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With the ever-present threat of pandemics caused by emerging infectious diseases, the ability to quickly and effectively design, produce and administer novel vaccines is crucial. Inovio Pharmaceuticals is focusing on next-generation DNA vaccines and delivery pathways for complex diseases, such as HIV and cancer. Dr Kate Broderick is the Vice President, Preclinical R&D, for Inovio Pharmaceuticals and her role is to oversee the development of enhanced delivery techniques for these gene-based therapeutics.
Inovio Pharmaceuticals is committed to revolutionising healthcare across the globe using next-generation vaccine development approaches and novel delivery methods. Whilst most current vaccines are given intramuscularly, there may be alternative target tissues equally able to invoke a strong immune response. Vaccines have already been developed for many common diseases, but despite this, there are many complex and devastating diseases with no available vaccines.
Figure 1: Panoramic image of the dermal tissue after pDNA delivery in the presence of surface electroporation (SEP).
Guinea pigs were treated intradermally with 50 µg pRFP and the injection site immediately electroporated with the SEP device. The mapped and stitched multipanel and multisection confocal image reconstruction revealed reporter gene (red fluorescent protein (RFP) expression in the epidermal layer of guinea pig skin aligned to the site where plasmid injection and electroporation with SEP was performed. Image acquired six hours after treatment. Magnification 10×.D, dermis; ED, epidermis; SC, stratum corneum. RFP (red), K10 stain (green), Hoechst (blue). Scale bar represents 1mm. This figure was previously published in Molecular Therapy – Methods & Clinical Development ‘DNA vaccination strategy targets epidermal dendritic cells, initiating their migration and induction of a host immune response ‘(2014) Volume 1, Issue 4054; doi:10.1038/mtm.2014.54 and is under the Creative Commons licence CC BY-NC-ND 3.0.
The next generation of vaccines
DNA vaccines are different to conventional vaccines which can be made from the whole virus or bacteria itself, e.g., a whole inactivated virus-based vaccine. These are often referred to as first-generation vaccines. Other conventional vaccine approaches commonly involve a subunit of the pathogen-containing specific protein antigens, which alter the cells themselves. Next-generation vaccines consist of DNA which codes for specific proteins from a pathogen. DNA enters the cells and the cells use their own replication machinery to synthesise the pathogen proteins. Because these proteins are recognised as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers immune responses. The DNA must be delivered in a vector, but there is no immune response to the vector itself, only the foreign proteins processed and displayed by the cells. Due to the inherent stability of DNA vaccines and Inovio’s optimised DNA storage formulation, they do not require storage at low temperatures. This is a major advantage over many current vaccines, as this issue often limits the distribution and use of vaccines in developing countries.
Inovio’s DNA vaccine platform can overcome many of the issues surrounding conventional vaccine platforms. These include the ability to induce robust immune responses, the flexibility to combine multiple antigens in a single vial, rapid design and production and improved storage requirements, e.g., they do not necessarily need to be kept cold and can instead be stored at room temperature. The advantage of this is that specialised equipment is not required to store the vaccine, making it more accessible to clinicians and patients.
Figure 2: Pathologic analysis of selected tissues in LASV-exposed NHPs. A) Spleen, peri-arteriolar lymphoid sheath (PALS) hyperplasia in a vaccinated macaque that survived LASV challenge, 4X magnification; Inset, spleen, PALS hyperplasia with a complete absence of LASV immunoreactivity; B) Kidney, positive cytoplasmic immunoreactivity in islet and exocrine epithelial cells Of an NHP that succumbed on Day 11 post-exposure, 20x; C) Lung, cytoplasmic immunoreactivity in low numbers of alveolar macrophages, pneumocytes and endothelial cells in an NHP that succumbed on Day 17 post-exposure, 20x; D) Liver, positive apical to membranous hepatocyte and cytoplasmic.This endothelial immunoreactivity in an NHP that succumbed on Day 11 post-exposure, 40x. This figure was previously published in Human Vaccines & Immunotherapeutics, Volume 13, Issue 12, 2017, 2902–2911, https://doi.org/10.1080/21645515.2017.1356500 and is under the Creative Commons licence CC BY-NC-ND 4.0.
Existing DNA vaccines
Although there are currently no approved DNA vaccines for human use, three DNA-based vaccines are already licensed for veterinary use. These vaccines target West Nile virus in horses, infectious haematopoietic necrosis virus in salmonid fish and malignant melanoma in dogs. Inovio and their collaborators, as well as many colleagues in the field at companies, universities and institutes all over the world, have worked diligently towards the goal of the first DNA vaccines approved for human use. Inovio and their collaborators at USAMRIID have conducted several studies using a novel DNA vaccine against Lassa virus, which has shown to be 100% effective in both guinea pig and macaque animal models in protecting against disease. Inovio and their collaborators GeneOne have also recently been involved in the development of a vaccine against Zika virus and have tested this vaccine in a Phase I safety and immunogenicity trial. The vaccine was administered intradermally (into the skin), followed by an enhancing delivery process called electroporation (EP) which significantly increases the uptake of the DNA into the cells. The vaccine administration was well tolerated and 100% immunogenic.
Remarkably, from receipt of the viral sequence to dosing a patient in the clinic, the Inovio trial was able to accomplish this in six months. This is an unprecedented timeline for vaccine development which speaks to the applicability of Inovio’s platform for meeting the challenges of emerging infectious diseases. Further work is still needed to assess the efficacy of the vaccine to protect humans against the virus, but it provides a promising solution to a disease which has devastating effects.
Vaccination against cancer is also getting closer to becoming a reality. In the case of anti-cancer vaccines, the DNA constructs will code for one or more tumour associated antigens with the aim of being to elicit a tumour-specific immune response.
Enhancing vaccine delivery technology
Development of a next-generation vaccine requires a next-generation delivery approach to administer the vaccine. One of the many advantages of using DNA vaccines is that they can be designed and manufactured extremely quickly, in a matter of weeks compared to the several years it may take to develop and create the same solution using a conventional vaccine approach. This gives Inovio real-time tools to combat emerging diseases and pandemics.
Figure 3: Survival curve, serum viremia, morbidity scores, and body weight and temperature changes for NHPs post-exposure. A) All of the DNA-vaccinated NHPs survived, while 2 of the 4 mock-vaccinated NHPs survived to the study end point. B) Overall morbidity scores assigned daily for each NHP as a subjective measure of observed responsiveness and presence or absence of disease signs; C) Serum viremia post-exposure as measured by plaque assay and expressed as the Log10 pfu/ml per blood sample collection day; D) Changes in body weight expressed as a percentage change from baseline weights for each NHP, then averaged per group; and E) Changes in body temperature as measured by rectal and/or temperature transponder chips and expressed as a percentage change from baseline temperatures for each NHP, then averaged per group. This figure was previously published in Human Vaccines & Immunotherapeutics, Volume 13, Issue 12, 2017, 2902–2911, https://doi.org/10.1080/21645515.2017.1356500
and is under the Creative Commons licence CC BY-NC-ND 4.0.
The Inovio CELLECTRA® delivery device platform represents alternative vaccine delivery tissue targets. The technology which the CELLECTRA® platform is based on, is called electroporation (EP). Using this technology, Inovio has so far conducted multiple clinical trials with over 1,700 subjects and administered approximately 6,000 doses of vaccine using electroporation.

Genetic-based vaccines will revolutionise healthcare by presenting potential solutions to global health issues, such as cancer, HIV and flu.

Electroporation was first used in 1982 and is the process of using an electrical current to make the cell membrane temporarily more permeable, allowing better uptake of a vaccine. An attractive target for vaccine delivery is the skin as it is easily accessed and easily monitored. Furthermore, it has many resident antigen-presenting cells, crucial for eliciting robust and long-lasting immune responses.

Figure 4: Histological analysis reveals reporter gene expression localised to cells in the epidermis
Histological analysis of GFP and RFP expression after ID plasmid administration followed by SEP in guinea pig skin. A). GFP treated skin biopsies were removed four hours post treatment, cryosectioned, DAPI stained and visualised using fluorescence microscopy (20x and 40x). An injection only control (no EP) is also shown. B). RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualised using confocal imaging. This figure was previously published in Vaccines, ‘Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporatio’, 2013, Volume 1, Issue 3, 384-397; doi:10.3390/vaccines1030384 and is under the Creative Commons licence CC BY-NC-SA 3.0.
The naked delivery of nucleic acid vaccines, in this case, DNA vaccines, is notoriously inefficient. Previous studies have shown that this often results in a weak or non-existent immune response to the vaccine. Therefore, EP offers an attractive enhancement to DNA vaccine administration. EP has been used extensively in the clinic targeting primarily either the muscle or the skin as target tissues. Inovio’s CIN EP-enhanced DNA vaccine Phase II trial was the first to demonstrate clinical efficacy.
Figure 5: Efficient plasmid transfection of Guinea pig skin by in vivo electroporation.
Guinea pig skin images 24 hours after treatment with GFP reporter plasmid by intradermal injection only (top panel), or intradermal injection with electroporation (lower panel).
As it has a local effect, EP can also be used to improve delivery to a target tissue of interest. Most importantly, DNA vaccines administered with EP can generate both T-cell and antibody immune responses.
Inovio’s goal
Genetic-based vaccines will revolutionise health care by presenting potential solutions to global health issues, such as cancer, HIV and flu and in addition to this, the preclinical development of a DNA-based vaccine delivery platform would make the technology suitable for mass vaccinations.
Inovio’s future aim is ultimately to use immunisation with DNA vaccines to protect against emerging infectious diseases in both developed populations and low and middle-income populations who may not currently have access to vaccines, whether this is for climatic, economic or technological reasons.

Following on from DNA vaccines, what do you think the next generation of vaccines and vaccine delivery platforms will look like?
At Inovio, we are intensely focused on harnessing the power of DNA vaccines to revolutionise the vaccine development and vaccine delivery realms both for infectious diseases and cancers. We believe that many attributes of DNA vaccines such as their speed of manufacture, ease of storage and distribution (i.e., not requiring a frozen cold chain) and most importantly, ability to generate both B and T cell immune responses position them as next-generation vaccine approaches to pressing public health concerns.

References

Research Objectives
Inovio’s goal is to facilitate preventive immunisation using its DNA vaccines against critical infectious diseases with unmet needs in large populations.
Funding

  • NIH
  • US Department of Defense
  • US Department of Defense
  • DARPA
  • CEPI

Collaborators

  • The Wistar Institute – The Weiner Lab
  • USAMRIID – Dr Connie Schmaljohn and team

Bio
Dr Broderick received her PhD from the University of Glasgow in Scotland and performed her post-doctoral research at the University of California, San Diego. She joined Inovio Pharmaceuticals in 2006 where she currently leads a diverse research group focused on enhanced delivery techniques for gene-based therapeutics as the Vice President, Preclinical R&D.
Contact
Dr Kate E. Broderick, PhD
Vice President, Preclinical Research and Development
Inovio Pharmaceuticals
10480 Wateridge Circle
San Diego
CA 92121, USA
E: Kate.broderick@inovio.com
T: +1 858 410 3161
W: http://www.inovio.com

Delivery of gene-base vaccines by novel, next-generation enhancing technologies