Is electroporation decisive for the efficacy of DNA vaccine against house dust mite allergy?

Keywords::

• allergy
• DNA
• electroporation
• house dust mite
• vaccine

The prevalence of house dust mite (HDM) allergy increased significantly worldwide during the last decade, reaching up to 10–20% in industrialized countries [1]. To date, HDM allergen-specific immunotherapy (SIT), through the subcutaneous (SCIT) or sublingual (SLIT) administrations of increasing doses of HDM allergens, is the unique desensitizing treatment to down-regulate the Th2-biased allergic response [2]. Unfortunately, such immunotherapeutic treatments did not offer significant benefits for all the patients and can even lead to anaphylaxis triggered by prime/boosting of specific IgE response through the use of natural allergens displaying IgE reactivity [3]. Even if tremendous progress was made in the development of recombinant HDM hypoallergens, SLIT and SCIT are both currently based on the administration of commercially available HDM allergenic extracts. Standardization issues are commonly associated including inconsistent allergen content (more than twenty different HDM allergens were identified so far) and allergen concentration [3]. Moreover, such crude extracts contain non-allergenic proteins as well as microbial compounds (LPS, chitin, β-glucan) which could drastically influence the immune response through notably innate immunity activation [4]. Another disadvantage of SIT lies in the duration of the treatment requiring 50–80 injections within 2 to 5 years, explaining more likely why only 5% of the allergic populations adhere with such therapies [5].

By their ability to elicit mainly antigen-specific cytolytic T-lymphocyte (CTL), Th1-biased humoral and cellular immune responses (IgG/IFNγ), DNA vaccines could represent a promising approach in the development of safer and more effective anti-allergy immunotherapies [6]. Indeed, numerous studies evidenced the prophylactic as well as the therapeutic effects of vaccinations with allergen encoding DNA plasmids to prevent and to revert respectively the allergic response in small rodents through the induction of allergen-specific Th1 immunity as well as tolerance mediated by Treg cells [7]. Whether these anti-allergy effects can be induced in humans still remain to be fully demonstrated. But the failure to translate the promising results from preclinical evaluation of anti-infectious/cancer diseases DNA vaccines to clinical trials clearly highlighted that DNA-based vaccines are basically weak immunogens in humans [6,7]. The poor DNA vaccine immunogenicity results more likely from a combination of factors including the limitation of the DNA doses in humans as well as ineffective cellular DNA uptake when administered by conventional injection.

Consequently, the key question to be addressed for successful DNA vaccinations is how to improve the immunogenicity of these DNA-based vaccine candidates in humans. DNA vaccine optimization strategies were mainly focused on i) methods to enhance in vivo delivery of DNA vaccine plasmids, ii) development of both chemical and genetic adjuvants to modulate specific immune responses, iii) strategies to increase the gene expression through gene regulatory elements or codon usage optimization and iv) the heterologous prime/boost vaccination regimen [8].

The in vivo DNA vaccine delivery was shown to be drastically improved by electroporation (EP) [9]. This technology which combines short electric pulses with DNA injection, triggers transient pore formation at the level of the cell membrane to facilitate the entry of the genetic material into the intracellular space. Such improvement in the DNA uptake markedly amplified the antigen expression and the immunogenicity of DNA vaccines. Whereas the impact of in vivo EP for DNA vaccinations was clearly evidenced for the development of protective immune response against infectious diseases as malaria, influenza or HPV, the evaluation of EP was not yet investigated in the context of the design of DNA-based vaccine against HDM allergy.

In a recent paper, we first compared the immunogenicity of DNA encoding the major HDM allergen Der p 2 delivered in naive mice by EP or conventional intramuscular (im.) injection [10]. Although three average (20 μg) DNA doses triggered similar IFNγ production through im. or EP, the stimulation of Der p 2-specific IgG1 and IgG2a responses was critically dependent on EP as such antibody production was not detected following im. DNA administration. As no allergy markers including anti-Der p 2 IgE and IL-5 were elicited through DNA immunizations, we next characterized the prophylactic potential of DNA encoding Der p 2 delivered by EP in a Der p 2 sensitization mice model. Strikingly, the potent specific Th1-biased immune response induced by EP injections of our vaccine candidate prevented the development of HDM allergy following Der p 2 sensitizations and intranasal challenges with HDM extracts. Such protective immunity was also generated even at the lowest DNA dose tested (2 μg). Although the therapeutic effect of DNA encoding Der p 2 delivered by EP remains to be addressed, our promising results on immunoprophylaxis of HDM allergy provide the rationale for the evaluation of such DNA vaccine in humans.

Nevertheless, our data already raise questions on the importance of EP in the development of efficient anti-allergy DNA vaccine. Indeed, prevention and/or suppression of the allergic response by DNA encoding allergen can be triggered through the conventional intramuscular route but, usually, with higher DNA doses. Under our experimental conditions, EP improved only the production of allergen-specific IgG but had no effect on IFNγ stimulation.

Effective vaccines to prevent infectious diseases are based on the induction of the potent antibody response able to neutralize the invading microbia. In contrast, the mechanisms behind efficient anti-allergy SIT remain to be fully characterized but the induction of peripheral tolerance (IL-10)/TGF-β-producing Treg) and/or Th1 memory (Th1 cells) represent key factors in the suppression of allergic inflammation [11]. It must be pointed out that several recently discovered suppressive cell subsets as Breg cells secreting IL-10 and cytokines as IL-35 could also participate to the alleviation of the allergic response [11]. Although anti-allergy vaccines based on allergen-derived T cell epitopes or DNA plasmids forcing intracellular expression as ubiquitin-allergen fusion molecules or replicase-based DNA were able to revert the allergic response without the induction of allergen-specific IgG [3,12], the contribution of the specific IgG response (notably allergen-specific human IgG4) in the SIT efficacy was also evidenced through preclinical and clinical studies [13]. Such IgGs are called blocking antibodies as they are able not only to inhibit the IgE-mediated allergen presentation by antigen presenting cells to T cells but also the allergen-induced degranulation of mast cells/basophils. Recently, such blocking IgG response was shown to participate to the long term allergen tolerance achieved by SIT [14]. The relevance of these blocking antibodies was also emphasized by the efficacy of passive immunization with allergen-specific IgG antibodies for treatment and prevention of pollen allergy [15].

Although the exact contribution of anti-Der p 2 IgG response to the observed protective effect remains to be assigned, our data clearly showed the interest of EP in boosting the development of the allergen-specific IgG. Moreover, EP can achieve a dose sparing effect which may be critical for the development of safe and effective DNA-based vaccine against HDM allergy for human use.

It is accepted that the adaptive responses to DNA vaccinations result also from the potent activation of DNA innate immune pathways [16]. Initially, the adjuvant capacity of extracellular naked DNA was thought to result mainly from the endosomal TLR9 activation through their unmethylated CpG motifs. But TLR-independent innate immune DNA-sensing pathways as TANK-binding kinase-1(TBK-1)-stimulator of IFN genes (STING) axis were recently detected in the cytosol. Strikingly, it was observed that the humoral and cellular immune responses through DNA vaccination was not affected in TLR9 deficient animals [17] but was clearly dependent on TBK-1 [18]. The more prominent role of cytosolic DNA sensors in driving the responses mediated by DNA vaccination highlighted EP as the method of choice to potentiate the effectiveness of DNA vaccines through nonendocytic delivery.

In conclusion, our results demonstrated that DNA vaccination is an efficient approach to prevent HDM allergy. Although further experimental studies are required to ascertain the critical role of EP in the effectiveness of our HDM DNA vaccine, such method of vaccination makes the DNA vaccine platform to be as attractive and competitive as other vaccine strategies to halt the HDM allergy pandemic.

Financial & conflict of interests disclosure

This work was funded by National Science and Technology Development Agency (NSTDA, BIOTEC Grant P-09-00324), the National Research university Project of CHE and the Ratchadaphiseksomphot Endowment Fund (HR1164A), the CU-Cluster-Emerging H-8-68-53 and the Chula 100 yrs/CU-Health/CU56-HR06 grants. Dr. Pinya Pulsawat was sponsoredby a Post-doctoral fellowship from the Chulalongkorn University Graduate School. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.