gov NCT02431767.FUNDINGThis 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.

Intradermal SynCon® Ebola GP DNA Vaccine Is Temperature Stable and Safely Demonstrates Cellular and Humoral Immunogenicity Advantages in Healthy Volunteers.

BACKGROUND: Nonlive vaccine approaches that are simple to deliver and stable at room temperature or 2-8°C could be advantageous in controlling future Ebola virus (EBOV) outbreaks. Using an immunopotent DNA vaccine that generates protection from lethal EBOV challenge in small animals and nonhuman primates, we performed a clinical study to evaluate both intramuscular (IM) and novel intradermal (ID) DNA delivery. METHODS: Two DNA vaccine candidates (INO-4201 and INO-4202) targeting the EBOV glycoprotein (GP) were evaluated for safety, tolerability, and immunogenicity in a phase 1 clinical trial. The candidates were evaluated alone, together, or in combination with plasmid-encoded human cytokine interleukin-12 followed by in vivo electroporation using either the CELLECTRA® IM or ID delivery devices. RESULTS: The safety profile of all 5 regimens was shown to be benign, with the ID route being better tolerated. Antibodies to EBOV GP were generated by all 5 regimens with the fastest and steepest rise observed in the ID group. Cellular immune responses were generated with every regimen. CONCLUSIONS: ID delivery of INO-4201 was well tolerated and resulted in 100% seroreactivity after 2 doses and elicited interferon-γ T-cell responses in over 70% of subjects, providing a new approach for EBOV prevention in diverse populations. Clinical Trials Registration. NCT02464670.

A Synthetic DNA, Multi-Neoantigen Vaccine Drives Predominately MHC Class I CD8+ T-cell Responses, Impacting Tumor Challenge.

T-cell recognition of cancer neoantigens is important for effective immune-checkpoint blockade therapy, and an increasing interest exists in developing personalized tumor neoantigen vaccines. Previous studies utilizing RNA and long-peptide neoantigen vaccines in preclinical and early-phase clinical studies have shown immune responses predominantly driven by MHC class II CD4+ T cells. Here, we report on a preclinical study utilizing a DNA vaccine platform to target tumor neoantigens. We showed that optimized strings of tumor neoantigens, when delivered by potent electroporation-mediated DNA delivery, were immunogenic and generated predominantly MHC class I-restricted, CD8+ T-cell responses. High MHC class I affinity was associated specifically with immunogenic CD8+ T-cell epitopes. These DNA neoantigen vaccines induced a therapeutic antitumor response in vivo, and neoantigen-specific T cells expanded from immunized mice directly killed tumor cells ex vivo These data illustrate a unique advantage of this DNA platform to drive CD8+ T-cell immunity for neoantigen immunotherapy.

Zika-Induced Male Infertility in Mice Is Potentially Reversible and Preventable by Deoxyribonucleic Acid Immunization.

Background: Zika virus (ZIKV) infection has been associated with prolonged viral excretion in human semen and causes testicular atrophy and infertility in 10-week-old immunodeficient mice. Methods: Male IFNAR-/- mice, knockout for type I interferon receptor, were immunized with GLS-5700, a deoxyribonucleic acid-based vaccine, before a subcutaneous ZIKV challenge with 6 × 105 plaque-forming units at 13 weeks of age. On day 28 postinfection, testes and epididymides were collected in some mice for histological and functional analyses, whereas others were mated with naive female wild-type C57BL/6J. Results: Although all mice challenged with ZIKV developed viremia, most of them were asymptomatic, showed no weight loss, and survived infection. On day 28 postinfection, none of the unvaccinated, infected mice (9 of 9) exhibited abnormal spermatozoa counts or motility. However, 33% (3 of 9) and 36% (4 of 11) of mated males from this group were infertile, from 2 independent studies. Contrarily, males from the noninfected and the vaccinated, infected groups were all fertile. On days 75 and 207 postinfection, partial recovery of fertility was observed in 66% (2 of 3) of the previously infertile males. Conclusions: This study reports the effects of ZIKV infection on male fertility in a sublethal, immunodeficient mouse model and the efficacy of GLS-5700 vaccination in preventing male infertility.

Protective Efficacy and Long-Term Immunogenicity in Cynomolgus Macaques by Ebola Virus Glycoprotein Synthetic DNA Vaccines.

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.

In Vivo Delivery of Synthetic Human DNA-Encoded Monoclonal Antibodies Protect against Ebolavirus Infection in a Mouse Model.

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.

Gag and env conserved element CE DNA vaccines elicit broad cytotoxic T cell responses targeting subdominant epitopes of HIV and SIV Able to recognize virus-infected cells in macaques.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting inessential variable regions are hurdles in the development of an effective AIDS vaccine. We developed a DNA vaccine comprising conserved elements (CE) of SIV p27Gag and HIV-1 Env and found that priming vaccination with CE DNA is critical to efficiently overcome the dominance imposed by Gag and Env variable regions. Here, we show that DNA vaccinated macaques receiving the CE prime/CE+full-length DNA co-delivery booster vaccine regimens developed broad, potent and durable cytotoxic T cell responses targeting conserved protein segments of SIV Gag and HIV Env. Gag CE-specific T cells showed robust anamnestic responses upon infection with SIVmac239 which led to the identification of CE-specific cytotoxic lymphocytes able to recognize epitopes covering distinct CE on the surface of SIV infected cells in vivo. Though not controlling infection overall, we found an inverse correlation between Gag CE-specific CD8+ T cell responses and peak viremia. The T cell responses induced by the HIV Env CE immunogen were recalled in some animals upon SIV infection, leading to the identification of two cross-reactive epitopes between HIV and SIV Env based in sequence homology. These data demonstrate that a vaccine combining Gag and Env CE DNA subverted the normal immunodominance patterns, eliciting immune responses that included subdominant, highly conserved epitopes. These vaccine regimens augment cytotoxic T cell responses to highly conserved epitopes in the viral proteome and maximize response breadth. The vaccine-induced CE-specific T cells were expanded upon SIV infection, indicating that the predicted CE epitopes incorporated in the DNA vaccine are processed and exposed by infected cells in their natural context within the viral proteome.

DNA Vaccine-Induced Long-Lasting Cytotoxic T Cells Targeting Conserved Elements of Human Immunodeficiency Virus Gag Are Boosted Upon DNA or Recombinant Modified Vaccinia Ankara Vaccination.

DNA-based vaccines able to induce efficient cytotoxic T-cell responses targeting conserved elements (CE) of human immunodeficiency virus type 1 (HIV-1) Gag have been developed. These CE were selected by stringent conservation, the ability to induce T-cell responses with broad human leukocyte antigen coverage, and the association between recognition of CE epitopes and viral control in HIV-infected individuals. Based on homology to HIV, a simian immunodeficiency virus p27gag CE DNA vaccine has also been developed. This study reports on the durability of the CE-specific T-cell responses induced by HIV and simian immunodeficiency virus CE DNA-based prime/boost vaccine regimens in rhesus macaques, and shows that the initially primed CE-specific T-cell responses were efficiently boosted by a single CE DNA vaccination after the long rest period (up to 2 years). In another cohort of animals, the study shows that a single inoculation with non-replicating recombinant Modified Vaccinia Ankara (rMVA62B) also potently boosted CE-specific responses after around 1.5 years of rest. Both CE DNA and rMVA62B booster vaccinations increased the magnitude and cytotoxicity of the CE-specific responses while maintaining the breadth of CE recognition. Env produced by rMVA62B did not negatively interfere with the recall of the Gag CE responses. rMVA62B could be beneficial to further boosting the immune response to Gag in humans. Vaccine regimens that employ CE DNA as a priming immunogen hold promise for application in HIV prevention and therapy.

Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques.

We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

Clinical and Immunologic Biomarkers for Histologic Regression of High-Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 after Immunotherapy.

Purpose: As previously reported, treatment of high-grade cervical dysplasia with VGX-3100 resulted in complete histopathologic regression (CR) concomitant with elimination of HPV16/18 infection in 40.0% of VGX-3100-treated patients compared with only 14.3% in placebo recipients in a randomized phase IIb study. Here, we identify clinical and immunologic characteristics that either predicted or correlated with therapeutic benefit from VGX-3100 to identify parameters that might guide clinical decision-making for this disease.Experimental Design: We analyzed samples taken from cervical swabs, whole blood, and tissue biopsies/resections to determine correlates and predictors of treatment success.Results: At study entry, the presence of preexisting immunosuppressive factors such as FoxP3 and PD-L1 in cervical lesions showed no association with treatment outcome. The combination of HPV typing and cervical cytology following dosing was predictive for both histologic regression and elimination of detectable virus at the efficacy assessment 22 weeks later (negative predictive value 94%). Patients treated with VGX-3100 who had lesion regression had a statistically significant >2-fold increase in CD137+perforin+CD8+ T cells specific for the HPV genotype causing disease. Increases in cervical mucosal CD137+ and CD103+ infiltrates were observed only in treated patients. Perforin+ cell infiltrates were significantly increased >2-fold in cervical tissue only in treated patients who had histologic CR.Conclusions: Quantitative measures associated with an effector immune response to VGX-3100 antigens were associated with lesion regression. Consequently, these analyses indicate that certain immunologic responses associate with successful resolution of HPV-induced premalignancy, with particular emphasis on the upregulation of perforin in the immunotherapy-induced immune response. Clin Cancer Res; 24(2); 276-94. ©2017 AACR.

Broad cross-protective anti-hemagglutination responses elicited by influenza microconsensus DNA vaccine.

Despite the routine development and distribution of seasonal influenza vaccines, influenza remains an important pathogen contributing to significant human morbidity as well as mortality each year. The seasonal variability of influenza creates a significant issue for vaccine development of seasonal strains that can afford protection from infection or disease based on serotype matching. It is appreciated that the globular head of the HA antigen contained in the vaccines generates antibodies that result in HAI activity that are a major correlates of the protection against a particular strain. Due to seasonal genetic changes in the HA protein, however, new vaccine strains are needed to be developed continually to match the new HA antigen of that seasons virus. A distinct advantage in seasonal vaccine development would be if a small group of antigens could be developed that could span many seasons without needed to be replaced due to this genetic drift. Here we report on a synthetic microconsensus approach that relies on a small collection of 4 synthetic H1HA DNA antigens which together induce broad protective HAI immunity spanning decades of H1 influenza viruses in mice, guinea pigs and non-human primates. The protective HAI titers induced by microconsensus immunogens are fully functional in vivo as immunized ferrets were completely protected from A/Mexico/InDRE4487/2009 virus infection and morbidity associated with lethal challenge. These results are encouraging that a limited easy-to-formulate collection of invariant antigens can be developed which can span seasonal vaccine changes allowing for continued immune protection.

DMAb inoculation of synthetic cross reactive antibodies protects against lethal influenza A and B infections.

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.

Safety and Immunogenicity of PENNVAX-G DNA Prime Administered by Biojector 2000 or CELLECTRA Electroporation Device With Modified Vaccinia Ankara-CMDR Boost.

Background: We report the first-in-human safety and immunogenicity evaluation of PENNVAX-G DNA/modified vaccinia Ankara-Chiang Mai double recombinant (MVA-CMDR) prime-boost human immuonodeficiency virus (HIV) vaccine, with intramuscular DNA delivery by either Biojector 2000 needle-free injection system (Biojector) or CELLECTRA electroporation device. Methods: Healthy, HIV-uninfected adults were randomized to receive 4 mg of PENNVAX-G DNA delivered intramuscularly by Biojector or electroporation at baseline and week 4 followed by intramuscular injection of 108 plaque forming units of MVA-CMDR at weeks 12 and 24. The open-label part A was conducted in the United States, followed by a double-blind, placebo-controlled part B in East Africa. Solicited and unsolicited adverse events were recorded, and immune responses were measured. Results: Eighty-eight of 100 enrolled participants completed all study injections, which were generally safe and well tolerated, with more immediate, but transient, pain in the electroporation group. Cellular responses were observed in 57% of vaccine recipients tested and were CD4 predominant. High rates of binding antibody responses to CRF01_AE antigens, including gp70 V1V2 scaffold, were observed. Neutralizing antibodies were detected in a peripheral blood mononuclear cell assay, and moderate antibody-dependent, cell-mediated cytotoxicity activity was demonstrated. Discussion: The PVG/MVA-CMDR HIV-1 vaccine regimen is safe and immunogenic. Substantial differences in safety or immunogenicity between modes of DNA delivery were not observed. Clinical Trials Registration: NCT01260727.

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever.

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. 1,2 There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. 1,2 in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans.

An engineered bispecific DNA-encoded IgG antibody protects against Pseudomonas aeruginosa in a pneumonia challenge model.

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.

Novel prostate cancer immunotherapy with a DNA-encoded anti-prostate-specific membrane antigen monoclonal antibody.

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.

HIV Env conserved element DNA vaccine alters immunodominance in macaques.

Sequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of 'conserved' and 'variable' regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0-5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines.