Structural optimization of the novel subunit vaccine platform based on multimeric nanoparticles formed by the Measles Virus Nucleoprotein in yeast

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University of Pisa

Department of Biology

Curriculum Biology Applied to Biomedicine

Master thesis

Structural optimization of the novel subunit vaccine

platform based on multimeric nanoparticles formed by

the measles virus nucleoprotein in yeast

Candidate:

LUISA MANDORLI

Supervisors

Dr. Daria Jacob

Dr. Frederic Tangy

Dr. Monica Sala

Viral Genomics and Vaccination Unit Institut Pasteur, Paris, France

University supervisor

Dr. Mauro Pistello

University of Pisa

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ABBREVIATIONS AND ACRONYMS

Abs Antibodies

α-MoRE α-helical molecular recognition element

AOX1 Alcohol Oxidase 1

AOX2 Alcohol Oxidase 2

APCs Antigen-presenting cells AS03 Adjuvant System 03 BCRs B-cell receptors C C protein

CCR Central Conserved Region CS Circumsporosoite (protein) CTL Cytotoxic T-cell CWP Cell-Wall Protein Dc Dendritic cell EM Electron Microscopy F Fusion protein

FDA Food and Drug Administration G2VU Viral Genomics and Vaccination Unit GVAP Global Vaccine Action Plan

H Haemagglutinin protein HBsAg Hepatitis B surface Antigen HBV Hepatitis B virus

HCA Hydrophobic Cluster Analysis

HCV Hepatitis C virus

HIV Human Immunodeficiency Virus HKY Heat-killed (or heat-inactivated) yeast HPV Human Papilloma Virus

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Hsp72 Heat-shock protein 72

IBDV Infectious Bursal Disease Virus IDPs Intrinsically Disordered Proteins IgG Immunoglobulin G

IP Institut Pasteur

IUPs Intrinsically Unstructured Proteins L Large protein

M Matrix protein

MMR Measles, Mumps, and Rubella Vaccine

MR Mannose Receptor

M&R Initiative Measles and Rubella Initiative MV Measles virus

N Nucleoprotein

N° monomeric N

NK Natural Killer (cells) NNUC

assembled N

NQD double-mutant SL(228–229)QD of the measles virus nucleoprotein

P Phosphoprotein

PAMP Pathogen-Associated Molecular Pattern

Pb Plasmodium berghei

PONDR Predictor Of Naturally Disordered Regions

PRR Pattern-Recognition Receptor

RdRp RNA-dependent RNA polymerase

RNPs RiboNucleoParticles RSV Respiratory syncytial virus TLRs Toll-like Receptor

UNICEF United Nations Children's Fund V V protein

VAERS Vaccine Adverse Event Reporting System

VLPs Virus-like particles

WHO World Health Organization XD X Domain

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ACKNOWLEDGMENTS

I would like to thank all the people, and they are many, who made my internship at the Pasteur Institute possible and turned this period of stage into a wonderful experience under all points of view.

Firstly I would like to thank all the team of the Viral Genomic and Vaccinology Unit that welcomed me like a family with open arms.

First of all Monica, I mainly have to thank her: thanks for believing in me and for offering me the enormous opportunity to work for 8 months in one of the best infectious diseases research centres. A special thank-you goes to Dasha, who always followed me step by step in all my work with amazing patience; beyond her supervisor role she was for me a teacher, a guide, a behaviour model to follow and, most of all, a friend. I really need to thank Megane: between laughter and jokes, I spent with her unforgettable moments, which I will remember forever. Raul, Samira, Hai, Jibby, I could not imagine how the lab life would be like without you, with your smiles, your laughs and your good humour the time I spent at work became funny and pleasant. I would really like to thank all of you guys, especially for being my friends outside the Institute, too. A heartfelt thank-you to all the other members of our team who really welcomed me with a unique warmth; I thank you for helping me greatly not only with regard to the lab activities, but also in learning French. An immeasurable thank-you to Fred, an extraordinary leader: you have the esteem, the admiration and the faith of all people around you. I thank you from the bottom of my heart for having always supported us, both as researchers and single persons.

How could I forget to thank Italians of Pasteur Institute? I shall not. Got in touch almost by chance, we became very close friends in an incredible short time, companions of good and bad moments, with whom I shared most of the time spent in Paris and whose wonderful memories I will keep forever.

A huge thank to my roommate, Clio. From our cohabitation I learnt really a lot regarding all fields, music, food, critique, politics. You are one of the smartest persons I have ever met, a very

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special friend, a noteworthy warrior on the field as well as in life, but most of all, an outstanding cook!

I thank all my dearest friends in Forte dei Marmi and my beloved family. Although physically far from me, you all have always remained by my side; you always made me feel your love and your support, especially in the hardest moments.

I owe a special thank-you to my academic supervisor Mauro Pistello, always available to give me his precious advice and his points of view about scientific problems.

Last but not least, thanks to the University of Pisa and Erasmus that ensured this great opportunity become reality.

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SUMMARY

The impact of persisting and newly emerging infectious diseases is severe as many diseases do not have an efficient treatment. For some diseases treatments exist, but are highly expensive, or are partially inefficient due to drug-resistance. In this regard, research in the field of vaccination is fundamental. Vaccines offer long-term, sometimes life-long, protection against a disease. Routine pediatric vaccines help save up to 3 million lives every year, according to the World Health Organization. Due to their long-term efficacy, vaccines represent one of the most powerful and cost-effective tools in public health. However, vaccine development is a very long and complex process. Before a vaccine can be marketed, it is subjected to rigorous research and optimization, followed by several years of pre-clinical and clinical trials.

The Viral Genomics and Vaccination Unit (UG2V) of Pasteur Institute has been involved since years in the development of vaccines and new vaccine strategies against multiple infectious diseases. The UG2V has recently undertaken the development of a new subunit vaccine platform. This platform is based on recombinant Pichia pastoris yeasts that express antigens of choice, which are multimerized on measles virus nanoparticles inside the yeast cell.

Numerous studies showed that antigen multimerization is an important parameter for inducing an immune response. Indeed, multimeric, rather than monomeric, antigens are more efficiently recognized and processed by antigen presenting cells. The nucleoprotein (N) of the measles virus self-assembles around RNA molecules present in the cytoplasm of mammalian, bacterial or yeast cells, forming helical multimeric structures called ribonucleoparticles (RNP). The N protein is composed of two domains: a globular N-terminal domain Ncore (amino acids 1-400), which contains the multimerization site, and a flexible C-terminal domain Ntail (amino acids 401-525). The first 50 amino acids of the Ntail are incorporated in the globular core of the RNP, while the remaining C-terminal region (75 amino acids) is structurally disordered and overhangs from the surface of the RNP structure. This structurally disordered region of the Ntail represents an attractive linker for fusing antigens of interest and thus multimerizing them on the RNP particle.

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Yeasts are commonly used in pharmaceutical industry for producing and purifying proteins. However, several studies have also demonstrated that heat-inactivated yeasts can work as delivery vehicles, as they induce strong immune responses against the delivered antigen in the absence of accessory adjuvants. A number of such recombinant yeasts expressing cancer or viral antigens are currently being evaluated in Phase I and II clinical trials as therapeutic vaccines. UG2V is developing a novel vaccine platform using yeasts as both antigen production and delivery system. This platform is based on two major concepts: (i) Antigen multimerization by fusion to the measles virus N protein and reconstitution of recombinant antigen-carrying RNPs and (ii) Expression and delivery of antigen-carrying RNPs in whole heat-inactivated P. pastoris yeasts.

The vaccine platform was first tested in a rodent malaria infection model with the CS antigen of Plasmodium berghei (PbCS), a species of Plasmodium parasite that infects mice. Electron microscopy and immunofluorescence analyses revealed rod-shaped RNPs of N-PbCS in the cytoplasm of P. pastoris yeasts. Immunization of mice with heat-inactivated N-PbCS yeasts induced strong immune responses against the PbCS antigen and provided significant clinical benefits following a severe infection.

Despite good results, the vaccine platform need to be further optimized. It was observed that a certain amount of PbCS detaches from the RNP, which may decrease the immunogenicity of the vaccine. The structurally disordered C-terminal region of the N protein allows it to recruit and interact with a variety of host-cell proteins by adapting its tertiary structure. On the other hand, structurally disordered proteins are also known to be hypersensitive to proteolysis. Thus, the Ntail may attract yeast proteases and increase susceptibility of the fusion protein to proteolysis.

My master thesis focuses on optimization of RNP. My goal was to identify the optimal length of the Ntail for antigen fusion and thus to minimize partial dissociation of the antigen from the RNP. To evaluate whether Ntail C-terminal region induced proteolytic dissociation of the antigen, I will generate N protein mutants truncated at three different sites and fused PbCS antigen to these ends. I will then evaluate expression levels of modified N-PbCS proteins in P. pastoris yeast, their ability to assemble into RNPs and dissociation of PbCS from RNP. This work will provide insights into structural properties of antigen-carrying RNPs and significantly contribute to optimize the system for further vaccine applications of this platform.

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INTRODUCTION

1. VACCINES AND VACCINE STRATEGIES

Vaccination represents one of the main and the most effective medical interventions to eliminate or reduce the incidence and mortality of infectious diseases and thus to improve people's health. To search the origin of modern vaccinology, we have to go back in the 430 BC in ancient Greece, when Thucydides noticed that severe contagious diseases were not contracted twice. However, the idea of using attenuated forms of pathogens to protect against a real infection came only in the XVIII-XIX centuries with the smallpox vaccine of Edward Jenner and the rabies vaccine of Louis Pasteur. These were the greatest scientists at the roots of vaccinology, who introduced the concept of using live-attenuated or inactivated pathogens to induce protective immunity against an infectious disease [1].

During the XX century, vaccines were widely used to take control over the spread of infectious diseases. Global vaccine campaigns allowed worldwide eradication of smallpox, one of the most severe human diseases, as well as almost total disappearance of polio and a drastic reduction of other infectious diseases such as measles, mumps and rubella. National immunization programs are working to reduce a great number of viral and bacterial infections that affect mainly children. The World Health Organization (WHO) reports that today vaccines prevent 2.5 million deaths per year [1]. The 194 Member States of the World Health Assembly in May 2012 endorsed the Global Vaccine Action Plan (GVAP) with the aim to prevent millions of deaths by 2020. The GVAP gathers together health and immunization experts and stakeholders from all over the world and it acts through reinforcing routine immunization in order to meet vaccination coverage targets to control vaccine-preventable diseases, as well as improving the efficacy and safety of vaccines already on market and developing new ones 1.

Vaccination is a preventive medical intervention, therefore, in contrast with disease treatment, it

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is intended for the healthy population. The main principle in vaccinology is to induce an immune response that mimics natural infection, therefore vaccines are designed to have similar immunogenic characteristics to those of a specific pathogen, but without actually causing disease. Moreover, vaccination is more advantageous from the socio-economic point of view than the pharmacological treatment of already infected individuals. For instance, immunizing children against pneumonia, diarrhea and meningitis will not only prevent more than 100 million cases of illness and avert 3.7 million deaths in young children over the next ten years, but will also represent a significant economic benefit for families and governments, allowing to save millions of dollars2.

Even though world-scale vaccination has led to eradication or reduction of most of the viral and bacterial infections (especially in developed countries), some major pathogens, such as Mycobacterium tuberculosis, don't have effective vaccines or no licensed vaccines at all (Human Immunodeficiency Virus (HIV), malaria Plasmodium parasite).

Vaccines can be gathered in two main groups: the first one includes live-attenuated pathogens, while in the second category are grouped a wide range of vaccines, like inactivated pathogens, inactivated toxins, carbohydrates cocktails, subunit vaccines and conjugate ones [2]. Since conventional methods, such as inactivation or live-attenuation, fail in development of vaccines against some diseases, novel approaches, technologies and vaccination strategies are required.

In the last two decades, progress in immunology, microbiology, molecular biology and genetics provided new technologies for the development of so-called “next-generation vaccines”. New vaccine technologies aim at improving such key areas as

i) exploration of various administration routes to stimulate systemic and/or mucosal immunity;

ii) improvement of vaccine adjuvants in order to stimulate both branches of adaptive immune responses;

iii) optimization of antigen presentation in order to enhance adaptive immune responses; iv) improvement of vaccine production and delivery techniques [4].

2 http://www.gavi.org/about/ghd/dov/

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Induction of a specific adaptive response involves first of all activation of innate immunity, which induces and coordinates further immune responses. Thus, targeting innate immunity at different levels influences consecutive adaptive immune responses and, therefore, the protection provided by vaccination [2]. For instance, the scientific argument for choosing one vaccination route rather than another depends on various factors, such as the distribution of antigen-presenting cells (APCs) at the injection site as well as the pathogen portal of entry [2]. Thus, alternative vaccination routes are being developed, such as via mucosal tissues or cutaneous tissues, and are eventually more advantageous than classic administration modes, such as intramuscular and subcutaneous.

Another way to modulate immune responses is through vaccine adjuvants. Adjuvants play a main role in modulation of quantity and quality of specific immunity by various mechanisms, improving vaccine efficacy and increasing the duration of protective responses. Nevertheless, the safety profiles of new-generation chemical adjuvants necessitate particular attention; due to their remarkable ability to elicit immune system reactions, local and systemic adverse reactions can potentially occur [1]. For instance the Adjuvant System 03 (AS03) adjuvant in the influenza vaccine was shown to cause narcolepsy in teenagers [5], and the adjuvant AS04 in the newly licensed Human Papilloma Virus (HPV) vaccine may be associated with autoimmunity.

A lot of studies are focused on efficient antigen delivery strategies that target different branches of adaptive immune responses in the absence of potentially dangerous adjuvants. Firstly, numerous replicating and non-replicating vectors have been used to induce both strong T-cell responses, including cytotoxic T-cell (CTL) response, as well as antibody (Ab) responses against delivered antigens. These include attenuated poxviruses, adenovirus, picornaviruses, flaviviruses, and many others, such as live-attenuated bacteria, usually Salmonella or Listeria spp, and heat-killed yeasts [1][2][6]. Secondly, various multimeric carriers, usually proteins of viral origin, are used to deliver antigens within subunit vaccines.

Bachmann and Jennings summed up in a review article [7] the importance of these physical criterions of carrier particles. Nanoparticles (ranging from 10nm to 200nm) with charged hydrophobic surface or receptor-interacting structures are most efficiently recognized and captured by APCs and thus are most efficient in inducing adaptive immune responses. Moreover, geometry and organization of surface molecules assume great importance; therefore, antigens are often organized into repetitive structures. Mostly importantly, repetitive surfaces allow a

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high-14

avidity interaction with B-cell receptors (BCRs) leading to strong B-cell activation [7]. Thus, current vaccine development is based on nanotechnological approaches that allow customizing physical parameters of vaccine particles such as size, shape, charge, porosity and hydrophobicity (FIGURE 1), and thus improving their immunogenic properties. This gives rise to subunit vaccines that are made of antigens that are assembled on delivery nanoparticles. Joining tiny pathogenic components into a unique larger structure composed of repetitive, high-density displayed epitopes is highly beneficial for vaccine immunogenicity. Comparing to a single antigen, delivery of multimerized antigen was shown to enhance its immunogenicity and elicit stronger innate immune responses [7]. Antigen multimerization is therefore a fundamental concept in vaccinology, and various nanotechnological approaches are being developed in order to create new particles for multimerizing antigens.

Nature Reviews | Immunology

Cylindrical fullerene Micelle

Liposome Oil-in-water emulsion

<5 nm Dendrimer

Virus-like particle Synthetic virus-like particle a e i f g h b c d

Dendrimer Spherical fullerene

Partition like small through the kidney

Size Nanoparticle Bioactivity

10–20 nm Polymer

Escape the vasculature, lymphatics like proteins

50–100 nm DNA polyplex Penetrate the mucosal membranes and the skin and are taken up into cells.

>150 nm Liposome Taken up mainly into phagocytic cells

Synthetic VLPs. The development of fully synthetic

VLPs using chemically synthesized lipopeptide mono-mers28_{to enhance nanoparticle assembly and to}

stabi-lize the three-dimensional conformational structure of protein antigens provides an intriguing vaccine strategy to stimulate neutralizing antibodies against HIV-1. In contrast to enveloped HIV-1 VLPs, which are limited by low Env density per virion4,29,30_{, or to the self-assembling}

peptide nanoparticles (SAPNs) described below, the synthetic approach does not require recombinant DNA technology or the expression and the purification of the monomer proteins from producer cells. Lipopeptide-based synthetic VLPs (20–30 nm in size) have been used to repetitively display a peptide-mimetic epitope derived from the V3-variable loop of gp120 (REF. 31)_{. This}

engineered epitope was designed by modelling the stable three-dimensional β-hairpin conformation that is formed after the binding of a broadly cross-neutralizing human monoclonal antibody to the gp120 antigen. This synthetic VLP also incorporated a universal T_H cell epitope32_(this

immunogenic peptide promiscuously binds to multiple different MHC class II molecules to improve the induc-tion of TH cells) and a tripalmitoyl-S-glyceryl cysteine

(Pam3Cys) lipid moiety, which induces TLR2 activation. Interestingly, the immunization of New Zealand white rabbits with these synthetic VLPs alone induced the pro-duction of neutralizing antibodies against the envelope proteins of multiple HIV-1 strains31_{. Thus, structural}

vac-cinology and other strategies for immunogen design29,33_,

in combination with repetitive antigen display using nanoparticle-based technologies as a vaccine platform, might enable the induction of responses against poorly accessible but conserved neutralizing epitopes rather than against more readily accessible immuno dominant non-neutralizing epitopes expressed on the native gp120 spike29_{. Responses against these poorly}

accessi-ble epitopes are required to overcome the extraordinary mutation rate and the diversification of HIV-1 during the course of infection and to prevent viral escape.

The design of synthetic nanoparticles to incorpo-rate lipid moieties for the conformational stabilization of protein antigens, such as the membrane-proximal external region of gp41, is also of great interest as certain

Figure 1 | Examples of nanotechnologies applied to immunoregulation. Nanotechnologies that can be applied to immunoregulation include nanoparticles (parts a–c), nanoemulsions (parts d–f) and virus-like particles (parts g–h). Nanoparticles include dendrimers which branch out (part a), carbon molecules known as spherical fullerenes (part b) and cylindrical carbon molecules known as cylindrical fullerenes (part c). Nanoemulsions incorporate immiscible components such as oil and water that might form amphiphilic molecules such as micelles (part d), liposomes with a lipid bilayer (part e) and oil-in-water emulsions (part f). Virus-like particles are self-assembled structures composed of one or more viral capsid proteins (part g), whereas synthetic virus-like particles are self-assembled from chemically synthesized components

h). Examples of the relationship between nanoparticle size and bioactivity are shown in (part i).

Box 1 | Defining nanotechnology

Definitions of the exact size range that the field of nanotechnology covers have been determined on the basis of size as well as function. The US National Nanotechnology Initiative aims to expedite the discovery, the development and the deployment of nanoscale technology for public benefit, and defines nanotechnology on the basis of size alone, using the range of 1 to 100 nm122_{. Other groups, including the US Food and Drug} Administration (FDA), define nanotechnology on the basis of scale and function, using the range of 1 to 1,000 nm, provided that the physical, chemical or biological effects of the material in question are attributable to its dimensions123_{. The European Medicines Agency} (EMA) initially defined nanotechnology in the range of 0.2 to 100 nm in size but has broadened the definition to less than 1,000 nm in size124_.

R E V I E W S

FIGURE 1

Examples of how nanotechnology applied to vaccinology [3]

Structures (a)-(h) are used to incorporate and multimerize antigens. They enhance the immunogenicity of the carried antigen through assembled delivery.

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One of the examples of such nanotechnology applied to immune regulation are Virus-like particles (VLPs) already widely used in vaccinology. VLPs used in vaccines can be both natural, comprising the natural viral subunits of the viral capsid (such as defective particles of Hepatitis B virus HBV or Hepatitis C virus HPV), or chemically synthesized pre-designed subunits (“self-assembling peptide nanoparticles”). VLPs are hollow as they don’t incorporate genetic material and are thus unable to replicate or to undergo genetic recombination, which makes them particularly safe. Due to their close resemblance to native viruses, VLPs are very effective in enhancing antigen uptake by immune system cells as well as in promoting dendritic cells (DCs) activation and maturation. A well-organized and repetitive external structure not only facilitates its effective capture by BCRs on B cells, but also allows antigen presentation on major histocompatibility complex (MHC) molecules class I and II (FIGURE 2). Through these mechanisms nanoparticles are able to induce long-lasting humoral and cellular immune responses [3, 8].

FIGURE 2

Immune system mechanisms involved in nanoparticles recognition, capture and processing [3]

Multimeric structures are efficiently recognized by APCs (macrophages and DCs) and B cells. Antigens are processed and presented in MHC class II molecules on

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cross-presentation.

For antigen delivery, VLPs can act like as a scaffold for multivalent surface presentation of antigens. Genetic or chemical engineering allows surface modification of these particles. An example of such chimeric VLPs that carry heterologous antigens on their surface is the RTS,S malaria vaccine composed of the circumsporosoite (CS) antigen fused on top of the HBV VLPs [9]. A wide range of VLPs are used for vaccine and vaccine development (HBV, HPV, Influenza, HCV, HIV, Ebola virus) and a long list of vaccine platforms are based on chimeric VLPs (TABLE 1).

TABLE 1

VLPs used for vaccine development [8]

Other viral components than VLPs have multimeric origin. The ribonucleoparticles (RNPs) based on the respiratory syncytial virus (RSV) nucleoprotein were shown to be highly immunogenic because of the presence of a well-organized and repeated structure [10]. These recombinant RNPs were able to elicit a strong immune responses resulting from uptake by DCs and

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development of specific CD8+

T cells as well as local and systemic Abs responses.

The Viral Genomics and Vaccination Unit (G2VU) of Institut Pasteur (my host laboratory) recently developed a novel nanoparticle for antigen delivery. It is based on the measles virus (MV) nucleoprotein (N), which forms the virus nucleocapsid. It is a multimeric protein to which antigens of choice are attached [11]. The following chapter will describe the advantages of using the MV protein N as a novel nanoparticle for antigen delivery.

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2. MEASLES VIRUS NUCLEOPROTEIN FOR ANTIGEN MULTIMERIZATION

2.1. THE MEASLES VIRUS

2.1.1. EPIDEMIOLOGY, TRASMISSION, PATHOLOGY

Measles is one of the most severe directly-transmitted infectious diseases of mankind. This highly contagious disease still remains one of the major causes of death worldwide, especially among children, despite the existence of a safe effective vaccine [12]. It is caused by the measles virus of the Mononegavirales order, Paramyxoviridae family, that includes several other human pathogens such as mumps and parainfluenza.

The availability of measles vaccine, developed and widely used since the 1960's [13], led to an important decline of the global mortality due to this severe pathology (decreasing by 78% in the recent years) 3. Nevertheless, 45 million cases and 800,000 deaths are still counted among children of all ages and regularly outbreaks occur due to insufficient vaccine uptake [14][15]. This severe disease still remains one of the main causes of preventable deaths worldwide. The Measles and Rubella Initiative (M&R Initiative), in collaboration with WHO, UNICEF and other health organizations, took the lead on that issue and launched a new Global Measles and Rubella Strategic Plan. The plan set a number of goals to achieve by 2020 to progress toward measles control; it resolves to expand routine immunization and vaccine campaigns. The challenge is the measles elimination at least from five WHO regions, following the example of the WHO Region of Americas that completely eliminated measles transmission in 20024 [16]. Unfortunately, achievement of this goal has to face problems such as high population density, increasing international travel or weakness in public health systems. Armed conflicts and natural disasters can make the situation even worse, causing population spread and interruption of health services. On the other hand, the major obstacle to improving measles control and to maintaining high levels of immunity in both developed and developing countries is the misperception of measles as

3 http://www.who.int/immunization/topics/measles/en/ 4 http://www.who.int/mediacentre/factsheets/fs286/en/

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a real threat aggravated by the fear of scientifically contested risks associated with the measles vaccine 5.

MV is spread by airborne transmission, and humans are its only natural reservoir. Due to high contagiousness, measles persists in the human population simply through sequential infections. Furthermore MV is contagious some days before and after the onset of the rash, and can survive up to two hours in the environmental media. In addition, individuals co-infected with HIV-1 and lacking an efficient cellular immune response may not develop the typical measles rash responsible of this symptom 6 [16].

MV enters into a new susceptible host through droplets derived from coughing and sneezing. Once in the epithelial cells of the upper respiratory tract - the entry gate - it replicates and spreads into the host tissues. It reaches the local lymphatic tissues where it replicates in lymphocytes, monocytes and macrophages. Thus viraemia occurs and it leads to the dissemination of measles in several organs such as skin, liver, kidney. There the virus keeps replicating and infects epithelial and endothelial cells [12]. Clinical manifestations of the measles disease usually begin 10-14 days after infection with a prodrome characterized by high fever, running nose, cough, red bloodshot eyes and tiny white spots inside the oral cavity. These symptoms intensify up to the onset of the typical rash that lasts for several days and then fades away. The characteristic erythema stars from the face and upper neck and propagates on the whole body. Recovery starts after appearance of the rash, but complications occur in 10%-40% of cases and they are linked to age, malnutrition (especially vitamin-A deficiency), and other causes when the immune system is compromised, such as HIV-1 infection. Since MV spreads all over the host organism, complications can involve every organ (blindness, mouth ulcers, severe diarrhea, ear infection, pneumonia – responsible for the most measles-related deaths), however deadliest ones are due to the impact on the central nervous system (different types of encephalitis).

5 http://www.who.int/immunization/newsroom/Measles_Rubella_StrategicPlan_2012_2020.pdf?ua=1 6 http://whqlibdoc.who.int/publications/2009/9789241597555_eng.pdf?ua=1

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2.1.2. MEASLES VIRUS STRUCTURE AND LIFE CYCLE

Measles is a spherical virus enclosed in a lipid envelope of host cell origin (FIGURE 3). The RNA genome of the virus is non-segmented, single-stranded, negative-sense; it is enclosed in a helical nucleocapsid and encodes for eight proteins: six structural proteins and two non-structural ones (V and C proteins, pathogenicity factors) [12]. Among the non-structural proteins, the nucleoprotein N encapsidates the RNA genome forming a helical nucleocapsid, and associats with the phosphoprotein (P) and the large protein (L) for transcription and replication. Nucleocapsid is enclosed in turn into an envelope composed of the lipid bilayer in which haemagglutinin protein (H) and fusion protein (F) are nestled. The inner side of the envelope is contoured by the matrix protein (M).

The two transmembrane glycoproteins H and F protrude from the viral surface and are responsible for the pathogenic and immunogenic properties of measles virus. While H protein binds the host cellular receptors CD46 and CD150, F protein mediates the entry into host cell. CD150 is expressed on activated T and B lymphocytes and CD46 is ubiquitously expressed on all nucleated cells, which explains the systematic nature of measles infection [16].

a b c Fusion (F) Haemagglutinin (H) Nucleocapsid (N) Matrix (M) Large protein (L) Phosphoprotein (P) Lipid bilayer N P/C/V M F H L RNA H,F,N proteins Capsid proteins Nucleus Replication Replication vcRNA (+) vcRNA (–) mRNAs mRNA synthesis

Fusion

Translation

and modification Progeny RNA

genomes (–) Budding

Lipid membrane CD46

CD150

Recall antigen

An antigen to which a host has previously been exposed.

response to MV infection and secrete cytokines capable of directing humoral and cellular immune responses. Plasma cytokine profiles show increased levels of IFN-γ during the acute phase, followed by a shift to high levels of interleukin (IL)-4 and IL-10 during convalescence8_.

The initial predominant T-helper-1 (Th1)-response is essential for viral clearance, and the later Th2 response promotes the development of protective MV-specific antibodies.

The immune responses induced by MV infection are paradoxically associated with depressed responses to non-MV antigens, and this effect continues for several

weeks to months after resolution of the acute illness. Following MV infection, delayed-type hypersensitivity (DTH) responses to recall antigens, such as tuberculin, are suppressed9_{and cellular and humoral responses to}

new antigens are impaired10_{. This MV-induced immune}

suppression renders individuals more susceptible to secondary bacterial and viral infections that can cause pneumonia and diarrhoea, and is responsible for much of the measles-related morbidity and mortality11,12_.

Pneumonia, the most common fatal complication of measles, occurs in 56–86% of measles-related deaths13_.

Abnormalities of both the innate and adaptive Box 1 | Measles virus – the basics

The measles virus (MV) is a spherical, non-segmented, single-stranded, negative sense RNA virus (see the figure part a) and is a member of the Morbillivirus genus in the family of Paramyxoviridae. Other members of the Morbillivirus genus that are not pathogenic to humans include rinderpest virus and canine distemper virus. MV is killed by ultraviolet light and heat. Attenuated measles vaccine viruses retain these characteristics, necessitating a cold chain for the transport and storage of measles vaccines.

The MV RNA genome comprises approximately 16,000 nucleotides (see the figure part b) and is enclosed in a lipid-containing envelope that is derived from the host cell. The genome encodes eight proteins, two of which (V and C) are non-structural proteins and are alternatively translated from the RNA, or an edited RNA, coding for the phosphoprotein (P).Of the six structural proteins, P, large protein (L) and nucleoprotein (N) form the nucleocapsid that encloses the viral RNA. The haemagglutinin protein (H), fusion protein (F) and matrix protein (M), together with lipids from the host cell membrane, form the viral envelope.

The H protein interacts with F to mediate attachment and fusion of the viral envelope with the host cell membrane, enabling viral entry into the cell80_{(see the figure part}_c_{). The primary function of the H protein is to bind to the host cellular}

receptors for MV. The two identified receptors are CD46 and CD150 (also known as SLAM). CD46 is a complement regulatory molecule expressed on all nucleated cells in humans. SLAM (signalling lymphocyte activation molecule) is expressed on activated T and B lymphocytes and antigen-presenting cells. The binding sites on H for these receptors overlap and strains of MV differ in the efficiency with which each receptor is used. Wild-type MV binds to cells primarily through the cellular receptor SLAM, whereas most vaccine strains bind to CD46, as well as to SLAM81_{. Other unidentified}

receptors for MV probably exist on human endothelial and epithelial cells82_.

Remaining MV proteins are involved in viral replication. The P protein regulates transcription, replication and the efficiency with which the nucleoprotein assembles into nucleocapsids83_{. The M protein links ribonucleoproteins with}

envelope proteins during virion assembly. The functions of the V and C proteins have not been clearly defined, but both proteins seem to contribute to the virulence of MV by regulating transcription and sensitivity to the antiviral effects of IFNα/β84,85_{. Part}_a_{of the figure is modified with permission from}_{REF. 86}_{© (2002) Cambridge press.}

R E V I E W S

FIGURE 3

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MV life cycle begins with the attachment of its glycolipids or proteins to the host membrane by means of it H protein. Then, the F protein is recruited and after it undergoes conformational change, the virus enters the host cell through fusion of the viral envelope and the cytoplasmic membrane. The RNA viral genome is released in the cytoplasm, where transcription and replication occur through the action of the viral RNA-dependent RNA polymerase (RdRp), made of the L and P proteins. As soon as the new viral genomes start to be synthesized, they associate simultaneously to the N protein to form the nucleocapsid that leaves the host cell enveloped in a part of its cellular membrane [16]. Transcription and replication are regulated by the N protein that exists in two different forms: a monomeric form, important for the encapsidation of the virus genome during replication, and an assembled one which interacts with the RdRp allowing transcription [17].

2.1.3. IMMUNOLOGICAL RESPONSES TO MEASLES VIRUS

MV infection leads to a strong activation of host immune system. A strong specific response is needed for viral clearance, and a life-long protective immunological memory (both humoral and cellular) is established after clinical recovery.

In the first stage of infection, the virus replication is countered exclusively through innate immunity and, in particular, through natural killer (NK) cells activation and production of a great amount of antiviral signaling proteins, such as interferon 7 (Hahm 2009). Several days later with the activation of the adaptive immune system, MV-specific humoral and cellular responses arise. Indeed, measles virus-specific antibodies are detectable in the sample only once rash appeared. Antibodies against nucleoprotein (N) are the most abundant and the first produced, nevertheless neutralizing antibodies that provide protection against future re-infection are directed against measles surface proteins, especially against H [18] 7

. Cellular immunity responses are directed mainly by CD8+

T cells, but CD4+

T cells are also activated in order to enhance specific lymphocytes B cells for antibody production.

Remarkably, despite being an RNA virus, the genome of the MV has shown to be impressively

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stable [16]. Surface proteins remain unchanged and this high degree of conservation ensures that once infected in the life, an individual developed enduring and definitive immunity. From a practical point of view, the great antigenic stability was a major precondition for development of an ever-efficient vaccine. Indeed, the very first measles vaccine, developed from a single measles virus strain in the 1960's [13], is still nowadays protective worldwide [16] 7

.

2.2.MEASLES IN VACCINOLOGY

Measles live-attenuated vaccine is one of the most efficient (95%protection), safe and life-long protective vaccines in use nowadays. The vaccine has also shown to be very stable from the point of view of reversion to pathogenicity [19]. Several MV vaccines are available, both, as monovalent vaccines or in combination with other vaccines (mainly rubella and mumps, pediatric MMR vaccine). The first attenuated measles strain was isolated in 1954 by Enders and Peebles [20]. Live-attenuated viruses for vaccine production were obtained through subsequent passages in cell cultures; in this way some of the most well known vaccines strains were produced, including the Schwarz, the Edmonston-Zagreb, the Moraten strains [12, 21]. MV vaccines are usually administered via subcutaneous injection, but can also be also injected intramuscularly. The vaccine induces both antibodies and cellular immune responses comparable to those occurring in natural infection [12, 22]

MV vaccine presents a large number of advantages including great genome stability, tolerability, remarkable efficacy in inducing both branches of immune system and a life-long immunity. Furthermore, it has a large capacity of insertion. MV vaccine is therefore evaluated as an attractive vaccination vector to immunize simultaneously against several infectious diseases including measles [23], [19], [15]. Studies on recombinant MV expressing heterologous antigens proved its capacity to activate immune responses against heterologous antigen. Notably, pre-existing antibodies against MV do not influence the capacity of the recombinant vaccine to induce antibodies against the delivered antigen [23], [19]. Moreover, the MV vaccine was recently evaluated in a therapeutic approach to treat cancer by delivering cancer antigens and inducing cytotoxic responses against cancer cells [24].

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2.3. MEASLES VIRUS NUCLEOPROTEIN

2.3.1. STRUCTURE AND FUNCTION

The main role of the MV nucleoprotein (N) is encapsidation of the non-segmented, negative-sense, single stranded RNA genome. The very stable association between N and RNA genome creates a “herringbone-like” structure of the nucleocapsid, which is highly resistant both to dissociation by salt and nuclease digestion [17][25]. Multiple copies of N are needed to package the viral RNA; a precise number of RNA bases, different among Mononegavirales, interact with N monomers. In the Paramyxoviridae family, to which MV belongs, each nucleoprotein binds to 6 nucleotides resulting in a flexible 1µm-long and 18 nm-wide structure, with a central channel of 4-5 nm in diameter for the RNA [17][26] (FIGURE 4). Importantly, it is the RNA-N complex and not the naked RNA that is used as substrate for both viral genome replication and transcription. This fact can be explained by the impossibility for the viral polymerase L to bind directly to naked RNA genome, since L protein requires interaction with phosphoprotein P to perform its function, thus forming the whole virus-encoded RdRp [25][27][17][26]. The phosphoprotein plays a cardinal role by tethering, on the one hand, the L protein onto the nucleocapsid template, and on the other hand, by binding the C-terminal extremity of N protein (FIGURE 5). Both transcription and replication initiate at the 3'-end of the nucleocapsid RNA. Replication leads to the sequential synthesis of measles virus in their specific order: N, P/V/C, M, F, H, L. As the nascent genomic RNA chain is formed it is immediately encapsidate in the complex formed by the interaction between N and P proteins [25]. The replication promoter is constituted by the juxtaposition of successive helical turns of the nucleocapsid generating the functional unit [17] (FIGURE 4).

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Hsp72 on genome replication versus transcription remains to be shown, with template changes unique to a replicase versus transcriptase being a primary candidate. The latter could involve unique nucleocapsid ultrastructural morphologies, with Hsp72-dependent morphologies being well-documented for

canine distemper virus (Oglesbee et al., 1989, 1990), a

Morbillivirus member that is closely related to MeV.

As for the functional role of Hsp72 in the context of MeV infection, it has been proposed that the elevation in the Hsp72 levels in response to the infection could contribute to virus

clearance (Carsillo et al., 2004; Oglesbee et al., 2002). Indeed,

the stimulation of viral transcription and replication by Hsp72 is also associated to cytopathic effects leading to apoptosis and release of viral proteins in the extracellular compartment (Oglesbee et al., 1993; Vasconcelos et al., 1998a, 1998b). These would stimulate the adaptative immune response,

thereby leading to virus clearance (see Gerlier et al., 2005;

Laine et al., 2005and references therein cited).

Additional binding of cellular partners by NTAIL has the

potential to influence both innate and adaptive immunity. NTAIL

is involved in the interaction with the interferon regulator factor 3 (IRF-3). This interaction triggers the phosphorylation-dependent activation of IRF-3 and its consequent nuclear

import (tenOever et al., 2002). Thus, interaction between NTAIL

and IRF-3 might ultimately lead to stimulation of interferon production. Finally, after apoptosis of infected cells, the viral nucleocapsid is released in the extracellular compartment where it becomes available to cell surface receptors. While

NCORE specifically interacts with FggRII (Laine et al., 2005),

NTAILinteracts with a yet uncharacterized receptor (referred to

as Nucleoprotein Receptor, NR). This latter is expressed at the surface of dendritic cells of lymphoid origin (both normal and

tumoral) (Laine et al., 2003), and of T and B lymphocytes

(Laine et al., 2005). Flow cytofluorimetry studies carried out on

truncated forms of NTAIL allowed the identification of the

NTAILregion responsible for the interaction with NR (Box1, aa

401 – 420) (Laine et al., 2005). The NTAIL– NR interaction

triggers an arrest in the G0/G1 phase of cell cycle whereas the

NCORE– FggRII interaction triggers apoptosis (Laine et al.,

2005). Both mechanisms have the potential to contribute to

Fig. 7. (A) Negative stain electron micrographs of N and NCORE. The bar corresponds to 100 nm. Rings and herringbone structures are indicated by different arrows. (B) Cryo-electron microscopy reconstructions of MeV nucleocapsid (left) and schematic representation of the nucleocapsid (right) highlighting the structure of the replication promoter composed of two discontinuous units juxtaposed on successive helical turns (see regions wrapped by the red and blue N monomers). Background: electron micrographs of MeV nucleocapsid (courtesy of D. Bhella, MRC, Glasgow, Scotland). Reprinted from Virologie, 2005; 9(5). Copyright with permission from John Libbey Eurotext Ltd.

J.-M. Bourhis et al. / Virology 344 (2006) 94 – 110 103

FIGURE 4

Measles virus nucleocapsid. On the left, side view of the nucleocapsid herringbone structure. On the right, dissection of nucleocapsid with the replication promoter (in blue and red) [17]

FIGURE 5

Schematic representation of the nucleocapsid. Different interaction among its components

[17].

The regulatory mechanism by which MV controls the balance between transcription and replication is not yet fully understood, but it is suggested that the RdRp mode of operation is controlled by the spatial relationship between promoter elements on the viral genome, affected in its turn by the different conformations of N. Indeed, by binding viral cofactors or host cell proteins, N can be induced to assume a specific folding which influences the form of the whole

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nucleocapsid [26]. Two forms of N can be found in infected cells during a viral infection: a soluble monomeric form (referred to as N°), and an assembled form (referred to as NNUC

) [28]. By its ability to naturally assemble around RNA molecules, N protein in its monomeric form is responsible for encapsidation of newly generated RNA chains during replication. However, in the absence of the viral RNA, N° is able to self-assemble into nucleocapsid-like particles. Therefore a regulatory mechanism exists that prevents illegitimate self-assembly of N: interaction between P protein to N° generates a soluble N°-P complex that avoids self-assembly of N and also operates as template for the polymerase to begin genomic encapsidation. Hence, assembled form of N interacts with P and with the RdRp during transcription and replication [17][28][25][29].

2.3.2.N

_CORE

AND N

_TAIL

At least three functional sites are implicated in the functions of the N protein: i) the binding site for P protein, to form N°-P complex used to encapsidate newly-generated viral genomes during replication; ii) one or more sites for N-N interaction, essential for the self-assemblage of nucleoprotein around viral genome; iii) a site to interact with RNA for nucleocapsid initiation and its elongation [25].

N is composed of 525 amino acids and can be divided in two structural domains: an N-terminal domain, known as Ncore (amino acids 1–400) and a C-terminal domain, referred to as Ntail (amino acids 401–525) [25][30][29] (FIGURE 6). From the structural point of view, N protein appears as a globular body formed by Ncore from which Ntail protrudes toward the exterior of the viral nucleocapsid (FIGURE 7).

FIGURE 6

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FIGURE 7

Ntail overhangs from the RNP surface while the globular Ncore assembles to create the scaffold for the whole structure [31]

The N-terminal domain, Ncore, is a globular domain necessary for the self-assembly of N and the stability of the RNP. This region is characterized by a well-conserved sequence and its resistance to proteolysis. Deletion and mutational studies have shown the presence of two main regions within Ncore, which are necessary for the self-assembly and RNA binding [28][17][32].

The main region involved in the N-N interaction and in RNA binding is the region composed by the residues 258-357 (FIGURE 6), also called the central conserved region (CCR). It consists of highly conserved mainly hydrophobic residues. The region between amino acids 189 and 239 forms another well-conserved region folded as two α-helices, that turned out to be critical for self-assembly as well [28] [29]. As proof of concept, nucleoprotein mutants in which some hydrophobic residues were replaced with polar ones (i.e. double-mutant NQD, in which residues S(228)L(229) are replaced with QD) showed significantly hampered assembly into RNPs [29]. These studies helped to better understand the nature of bonds in the self-assembly. In particular it was shown that N-N interaction doesn't depend on ionic nature, but rather on hydrophobic bonds. The C-terminal Ntail domain was shown to be important for the interaction with P in order to act as template for RNA synthesis [28] [29] [33]. Electron microscopy (EM) showed the great influence exercised by Ntail in affecting the nucleocapsid conformation. Nucleocapsid-like

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structures of full-length N are herringbone-like particles ranging from 20 to 80 nm with an abundant presence of short nucleocapsid rings – a clear sign of intrinsic fragility (FIGURE 8, left). On the other hand, the truncated form of N devoid of its Ntail domain produces rigid helices of up to 400 nm long (FIGURE 8, right) [17]. This fact leads to a conclusion that removal of the Ntail domain stabilizes the helical nucleocapsid. Thus, the presence of Ntail relaxes the RNP structure and allows the polymerase to access the RNA [28].

Three regions can be founded within Ntail: Box1, Box2 and Box3 (FIGURE 6) [30]. Box2 was shown to possess an α-helical molecular recognition element (α-MoRE, amino acids 485-502). The α-MoRE keeps a dynamic equilibrium between a totally unfolded state and partially a-helical secondary conformation. This interconverting conformation enables the α-MoRE to efficiently undergo induced folding in a regular secondary structure once bound the molecular partners (disorder-to-order transition) [27]. Ntail binds to the C-terminal X domain of the phosphoprotein, called XD; once the binding occurs, the MoRE is induced to assume an a-helical conformation and transient non-specific interactions reduce the conformational flexibility. The great instability degree of this region also plays a central role in the progression of the polymerase along its template, due to consecutive cycles of α-MoRE-XD binding and releasing [34][31].

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FIGURE 8

Nucleoprotein in the absence of Ntail develops longer and rigid RNP structures [17] Left – RNPs of full-length N, right – RNPs of Ncore. Bar corresponds to100nm.

The Ntail is a flexible non-globular polypeptide chain with low compactness that protrudes from the nucleocapsid and is not visible by the electron microscopy. Contrarily to the Ncore, Ntail is highly variable among Paramyxoviridae members and hypersensitive to proteolysis [28]. These features characterize the Intrinsically Disordered Proteins (IDPs) or Intrinsically Unstructured Proteins IUPs. IDPs are fully functional proteins that carry out their function lacking any well-defined folded secondary and tertiary structure. The amino acids sequences of such proteins tend to be quite simple in composition, often depleted of “order-promoting amino acids” (W, C, F, I, Y, V, L, N), but rich in “disorder-promoting amino acids” (A, R, G, Q, S, P, E, K) frequently found on the surface of globular proteins [34][32][17][28]. Several quantitative and qualitative methods are available to characterize disordered proteins, like the PONDR (Predictor Of Naturally Disordered Regions), the hydrophobicity/net charge method, or the hydrophobic cluster analysis (HCA) [32]. Such analysis of the sequence and spectroscopic/hydrodynamic properties proved the unstructured nature of Ntail within the nucleocapsid. Within the IDPs family of proteins, Ntail belongs to the “premolten globule” subfamily. Proteins of this group

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neither have a totally random coil state, nor a well-defined globular structure, but are characterized by the presence of a conformational intermediate state between the two. Promolten globules, although lacking a well-defined secondary and tertiary structure, conserve a certain degree of compactness made of fluctuating secondary and tertiary structure regions. The maintenance of the unstructured state is kept through a dynamic process of breaking and reforming interaction and this action may facilitate the folding process induced by the binding partner, also called “induced folding” [17][30]. Hence, revisiting one of the main molecular biological dogmas is needed: the function of a protein and its three-dimensional structure are not necessarily strictly linked, accompanied with the question “folding for binding or binding for folding”? [35].

2.3.3.ADVANTAGES OF BEING A DISORDERED PROTEIN

In order to fulfill all its functions, Ntail possesses some specific characteristics. It protrudes from the nucleocapsid surface toward the “external” environment. Having a great “capture radius” [35] it can come in contact with a greater number of molecules, comparing to folded proteins. In addition, its flexible nature and the lack of a unique defined secondary structure allow the N protein to interact with different biological partners. Indeed, flexible structures have an advantage over the rigid ones, since high level of plasticity allows efficient interaction with structurally distinct partners. Custom folding of structurally disordered proteins allows binding partners both with high specificity and weak affinity, overcoming steric restrictions, which are a problem in interaction between rigid partners [17][28][32][26]. To better understand these advantages, Amedeo Caflish made an interesting metaphor: “Most children (and some research scientists, too) do not like to keep order in their rooms (desks), not only because it is tedious, but also because they can visually recognize and reach the toys they want to play with (papers and documents to read) more easily” [35]. As a result, “binding for folding” proteins is that they contribute to a more efficient control of the molecular pathways in which they are involved. The presence of the flexible Ntail allows the nucleocapsid to interact with a wide range of distinct partners: the phosphoprotein (both as N°-P and NNUC

-P),polymerase RpRd, matrix protein M, and various cellular factors (cytoskeleton components, interferon regulatory factor 3, etc) [28].

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Moreover, once released from the infected cells, the nucleocapsid can interact by means of Ntail with extracellular molecules such as FcγRII, Immunoglobulin G (IgG) receptors and heat-shock protein (Hsp72, which modulates the level of viral RNA synthesis) [17].

The involvement of Ntail in such fundamental viral processes like RNA transcription, replication and viral assembly is the proof of the great importance of structural disorder proteins. Interestingly, recent studies suggest that eukaryotes have more unstructured proteins that bacteria or archea [36]; therefore structural plasticity may be related to history and evolution.

2.3.4. RECOMBINANT MEASLES VIRUS NUCLEOPROTIN EXPRESSED

YEAST

In the absence of viral RNA and other viral proteins, nucleoprotein N has a peculiar capacity to self-assemble on cellular RNA molecules yielding nucleocapsid-like superstructures in different expression systems such as mammalian [17], bacterial [26] or yeast [37] cells. Studies of Slibinskas et al., 2004 performed N expression in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae [37]. The results of their work demonstrated that in both the yeast genera RNPs were formed (FIGURE 9), however P. pastoris produced a higher amount of N than S. cerevisiae (29% vs 18% of total soluble protein). These nanoparticles – ribonucleoparticles RNPs – highly resemble to those constituted in MV-infected cells.

FIGURE 9

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The flexibility and structural disorder of the Ntail domain led to its use as an anchor for attaching antigens and the development of the new sub-unit vaccine platform by the Viral Genomic and Vaccination Unit [11]. The team chose to express such antigen-carrying RNPs in

P. pastoris yeast and to use heat-killed yeast for antigen delivery.

3.YEAST AS ANTIGEN PRODUCTION AND DELIVERY SYSTEM

3.1 YEAST IN RESEARCH AND PHARMACEUTICAL INDUSTRY

Yeast is used in laboratories all around the world for studying biological systems since decades. Yeast is a widely accepted model organism for studying complex processes that occur in cells of higher eukaryotes. First of all, yeast is unicellular, which makes it particularly suitable for studying physiological and biochemical processes that are more difficult to study in highly evolved organisms. Furthermore, yeast is a eukaryote, so knowledge obtained from studying intracellular processes in yeast can be transferred, more or less widely, to other eukaryotic organisms due to the high conservation of genome sequences. Thus, studies of intracellular processes in yeast, such as DNA replication, recombination, cell cycle and metabolic pathways, have contributed to the knowledge of eukaryote biology, including the human one.

The yeast is also an organism that is easy to manipulate and breed even in poorly equipped laboratories. Most-widely used laboratory yeasts are not pathogenic and can be safely handled without special precautions. Yeasts are small in size (3–4 µm in average) (FIGURE 10) and have a relatively short division cycle providing a rapid culture growth (generation time about 2 hours). Genetically well characterized, yeast can be easily manipulated for gene deletion/insertion. As a further advantage, a lot of genetic tool kits ready to use are available on the market, and several classic procedures for genetic modification were developed (for example, “Invitrogen” Pichia pastoris and Saccharomyces cerevisiae expression kits). Therefore, gene

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eukaryotic genes and proteins within intracellular pathways.

FIGURE 10 Appearance of yeasts [38]

Thanks to such advancements in yeast biotechnology, as well as large-scale production at low cost, yeasts became one of the most important and widely used systems for heterologous protein expression. Several yeast genera, such as Saccharomyces, Pichia, Kluyveromyces and others, are widely used in both research and pharmaceutical industries for protein expression and purification [39]. Several proteins destined for medical use are produced in yeasts, for example hepatitis B surface antigen particles (HBsAg), recombinant interferon, insulin, human serum albumin (HSA) [40], human IgG antibodies [41] and others.

Among different yeast species, methylotrophic ones are emerging as an attractive host for heterologous gene expression, offering methanol-inducible controlled protein expression. This type of yeast is able to use methanol as the only carbon source by means of the methanol utilization pathway (Figure 11).

Structural optimization of the novel subunit vaccine platform based on multimeric nanoparticles formed by the Measles Virus Nucleoprotein in yeast

University of Pisa

Department of Biology

Curriculum Biology Applied to Biomedicine

Master thesis

Structural optimization of the novel subunit vaccine

platform based on multimeric nanoparticles formed by

the measles virus nucleoprotein in yeast

Candidate:

LUISA MANDORLI

Supervisors

Dr. Daria Jacob

Dr. Frederic Tangy

Dr. Monica Sala

University supervisor

Dr. Mauro Pistello

University of Pisa

TABLE OF CONTENTS

ABBREVIATIONS AND ACRONYMS

ACKNOWLEDGMENTS

SUMMARY

INTRODUCTION

1. VACCINES AND VACCINE STRATEGIES

R E V I E W S

2. MEASLES VIRUS NUCLEOPROTEIN FOR ANTIGEN MULTIMERIZATION

2.1. THE MEASLES VIRUS

2.1.1. EPIDEMIOLOGY, TRASMISSION, PATHOLOGY

2.1.2. MEASLES VIRUS STRUCTURE AND LIFE CYCLE

R E V I E W S

2.1.3. IMMUNOLOGICAL RESPONSES TO MEASLES VIRUS

2.2.MEASLES IN VACCINOLOGY

2.3. MEASLES VIRUS NUCLEOPROTEIN

2.3.1. STRUCTURE AND FUNCTION

2.3.2.N

AND N

2.3.3.ADVANTAGES OF BEING A DISORDERED PROTEIN

2.3.4. RECOMBINANT MEASLES VIRUS NUCLEOPROTIN EXPRESSED

YEAST

3.YEAST AS ANTIGEN PRODUCTION AND DELIVERY SYSTEM

3.1 YEAST IN RESEARCH AND PHARMACEUTICAL INDUSTRY