Trials Of DNA Vaccines Research Articles

Phase 1 clinical trial of Hantaan and Puumala virus DNA vaccines delivered by needle-free injection

Hantaan virus (HTNV) and Puumala virus (PUUV) are pathogenic zoonoses found in Asia and Europe, respectively. We conducted a randomized Phase 1 clinical trial of individual HTNV and PUUV DNA vaccines targeting the envelope glycoproteins (GnGc), as well as a combined HTNV/PUUV DNA vaccine delivered at varying doses using the PharmaJet Stratis® needle-free injection system (NCT02776761). Cohort 1 and 2 vaccines consisted of 2 mg/vaccination of HTNV or PUUV plasmid, respectively. Cohort 3 vaccine consisted of 2 mg/vaccination of 1:1 mixture of HTNV and PUUV vaccines. Vaccinations were administered on Days 0, 28, 56, and 168. The vaccines were safe and well tolerated. Neutralizing antibody responses were elicited in 7/7 (100%) subjects who received the HTNV DNA (Cohort 1) and 6/6 (100%) subjects who received the PUUV DNA (Cohort 2) vaccines alone. The combination vaccine resulted in 4/9 (44%) seroconversion against both viruses. After the first two vaccinations, the seroconversion rates for the HTNV and PUUV vaccines were >80%.

A brief review on DNA vaccines in the era of COVID-19.

This article provides a brief overview of DNA vaccines. First, the basic DNA vaccine design strategies are described, then specific issues related to the industrial production of DNA vaccines are discussed, including the production and purification of DNA products such as plasmid DNA, minicircle DNA, minimalistic, immunologically defined gene expression (MIDGE) and Doggybone™. The use of adjuvants to enhance the immunogenicity of DNA vaccines is then discussed. In addition, different delivery routes and several physical and chemical methods to increase the efficacy of DNA delivery into cells are explained. Recent preclinical and clinical trials of DNA vaccines for COVID-19 are then summarized. Lastly, the advantages and obstacles of DNA vaccines are discussed.

DNA Vaccine Delivery and Improved Immunogenicity.

The promise of DNA vaccines is as compelling today as it was more than a decade ago. Ease of manufacture, stability at ambient temperatures without the need for a cold chain and its ability to mimic natural infections and elicit appropriate immune responses makes this vaccine platform extremely attractive. Although, human clinical trials of DNA vaccines have yielded less than optimal results, the approval and licensing of a few veterinary vaccines is testimony to the proof-of-concept and the hope that licensed DNA vaccines for human use may not be too far away. Delivery and targeting of immunologically relevant cells appears to be the major hurdle in maximizing the immunogenicity of DNA vaccines. Several different approaches that are currently pursued in achieving this objective are discussed.

Abstract A021: Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) expressed during development in the nervous system. Aberrant expression of ALK drives several tumor malignancies. About 3-7% of non-small cell lung cancers (NSCLC) harbor ALK rearrangements. Treatment with the Tyrosine Kinase inhibitor (TKI) crizotinib results in a high objective rate response (ORR) of ∼60%, but median progression-free survival (mPFS) is only 8-10 months, owing to the rapid emergence of acquired drug resistance. Alectinib and ceritinib, a second generation of TKIs developed to overcome crizotinib resistance, induce a transient response followed by tumor relapses. Currently, there are at least 7 additional ALK TKIs in clinical development. Emergence of mechanisms of resistance to the new TKI is expected. Thus, innovative therapeutic approaches are needed to complement the partial success obtained by TKIs in ALK-positive NSCLC patients. Three properties make of ALK a rather unique shared antigen: 1) ALK is a driver oncogene that is expressed in all the tumoral cells; 2) ALK is not expressed by normal cells with only marginal expression in few cells in the nervous system of adults, an immuneprivileged tissue, anticipating limited if any autoimmune side effects by an ALK-specific immunotherapy; 3) ALK is immunogenic in humans because ALK-specific antibodies and T cell responses are detected in ALK-positive lymphoma and lung cancer patients. As an additional advantage, the availability of ALK TKI to reduce tumor might facilitate immunotherapeutic approaches. Therefore, we aim to develop a strategy of vaccination in human patients to treat ALK-positive tumors in combination with TKIs and/or immunocheckpoints blockade. We recently developed an ALK vaccine based on a DNA plasmid that generates an efficacious anti-ALK immune response in pre-clinical mouse models. This vaccine potentiates the effects of ALK TKI and can be combined with immune checkpoint blockade therapy. However, a consistent theme in human DNA vaccine trials has been their suboptimal immunogenicity when compared with protein-based vaccine approaches. To move towards a peptide-based vaccine, we sought to identify antigenic peptides responsible for these immune responses by in vitro screening with ALK overlapping peptides in splenocytes from ALK vaccinated mice and by MHC-I binding predictive algorithms. Four peptides induced IFN-γ production in splenocytes and one of these peptides contained a predicted CD8+ T-cell epitope. To validate these results in vivo, mice were vaccinated with these peptides. The peptide containing the predicted CD8+ 9mer was confirmed to induce CD8+ T-cell responses while the others induced CD4+ T-cell responses. To increase immunogenicity, peptides were modified with an amphiphilic tail. As we recently showed, this conjugation promotes binding to endogenous albumin, increasing lymph node accumulation and T-cell priming. In prophylactic experiments, mice vaccinated with the CD8+ T-cell epitope, but not the CD4+ T-cell epitopes, almost completely rejected ALK-expressing lung orthotropic tumors. To validate the efficacy of the vaccine in a therapeutic protocol, EML4-ALK transgenic mice that spontaneously develop ALK-positive lung tumors were vaccinated with a combination of these peptides. The vaccine significantly delayed the progression of primary lung tumors. We propose that a peptide-based ALK vaccine, combined with ALK TKI treatment and/or immune checkpoint blockade therapy, could represent a novel therapeutic option for ALK-positive lung cancer patients. A Phase I clinical trial with a peptide-based ALK vaccine in NSCLC patients is currently under preparation. Citation Format: Rafael B. Blasco. Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A021.

DNA vaccines against Zika virus speed into clinical trials.

Less than 6 months after Zika infection was declared a public health emergency, the first clinical trials of DNA vaccines against the virus are beginning. Less than 6 months after Zika infection was declared a public health emergency, the first clinical trials of DNA vaccines against the virus are beginning.

Advances and challenges in the development of therapeutic DNA vaccines against hepatitis B virus infection.

Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice.

BackgroundHuman papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer.MethodsIn this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems.ResultsHigh-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression.ConclusionsThe fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.

Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.

A Phase 1 clinical trial of Hantaan virus and Puumala virus M-segment DNA vaccines for hemorrhagic fever with renal syndrome

Candidate DNA vaccines for hemorrhagic fever with renal syndrome expressing the envelope glycoprotein genes of Hantaan (HTNV) or Puumala (PUUV) viruses were evaluated in an open-label, single-center Phase 1 study consisting of three vaccination groups of nine volunteers. The volunteers were vaccinated by particle-mediated epidermal delivery (PMED) three times at four-week intervals with the HTNV DNA vaccine, the PUUV DNA vaccine or both vaccines. At each dosing, the volunteers received 8 μg DNA/4 mg gold. There were no study-related serious adverse events, and all injection site pain was graded as mild. The most commonly reported systemic adverse events were fatigue, headache, malaise, myalgia, and lymphadenopathy. Blood samples were collected on days 0, 28, 56, 84, 140, and 180, and assayed for the presence of neutralizing antibodies. In the single vaccine groups, neutralizing antibodies to HTNV or PUUV were detected in 30% or 44% of individuals, respectively. In the combined vaccine group, 56% of the volunteers developed neutralizing antibodies to one or both viruses. These results demonstrate that the HTNV and PUUV DNA vaccines are safe and can be immunogenic in humans when delivered by PMED.

Plasmid DNA Gene Therapy by Electroporation: Principles and Recent Advances

Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

Development of dengue DNA vaccines

Vaccination with plasmid DNA against infectious pathogens including dengue is an active area of investigation. By design, DNA vaccines are able to elicit both antibody responses and cellular immune responses capable of mediating long-term protection. Great technical improvements have been made in dengue DNA vaccine constructs and trials are underway to study these in the clinic. The scope of this review is to highlight the rich history of this vaccine platform and the work in dengue DNA vaccines accomplished by scientists at the Naval Medical Research Center. This work resulted in the only dengue DNA vaccine tested in a clinical trial to date. Additional advancements paving the road ahead in dengue DNA vaccine development are also discussed.

Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection.

BackgroundChronic HBV infects 350 million people causing cancer and liver failure. We aimed to assess the safety and efficacy of plasmid DNA (pSG2.HBs) vaccine, followed by recombinant modified vaccinia virus Ankara (MVA.HBs), encoding the surface antigen of HBV as therapy for chronic HBV. A secondary goal was to characterize the immune responses.MethodsFirstly 32 HBV e antigen negative (eAg–) participants were randomly assigned to one of four groups: to receive vaccines alone, lamivudine (3TC) alone, both, or neither. Later 16 eAg+ volunteers in two groups received either 3TC alone or both 3TC and vaccines. Finally, 12 eAg– and 12 eAg+ subjects were enrolled into higher-dose treatment groups. Healthy but chronically HBV-infected males between the ages of 15 – 25 who lived in the western part of The Gambia were eligible. Participants in some groups received 1 mg or 2 mg of pSG2.HBs intramuscularly twice followed by 5×107 pfu or 1.5×108 pfu of MVA.HBs intradermally at 3-weekly intervals with or without concomitant 3TC for 11–14 weeks. Intradermal rabies vaccine was administered to a negative control group. Safety was assessed clinically and biochemically. The primary measure of efficacy was a quantitative PCR assay of plasma HBV. Immunity was assessed by IFN-γ ELISpot and intracellular cytokine staining.ResultsMild local and systemic adverse events were observed following the vaccines. A small shiny scar was observed in some cases after MVA.HBs. There were no significant changes in AST or ALT. HBeAg was lost in one participant in the higher-dose group. As expected, the 3TC therapy reduced viraemia levels during therapy, but the prime-boost vaccine regimen did not reduce the viraemia. The immune responses were variable. The majority of IFN-γ was made by antigen non-specific CD16+ cells (both CD3+ and CD3–).ConclusionsThe vaccines were well tolerated but did not control HBV infection.Trial RegistrationISRCTN ISRCTN67270384

DNA Vaccines against Infectious Diseases and Cancer

Progress in the development of DNA vaccines and their delivery strategies has been made since their initial concept as a next generation vaccine. Since DNA vaccine includes non-infectious DNA parts of pathogens, it can't cause disease yet it closely mimic the natural process of infection and immune responses. Despite their early promising results of controlling infectious diseases and cancer in small animal models, DNA vaccines failed to display a level of immunogenicity required for combating these diseases in humans, possibly due to their lower protein expression levels. However, increasing evidence has shown that DNA vaccines are clinically well-tolerated and safe. Furthermore, one notable advantage of DNA vaccines includes convenient utilities of plasmid DNAs coding for antigens. For instance, any emerging pathogens could be prevented easily and timely by allowing the simple exchange of antigen-encoding genes. In this review, newly developed DNA vaccine strategies, including electroporation, which has emerged as a potent method for DNA delivery, targeting infectious diseases and cancer will be discussed with a focus on any on-going DNA vaccine trials or progress made pre-clinically and in clinics.

FDA guidance on prophylactic DNA vaccines: Analysis and recommendations

The FDA has been regulating the conduct of prophylactic DNA vaccine trials in the US for nearly 15 years. This work describes the evolution of FDA policy over that period, the status of current regulatory guidance, and provides recommendations for further changes to facilitate development in this field.

DNA vaccines for autoimmune diseases

Autoimmune diseases represent a group of disorders in which there exists a large unmet medical need for effective treatments, but also where there exists a tremendous responsibility among physicians and drug developers to maintain adequate and acceptable patient safety. Several drugs have been approved and many others are about to be approved for the treatment of autoimmune diseases, but in pushing the envelope of therapeutic efficacy, concerns have been raised about the long-term safety of these new therapies. DNA vaccines provide a method of treating autoimmune diseases in a highly specific manner, and could therefore overcome these safety concerns while still maintaining comparable efficacy. The numerous reports of DNA vaccines in animal models of autoimmune diseases and results from three recent human trials of DNA vaccines in autoimmune diseases are reviewed here.

Selection of appropriate serological tests to measure the incidence of natural Leishmania infantum infection during DNA/MVA prime/boost canine vaccine trials

In response to the increasing need for field trials of experimental DNA vaccines against zoonotic visceral leishmaniasis in dogs, our aim was to validate the use of ELISA protocols which will be suitable for detection of natural infection in vaccinated dogs. We have previously demonstrated that DNA/modified vaccinia virus Ankara (MVA) vaccine expressing tryparedoxin peroxidase (TRYP) induced high titres of TRYP antigen-specific IgG in immunized dogs. Here we report our findings that seroconversion to an unrelated diagnostic antigen rK39 did not occur in vaccinated dogs, and that responses to crude Leishmania infantum promastigote antigen (CLA) were weak and short-lived. This is in contrast to strong responses to both antigens shown in naturally infected dogs. To select an appropriate serological test for measurement of infection incidence, we also tested longitudinal samples from an immunologically well-characterized cohort of naturally infected dogs. The sensitivity of CLA ELISA was superior to that of rK39 in early stage infection (from 2 months before, to 2 months after the first detection of infection by PCR or parasitological culture), and more sensitive than rK39 in cross-sectional sampling (81.0% vs 61.9%). We conclude that CLA ELISA will provide sensitive estimates of L. infantum infection incidence in DNA/MVA vaccinated dogs, though optimal testing would include rK39, or a similar recombinant antigen, to improve overall specificity.

The First Antibody Responses to DNA Vaccines in Humans

To the Editor—In their recent article, Martin et al. [1] report an important phase 1 clinical trial of a DNA vaccine for West Nile Virus. They conclude that “[t]his clinical study represents the first report of neutralizing antibody activity elicited by a DNA vaccine in humans” [1, p. 1739]; the accompanying editorial commentary, by Peterson and Roehrig, repeats this claim [2, p. 1722]. Although Martin et al. elegantly demonstrate neutralizing-antibody responses by using plaque-reduction neutralization and reporter-virus particles, multiple earlier clinical trials demonstrating putative neutralizing-antibody responses have been reported. To our knowledge, the first report of a protective antibody response induced by a DNA vaccine was published in 2000 by Roy et al. [3]. DNA encoding the surface antigen of hepatitis B virus (HBV) was administered to 12 healthy, hepatitis-naive human volunteers via particle-mediated epidermal delivery (PMED). All volunteers developed protective antibody responses, defined as 10 mIU/mL. This level of antibody response against HBV has long been considered sufficient to protect against HBV infection [4]. Subsequent studies confirmed that this HBV DNA vaccine could elicit antibody responses in those who do not respond to conventional vaccine [5] and could increase antibody titers when administered via a disposable, commercial prototype device [6]. These reports, in combination with other clinical studies of DNA vaccines for a variety of pathogens, provide considerable evidence to support further human trials of DNA vaccines. Both the PMED system used in these HBV DNA vaccine studies and the Biojector 2000 NeedleFree Injection Management System that Martin et al. use for West Nile virus are excellent examples of innovative needlefree methods for the delivery of DNA vaccine. These novel delivery methods provide increased hope for the development of a commercially feasible DNA vaccine for use in humans.

DNA vaccines: are they still just a powerful tool for the future?

Vaccination is historically one of the most successful strategies for the prevention of infectious diseases. For safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. The antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. Differing ratios of various proportions between specific CD4+ and CD8+ T cell responses are essential for conferring the required protection in the case of individual vaccines. To stimulate both CD4+ and CD8+ T cells, the antigens must be processed and presented to both antigen-presentation pathways, MHC I and MHC II. Protein antigens delivered by vaccination are processed as extracellular antigens. However, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-DNA vaccination. In this overview, current knowledge of the host immune response to DNA vaccines is summarized in the introduction. The subsequent sections discuss techniques for enhancing DNA vaccine efficacy, such as DNA delivery to specific tissues, delivery of DNA to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. Finally, the prospects of DNA vaccination and ongoing clinical trials with various DNA vaccines are discussed.

Safety and Immunogenicity of Tyrosinase DNA Vaccines in Patients with Melanoma

Immunity to self antigens on cancer is constrained by tolerance/ignorance. DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), mediate tumor protection and regression in implantable mouse models, and dogs with spontaneous melanoma. We conducted a trial of mouse and human TYR DNA vaccines in stage III/IV melanoma patients. Eighteen human leukocyte antigen (HLA)-A*0201(+) melanoma patients were randomized as follows: one group received three mouse TYR DNA injections followed by three human TYR DNA injections; the other group received the same vaccines in opposite sequence. The study was conducted at three dose levels: 100, 500, and 1,500 microg DNA/injection, administered intramuscularly (IM) every 3 weeks. Most toxicities were grade 1 injection site reactions. Seven patients developed CD8(+) T-cell responses, defined by a >3 SD increase in baseline reactivity to TYR peptide in tetramer or intracellular cytokine staining (ICS) assays. There was found to be no relationship between dose, assigned schedule, and T-cell response. At a median of 42 months follow-up, median survival has not been reached. Mouse and human TYR DNA vaccines were found safe and induced CD8(+) T-cell responses in 7 of 18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells.

In Vivo Antigen Stability Affects DNA Vaccine Immunogenicity

The factors that determine the immunogenicity of Ags encoded by viral vaccines or DNA vaccines in vivo are largely unknown. Depending on whether T cell induction occurs via direct presentation of vaccine-encoded epitopes or via one of the different proposed pathways for Ag cross-presentation, the effect of intracellular Ag stability on immunogenicity may possibly vary. However, the influence of Ag stability on CD8(+) T cell induction has not been addressed in clinically relevant vaccine models, nor has the accumulation of vaccine-encoded Ags been monitored in vivo. In this study, we describe the relationship between in vivo Ag stability and immunogenicity of DNA vaccine-encoded Ags. We show that in vivo accumulation of DNA vaccine-encoded Ags is required for the efficient induction of CD8(+) T cell responses. These data suggest that many of the currently used transgene designs in DNA vaccination trials may be suboptimal, and that one should either use pathogen-derived or tumor-associated Ags that are intrinsically stable, or should increase the stability of vaccine-encoded Ags by genetic engineering.