Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer

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Fate of plasmid DNA after administration

After intramuscular or intradermal injection, the plasmid DNA will transfect somatic cells, such as myocytes and keratinocytes, and/or resident Antigen Presenting Cells (APCs) like dendritic cells (DCs) and macrophages located in the lamina propria (Kutzler and Weiner, 2008; Liu, 2011). DNA enters the cell through two main mechanisms. One is known as fluid‐phase endocytosis (not mediated by a receptor), which involves the ingestion of small molecules and/or fluids surrounding the cell (pinocytosis) (Levy et al., 1996; Budker et al., 2000; Varkouhi et al. 2011). Another is known as adsorptive endocytosis (not mediated by a receptor as well) in which the DNA is taken in by a cell by splitting off small vesicles from the cell surface (Budker et al., 2000; Faurez et al., 2010; Varkouhi et al. 2011). The plasmid sequence can also be recognized simultaneously by different receptors located at the plasma membrane (PM) from eukaryotic cells (Lehmann and Sczakiel., 2005).
Once inside the cell, DNA faces another obstacle: it needs to migrate to the cell cytoplasm. The endocytic vesicles containing the plasmid vaccine fuses with the lysosome, whose function is to digest molecules originally incorporated into the endosome through the activity of many hydrolytic enzymes (Varkouhi et al., 2011). It is still not well understood how the DNA escape from the phagolysosome and reaches the cell cytoplasm (Faurez et al., 2010). Continuously, once within the cytoplasm, vectors that survive from the endonucleases « attack » will reach the cell nucleus and, finally, initiate transcription of the gene of interest. Vacik and colleagues demonstrated that DNA located in the cytoplasm binds to both microtubules and microfilaments that form the cytoskeleton of cells through adapter proteins named dynein, and thus reach the cell nucleus (Vacik et al., 1999). It has been also suggested that DNA molecules located in the cytoplasm can create associations with polypeptides, such as transcription factors, that contains a nuclear localization signal (NLS) required to enter the nucleus (Hebert, 2003).
The third and last obstacle blocking the expression of the antigenic protein is the nuclear membrane. Some studies demonstrated that the plasmid vaccine is able to cross the nuclear membrane by two different ways: (i) passage of the DNA by diffusion through nuclear pore complexes, and (ii) during mitosis, when nuclear envelope is disassembly in dividing cells (Faurez et al, 2010). The DNA located in the nucleus can have access to the transcription machinery turning possible the transcription of the gene of interest. Later, the RNA transcript can be translated into protein in the cytoplasm of the cell (Faurez et al, 2010; Liu, 2011). The host cell provides necessary post‐translational modifications mimicking a real infection, this feature being one of the biggest advantage of the genetic immunization (Kutzler and Weiner, 2008; Liu, 2011). Figure 4 illustrates both intra and extracellular barriers that the DNA vaccine needs to face before reaching the cell nucleus.

Adaptive immune response: Cellular and Humoral Immunity

The mammalian immune system comprises of innate and adaptive branches that mount integrated protective responses against intruding foreign antigens. The innate immune system includes DCs, macrophages, granulocytes, and natural killer (NK) cells that mediate fast but ŶŽŶƐ ĞĐŝĮĐ ƌĞƐ ŽŶƐĞƐ ĂĨƚĞƌ ƌĞĐŽŐŶŝnjŝŶŐ ŐĞŶĞƌŝĐ ŵŝĐƌŽ ŝĂů Ɛƚƌ Đƚ ƌĞƐ͘ /Ŷ ĐŽŶƚƌĂƐƚ͕ ƚ Ğ Ă Ă ƚŝǀĞ ŝŵŵ ŶĞ ƐLJƐƚĞŵ ŝŶĐů ĞƐ d ĂŶ ĐĞůůƐ ƚ Ăƚ ŵĞ ŝĂƚĞ Ɛ ĞĐŝĮĐ ƚ ƚĞŵ ŽƌĂůůLJ ĞůĂLJĞ ƌĞƐ ŽŶƐĞƐ after recognizing antigenic epitopes (Cerutti et al., 2012). Therefore, adaptive immune responses depend on antigen activation of B and T lymphocytes into antibody‐producing plasma (humoral immunity) and T effector (cellular immunity) cells, respectively (Malek and Castro, 2010). Antigens administered in the form of DNA can stimulate both humoral and cellular immunity as they have been shown to be protective against viral, bacterial, and tumor challenge (Howarth and Elliott, 2004).
In order to stimulate both types of adaptive immune responses, APCs firstly present antigens through different pathways, as described in the previous section, activating CD8+ T cells (cytotoxic lymphocytes, CTLs) and CD4+ T helper cells (T helper lymphocytes). Inside the draining lymph nodes, B cells in contact with protein antigens, are stimulated by certain cytokines, such as IL‐21, IL‐4, and IL‐10, released mainly by CD4+ T cells (T‐dependent B cell activation) (Cerutti et al., 2012).

Preclinical and clinical progress of DNA vaccines

To date, there have been several preclinical and clinical studies on DNA vaccines. Although US FDA still did not approved DNA vaccines for use in humans, phase I clinical studies have been reported for the prevention and/or treatment of HIV, malaria, hepatitis B, SARS and many other infectious agents (Klinman et al., 2010). In addition to the initial demonstration of the efficacy of DNA vaccines to protect against infectious challenge in a mouse model of influenza virus, DNA vaccines have been shown to protect against influenza in ferrets, and primates; lymphocytic choriomeningitis virus; herpes simplex virus in guinea pigs and mice; rabies virus; cottontail rabbit papillomavirus; hepatitis B virus; malaria (Plasmodium falciparum); HIV in nonhuman primates; and against other several bacterial pathogens (Srivastava and Liu, 2003).
What is remarkable is that in the past 6 years, four DNA vaccines for larger animals have been licensed for use in the veterinary field. Two of them target infectious diseases such as West Nile virus in horses, authorized for use in the United States (US); and aquatic rhabdovirus (also termed as infectious hematopoietic necrosis, IHNV) in salmon, approved for use in Canada. Another one is a cancer vaccine for melanoma in dogs; and the last one has a therapeutic purpose in swine in which a plasmid encoded the growth hormone, when delivered before specific vaccination, demonstrated enhanced protection against Mycoplasma hyopneumoniae; both of them are authorized for use in the US and Australia. Although these animal disease models are not completely similar to humans, past success with DNA vaccines turns this vaccine platform very promising for use in humans (Faurez et al., 2010; Ingolotti et al., 2010; Findik and Çiftci, 2012) (Table 1).

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Improvement of DNA vaccines immunogenicity

The low immunogenicity of early DNA vaccines is hypothesized to stem, in part, from inefficient uptake of the plasmids by cells due to inefficient delivery (Ferraro et al., 2011). Nonetheless, the reasons for the failure of DNA vaccines to induce potent immune responses in humans have not been elucidated (Bolhassani et al., 2011). Therefore, research has focused on developing novel strategies to enhance transfection efficiency and improve other facets of the DNA vaccination platform using several strategies (Kutzler and Weiner, 2008; Ferraro et al., 2011).
Reasonably, vector modifications that improve antigen expression are highly correlative with improved immune responses. It has been shown that the use of altered transcriptional elements, like (i) the modified CMV promoters (chimeric SV40‐CMV promoters), (ii) mRNA containing introns, (iii) gene of interest codon optimized to match with the target organism, (iv) the use of a dual stop codon to limit read through translation, and (v) transcription terminators/polyadenylation signals can significantly improve either antigen transcription or translation by the host cell (Kutzler and Weiner, 2008; Williams et al., 2009). Optimization of the initiation start site for protein synthesis (kozak consensus sequences) is also desirable as endogenous sites of viruses and bacteria might not be optimal for expression in mammalian cells (Kutzler and Weiner, 2008). The addition of leader sequences can also enhance the stability of mRNA and contribute to translational efficiency. Considering the commercialization of DNA vaccines, the use of high‐efficiency origins of bacterial replication can also markedly improve the quantity of plasmid product (Williams et al., 2009). Of course the right choice of antigens is crucial for obtaining a satisfactory immune response as well as the use of unmethylated CpG, which should be included in the plasmid backbone in order to enhance the potency of a DNA vaccine (Glenting and Wessels, 2005). Another strategy attempt to increase the magnitude of immune responses by co‐expression of cytokines, chemokines or co‐stimulatory molecules, that can have a substantial effect on the immune response (Liu, 2003; Kutzler and Weiner, 2008; Ferraro et al., 2011). Dam and dcm methylation performed by bacteria should also be taken into account as this may affect antigen expression or make it prone to recognition as foreign DNA by the host innate immune system (Kutzler and Weiner, 2008; Williams et al., 2009). Another important effort to improve gene expression includes the delivery method employed to introduce the DNA vaccine in the organism (Bolhassani et al., 2011). These methods comprise physical and chemical approaches or the use of viral and bacterial vectors. Figure 7 presents some of the improvements proposed to increase the immunogenicity of DNA vaccines.

Table of contents :

CHAPTER 1‐THESIS INTRODUCTION
1.DNA VACCINES
1.1 Historical perspective of DNA vaccines
1.2 Structural features of DNA vaccines
1.3 Safety issues
1.4 Immunological aspects of DNA vaccines
1.4.1 Routes of administration
1.4.2 Fate of plasmid DNA after injection and antigen presentation
1.4.3 Antigen presentation
1.4.4 Adaptive immune response: Cellular and Humoral Immunity
1.4.5 Immune memory
1.5 Preclinical and clinical progress of DNA vaccines
1.6 Improvement of DNA vaccines immunogenicity
1.7 Non‐biological delivery systems for DNA vaccines
1.7.1 Physical approaches
1.7.2 Chemical vectors
1.8 Biological delivery systems for DNA vaccines
1.8.1 Virus as DNA delivery vehicles
1.8.2 Bacteria‐based vectors
2.BACTERIAL VECTORS AS DNA DELIVERY VEHICLES
2.1 Basic Principles for Bacteria‐Mediated DNA delivery at mucosal surfaces
2.1. 1 Intestinal mucosa
2.1.2 Commensal bacteria and the intestinal mucosa
2.3 Bacterial vectors used for gene transfer
2.3.1 Pathogenic bacterial DNA delivery
2.3.1.1 Extracellular pathogens
2.3.1.2 Intraphagosomal pathogens
2.3.1.3 Intracytosolic pathogens
3.LACTIC ACID BACTERIA (LAB)
3.1 Taxonomy and characteristics
3.1.1 Physiology of lactic acid bacteria into the human gastrointestinal tract
3.1.2 The probiotic action
3.2 The model LAB: Lactococcus lactis
3.3 Lactococcus lactis: From cheese making to Heterologous protein Delivery
3.3.1 Gene expression systems and heterologous protein production in Lactococcus lactis
3.3.2 Lactococcus lactis as mucosal delivery vectors for therapeutic proteins … Error! Bookmark not defined.
3.4 Lactococcus lactis: From protein to DNA delivery
3.4.1 Native L. lactis as DNA delivery vectors
3.4.2 Recombinant invasive L. lactis as plasmid DNA delivery vehicles
CHAPTER 2‐Aim of the study
CHAPTER 3‐In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A
2.1 Introduction
2.2 Materials and methods
2.3 Results and Discussion
2.4 Conclusions
CHAPTER 4‐Immune response elicited by DNA vaccination using Lactococcus lactis is modified by the production of surface exposed pathogenic protein
3.1 Introduction
3.2 Materials and methods
3.3 Results
3.4 Discussion
3.5 Conclusions
CHAPTER 5‐Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4 Discussion
4.5 Conclusions
CHAPTER 6‐GENERAL DISCUSSION, MAIN CONCLUSIONS AND DIRECTIONS FOR FUTURE WORK
CHAPTER 7‐REFERENCES
APPENDICE 1‐Recombinant Lactococcus lactis expressing both Listeria monocytogenes Lysteriolysin O and mutated internalin A applied for DNA vaccination
APPENDICE 2‐Immunotherapy of allergic diseases using probiotics or recombinant probiotics

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