The present invention relates to an anti-ZrfC antibody and the ZrfC protein of Aspergillus and the uses thereof in immunotherapy against fungal infections such as invasive pulmonary aspergillosis.

In particular, the invention relates to an anti-ZrfC antibody to be used in the treatment of infections caused by fungi that express ZrfC. Furthermore, the invention concerns the use of the ZrfC protein itself as a vaccine against fungal infections, such as fungal infections by the genus Aspergillus, e.g. invasive pulmonary aspergillosis.

It is known that invasive aspergillosis is an infection produced by the fungus Aspergillus in patients with a severe impairment of the immune system, which can manifest itself in leukaemia, AIDS or bone marrow transplants.

Aspergillosis clinically manifests itself as a pulmonary pathology, with a rapid course, and it is often fatal if the fungal hyphae disseminate through the bloodstream to other organs and tissues.

In particular, Aspergillus fumigatus is a saprophytic filamentous fungus that invades the lungs of immunocompromised individuals, causing invasive pulmonary aspergillosis (IPA) [23]. The infection cycle of Aspergillus begins with the production of conidia which scatter in the air and are easily inhaled [24] In a healthy individual, conidia are efficiently removed by epithelial cells or alveolar macrophages; in greatly immunocompromised subjects, by contrast, the conidia germinate and a fungal invasion of blood vessels occurs, with a possible dissemination to other organs [25].

The innate immune system plays a primary role of defence and at the same time an anti-inflammatory function which slows down and limits the infiltration of leukocytes in the inflamed site, reducing their recruitment and thus regulating the inflammatory response [26]. For example, TNFa regulates the infiltration of neutrophils and the secretion of a series of chemokines [27] Furthermore, it has been demonstrated that some proteins produced by the innate immune system selectively bind microbial agents (such as, for example, conidia of Aspergillus fumigatus and Pseudomonas aeruginosa) and activate different effector pathways in response to infection by pathogenic agents. Some proteins, such as, for example, PTX3, are capable of inhibiting infection and increasing survival in rat models of invasive pulmonary aspergillosis [28]. Immunotherapy through the use of proteins normally produced by the immune system thus represents a concrete strategy.

The current therapy is pharmacological with the use of specific antifungals, but notwithstanding the progress in therapy, the invasive forms of aspergillosis are often associated with significant morbidity and mortality. The molecules presently available (triazoles), though effective, have a poor safety profile and are often a cause of many side effects, including severe ones. In particular, three types of drugs are presently used for the treatment of this infection: triazoles, polyenes and echinocandins. Given the strong similarity between the enzymatic systems of the fungus and host, the use of these drugs is very often associated with major side effects (nephro- and hepatotoxic) which impose the suspension of the treatment. Furthermore, the identification of multiple- triazole-resistant strains from agricultural areas of southern Italy, selected as a result of the selective pressure of the fungicides used in farming, is having significant repercussions on the management of patients with pathologies caused by Aspergillus or invasive fungal infections (IFIs) in general [1 , 2]

Notwithstanding antifungal therapies, the mortality due to invasive pulmonary aspergillosis remains high, between 25 and 80% of patients with a certain diagnosis [3].

Therefore, the generation of new therapeutic agents capable of combating and/or preventing invasive infections caused by Aspergillus in individuals who are intolerant or resistant to the traditional pharmacological treatment represents an important medical and health need.

It is known that monoclonal antibodies are widely used in new target-specific therapies in oncology, but their use also in infectious diseases is expanding. Numerous monoclonal antibodies are being developed for various infectious diseases, but three have been authorised to date: palivizumab for the prevention of respiratory syncytial virus in children at high risk, raxibacumab and obiltoxaximab for prophylaxis and treatment of anthrax [22] The production of monoclonal antibodies can be very useful in the treatment of infectious diseases of a fungal nature.

In the light of the above, it is apparent the need to have new therapies against fungal infections, such as invasive fungal infections (IFIs), in particular those caused by Aspergillus, which overcome the disadvantages of the known therapies.

As mentioned above, it is known that A. fumigatus is a pathogen capable of growing and invading lung tissue and causing invasive pulmonary aspergillosis [3].

Zinc transporters are of great relevance in the virulence of Aspergillus fumigatus. Zinc is essential for many biochemical processes, for the growth of fungi and the propagation thereof in the tissue of the host [29] Following the infection of an individual, Aspergillus finds itself in a hostile environment, with a limited presence of zinc. The ability of the fungus to retrieve zinc from the pulmonary environment is tied to the presence of specific transporters for this metal. In eukaryotic cells, the transport of zinc inside them is entrusted to proteins belonging to the Zrt-, Irt-like protein (ZIP) family [30]. Six ZIP transporters of the eight encoded by the genome of Aspergillus fumigatus are localised at the plasma membrane level (zrfA, zrfB, zrfC, zrfD, zrfE and zrfH) [31].

The adaptation of the fungus to grow in the lungs is possible through the cooperation of transcription factors that are activated in response to the change in the pH of the external environment [32] Lung tissue is slightly alkaline and under this condition zrfC is the only zinc receptor to be efficiently activated and for this reason it is essential to the transport of zinc in the alkaline environment [33]. The importance of the ZrfC receptor in favouring the invasion of lung tissue lies in its N-terminal portion, which possesses four putative zinc binding motifs which are absent in other receptors. In fact, the deletion of this portion of the protein renders ZfrC similar, in terms of virulence, to the ZrfA and ZrfB receptors [33]. This characteristic of ZrfC imparts to A. fumigatus the ability to overcome the inhibitory effects on fungal expansion of calprotectin, an antimicrobial protein with zinc and manganese chelating properties, released by neutrophils during a fungal infection.

In this perspective, therapeutic treatments aimed at blocking the function of ZrfC constitute an essential condition for inhibiting the growth of A fumigatus. ZrfC thus represents an essential therapeutic target for combating invasive infections produced by A. fumigatus or other fungi endowed with the same receptor.

The solution according to the present invention fits into this context, as it aims to provide new methods and tools for blocking the activity of zrfC in the fungi which express it, such as, for example, Aspergillus fumigatus.

The present invention provides new antibodies, derivatives or fragments thereof, capable of recognising, interfering with and/or inhibiting the function of the zinc transporter ZrfC, a known factor of virulence in invasive pulmonary infections caused by Aspergillus and in general by fungi that express ZrfC. Furthermore, the present invention provides a strategy for vaccinating against infections caused by fungi that express ZrfC, such as Aspergillus infections, using the ZrfC protein itself or recombinant DNA techniques (genetic vaccination) associated with innovative methods of in vivo electroporation aimed at generating a cell- mediated immune response against ZrfC.

The invention also concerns a method that enables antibodies to be generated through a gene therapy approach that uses one or more vectors, for example preferably, but not limited to, plasmid DNA, for in vivo expression of the protein of interest. This vector can be injected intramuscularly and, after administration, an electric field can be applied in order to favour the passage of DNA into the muscle cell (electroporation). In vivo electroporation of plasmid DNA (DNA-EP) is a safe method with a consequent high dissemination of DNA in cells, a high expression of the protein of interest and, if the application is for vaccines, a considerable long-term immune response against the target antigen in a variety of species, including rodents, but also large animals such as dogs, pigs, cattle and monkeys [4-8].

Therefore, the antibodies and nucleotide and amino acid sequences according to the present invention advantageously show an inhibitory activity on the zinc transport function of the ZrfC protein and against fungal growth. In particular, the amino acid or nucleotide sequences according to the present invention are capable of inhibiting the proliferation of fungi that express ZrfC, such as Aspergillus fumigatus, when zinc is lacking.

According to the present invention, therefore, a new method for actively inducing an immune response against zrfC is proposed. In particular, the method consists in a genetic vaccination that uses a genetic vector, for example plasmid DNA, containing the optimised cDNA of zrfC. As mentioned above, the vector can be administered intramuscularly and the tissue can be subsequently subjected to electroporation. In this manner, an antibody- and/or cell-mediated immune response is generated in the host against the ZrfC zinc receptor. This strategy thus allows the host to be vaccinated against a fungal antigen that is essential to the growth and propagation of the fungus. The vaccine can have application as a monotherapy or in combination with other antifungal agents, belonging, but not only, to the pharmacological classes of the Polyenes, (e.g. Amphotericin-B), Triazoles (e.g. Voriconazole) and Echinocandins (e.g. Caspofungin).

It is therefore a specific object of the present invention an antibody, bi-specific antibody BiTE (bi-specific T cell engager) or chimeric antigen receptor (CAR) capable of recognising the extracellular domain of the fungal ZrfC protein, for example of Aspergillus, preferably of Aspergillus fumigatus, wherein the extracellular domain of the ZrfC protein comprises or consists in SEQ ID NO:2.

The extracellular domain of the ZrfC protein having the sequence SEQ ID NO:2 comprises four putative ZBM subdomains for binding zinc, shown here below:

The antibody, bi-specific antibody BiTE or CAR according to the present invention can recognise the extracellular domain of the fungal ZrfC protein and bind to one or more subdomains of the extracellular domain of the fungal ZrfC protein.

The antibody according to the present invention can be an antibody produced by any mammal or suitable for administration in any mammal, for example a human or humanised antibody.

The antibody can be a polyclonal antibody produced by the mammal following the administration of the extracellular domain of ZrfC in the form of an amino acid or nucleotide sequence. Furthermore, the antibody according to the invention can be a monoclonal antibody obtained from a hybridoma selected following the administration of the extracellular domain of ZrfC in the form of an amino acid or nucleotide sequence in a mammal, for example in mouse, and subsequently rendered suitable for administration in any other mammal for which the treatment is intended.

In particular, the antibody, BiTE or CAR according to the present invention can comprise a VFI sequence (variable region of the heavy chain) comprising or consisting of SEQ ID NO:6 or of an amino acid sequence at least 80% identical to SEQ ID NO:6 and a VL sequence (variable region of the light chain) comprising or consisting of SEQ ID NO:4 or an amino acid sequence at least 80% identical to SEQ ID NO:4.

According to one embodiment, the antibody, BiTE or CAR can be humanised.

In such a case, the antibody, BiTE or CAR according to the present invention can comprise a VFI sequence comprising or consisting of SEQ ID NO:7, SEQ ID NO:8 or of an amino acid sequence at least 80% identical to SEQ ID NO:7 or SEQ ID NO:8 and a VL sequence comprising or consisting of SEQ ID NO:9, SEQ ID NQ:10 or of an amino acid sequence at least 80% identical to SEQ ID NO:9 or SEQ ID NO:10.

It is a further object of the present invention a nucleotide sequence that encodes for an antibody, bi-specific antibody BiTE or chimeric antigen receptor (CAR) as defined above.

In particular, the nucleotide sequence according to the present invention can comprise a nucleotide sequence encoding a VH comprising or consisting of SEQ ID NO:6 or of an amino acid sequence at least 80% identical to SEQ ID NO:6 and a nucleotide sequence encoding a VL comprising or consisting of SEQ ID NO:4 or of an amino acid sequence at least 80% identical to SEQ ID NO:4.

According to specific embodiments, the nucleotide sequence encoding the VH can comprise or consist of SEQ ID NO:5, (VHI nucl) SEQ ID NO:11 or (VH2nucl) SEQ ID NO:12 and the nucleotide sequence encoding the VL comprises or consists of SEQ ID NO:3, (VL1 nucl) SEQ ID NO:13 or (VL2nucl) SEQ ID NO:14.

The invention further concerns an expression vector comprising a nucleotide sequence as defined above.

The subject matter of the present invention further relates to a cell, for example of a mammal, comprising a vector as defined above.

The present invention also concerns a pharmaceutical composition comprising or consisting of an antibody, BiTE or CAR, a nucleotide sequence, a vector, a cell, as defined above, together with one or more pharmaceutically acceptable excipients and/or adjuvants.

The pharmaceutical composition can further comprise one or more antifungals, such as, for example, antifungals of the class of polyenes, such as Amphotericin-B, of the class of triazoles, such as Voriconazole, and of the class of echinocandins, such as Caspofungin.

A further aspect of the invention relates to an antibody, BiTE or CAR, a nucleotide sequence, a vector, a cell, and a pharmaceutical composition, as defined above, for use as a medicament.

Furthermore, the present invention concerns an antibody, BiTE or CAR, a nucleotide sequence, a vector, a cell, a pharmaceutical composition, as defined above, for use in the treatment or prevention of infections caused by fungi that express ZrfC, preferably infections caused by Aspergillus, more preferably infections caused by Aspergillus fumigatus.

A further aspect of the present invention concerns a combination of an antibody, BiTE or CAR, a nucleotide sequence, a vector and/or a cell, as defined above, with one or more antifungals, for separate or sequential use in the treatment and prevention of infections caused by fungi that express ZrfC, preferably infections caused by Aspergillus, more preferably infections caused by Aspergillus fumigatus. For example, the antifungals can belong to the class of polyenes, such as Amphotericin-B, to the class of triazoles, such as Voriconazole, and to the class of echinocandins, such as Caspofungin.

The present invention further concerns an amino acid sequence comprising or consisting of one or more subdomains of the extracellular domain of the fungal ZrfC protein, for example of Aspergillus, preferably of Aspergillus fumigatus, or of said extracellular domain of the fungal ZrfC protein of sequence SEQ ID NO:2, a nucleotide sequence that encodes for said amino acid sequence, preferably fused to the FC domain of an immunoglobulin, an expression vector that comprises said nucleotide sequence or a pharmaceutical composition that comprises said amino acid sequence, nucleotide sequence or vector, said pharmaceutical composition optionally comprising an immune response booster and/or one or more antifungals, for use as a medicament, wherein said one or more subdomains are selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20. According to one embodiment, said nucleotide sequence can comprise one or more nucleotide sequences selected from SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24. Preferably, said nucleotide sequence is SEQ ID NO:1 .

The above-mentioned ZBM (Zinc-binding motif) subdomains are comprised in the extracellular domain of the fungal ZrfC protein ofsequence SEQ ID NO:2.

The above-mentioned subdomains are shown in table 1 reported below, which shows both the amino acid sequences and nucleotide sequences encoding for said subdomains.

Table 1

The aforesaid subdomains (ZBMs) comprise the consensus sequence CHXHX5CX6E/D [31] characterised by the presence of cysteine, histidine and negatively charged residues (aspartic acid and glutamic acid) typical of zinc-binding motifs described in other classes of proteins [34]

Also known is a prediction of the secondary structure of the extracellular domain of the fungal ZrfC protein made with the online tool JPred [35], from which one deduces the presence of structural elements such as b filaments in the ZBM subdomains.

A further object of the present invention concerns an amino acid sequence comprising or consisting of one or more subdomains of the extracellular domain of the fungal ZrfC protein, for example of Aspergillus, preferably of Aspergillus fumigatus, or of said extracellular domain of the fungal ZrfC protein of sequence SEQ ID NO:2, a nucleotide sequence that encodes for said amino acid sequence, preferably fused to the FC domain of an immunoglobulin, an expression vector that comprises said nucleotide sequence or a pharmaceutical composition that comprises said amino acid sequence, nucleotide sequence or vector, said pharmaceutical composition optionally comprising an immune response booster and/or one or more antifungals, for use in the prevention and treatment of infections caused by fungi that express ZrfC, preferably infections caused by Aspergillus, more preferably infections caused by Aspergillus fumigatus, for example as vaccines, wherein said one or more subdomains are selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20. The antifungals can be, for example, antifungals of the class of polyenes, such as Amphotericin-B, of the class of triazoles, such as Voriconazole, and of the class of echinocandins, such as Caspofungin. According to one embodiment, said nucleotide sequence can comprise one or more nucleotide sequences selected from SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24. Preferably, said nucleotide sequence is SEQ ID NO:1.

The present invention further concerns a combination of an amino acid sequence comprising or consisting of one or more subdomains of the extracellular domain of the fungal ZrfC protein, for example of Aspergillus, preferably of Aspergillus fumigatus, or of said extracellular domain of the fungal ZrfC protein of sequence SEQ ID NO:2, a nucleotide sequence that codes for said amino acid sequence, preferably fused to the FC domain of an immunoglobulin, an expression vector that comprises said nucleotide sequence, with an immune response booster and/or an antifungal, for separate or sequential use in the treatment and prevention of infections caused by fungi that express ZrfC, preferably infections caused by Aspergillus, more preferably infections caused by Aspergillus fumigatus, wherein said one or more subdomains are selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20. The antifungals can be, for example, antifungals of the class of polyenes, such as Amphotericin-B, of the class of triazoles, such as Voriconazole, and of the class of echinocandins, such as Caspofungin. According to one embodiment, said nucleotide sequence can comprise one or more nucleotide sequences selected from SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24. Preferably, said nucleotide sequence is SEQ ID NO:1.

According to the present invention, “separate use” means the administration, at the same time (or moment), of the two or more compounds of the combination according to the invention, in two or more distinct pharmaceutical forms. “Sequential use” means the successive administration (at different times or moments) of the two or more compounds of the combination according to the invention, in two or more distinct pharmaceutical forms.

The expression vectors according to the present invention, which can comprise both the nucleotide sequences that encode for the antibodies and the nucleotide sequence that encodes for the extracellular domain of the ZrfC protein of Aspergillus, can be selected from bacterial plasmids, adenovirus, poxvirus, vaccinia virus, fowlpox, herpes virus, adeno-associated virus (AAV), alphavirus, lentivirus, lambda phage, lymphocytic choriomeningitis virus, Listeria sp and Salmonella sp.

The expression vectors according to the present invention can be administered by electroporation (EP).

The principles of electroporation are very simple. The lipid membrane of a cell can be considered as a dielectric element interposed between the extracellular environment and the cytoplasmatic environment, which are both conductive. An electric field applied to a cell induces a transmembrane potential: when the dielectric potential of the membrane is exceeded, transient pores appear in the membrane, a process called electroporation [9, 10]. If the electric field is maintained for a sufficiently long time, the membrane becomes permeable (electropermeabilization) because the transient pores are stabilised and become large enough to allow charged macromolecules, such as DNA, to access the cytoplasm. The cells remain in a porated state for a limited period of time and close rapidly after the electric treatment ends. The duration of the electric pulse must be sufficiently short to avoid irreversible damage to the cell membrane and cell death. The transmembrane potential increases linearly with the intensity of the applied electric field, but above a certain threshold (generally 0.5-2 V) it decreases, indicating that the conductivity of the membrane increases due to the formation of hydrophilic pores [11 , 12]. As the molecules of a nucleic acid such as DNA or RNA are too large to penetrate through hydrophilic pores simply by diffusion, an electrophoretic field must be maintained for a sufficient time in order to allow the polyanions to move and enter the cell. The DNA must be in proximity to the cell membrane before the electric field is applied [9]. The DNA molecules pass through the pores of the membrane by means of an electrodiffusive process.

It is postulated that the progression of the DNA towards the nucleus takes place through a combination of classic electrophoresis and passive diffusion according to a concentration gradient. Therefore, the pulses must be optimised so as to obtain the best combination of cell permeabilization followed by the desired electrophoretic effect. In general, the pulse parameters are arbitrarily divided into short high-voltage pulses, greater than 400V/cm with a duration in the gamma range of mebo and low-voltage pulses, less than 400 V/cm with a duration in the interval of msec. An efficient gene transfer has been demonstrated using either only a sequence of high-voltage pulses or only low-voltage pulses. However, in theory the most effective strategy seems to be a combination of short initial high-voltage pulses followed by a sequence of longer lasting low- voltage pulses.

Protein expression in muscle is usually improved 100-1000 times after electroporation compared to the injection of naked DNA, thanks above all to greater cellular absorption [13-15].

Various devices for DNA-EP exist. The most advanced technologies are the ones being developed by Inovio Pharmaceuticals [16], Ichor Medical Systems [17] and IGEA [18].

Skeletal muscle is the most frequent target organ for DNA-EP. Skeletal muscles are easily accessible beneath the skin and are made up of post-mitotic cells capable of long-term expression of the transgene after transfection [19]. Furthermore, the tissue damage is rapidly repaired, without signs of muscle degeneration [20]. Muscle DNA-EP is an invasive procedure that requires needles for injection of the nucleic acid, followed by the electric discharge. In small animals this is achieved with flat electrodes positioned on the skin around the injected volume, whereas in larger species, including humans, it requires an array of needles inserted into the tissue. In clinical studies, this procedure has demonstrated to be well tolerated and not to cause severe pain [21].

The expression vectors according to the present invention which comprise the nucleotide sequences encoding for the antibodies can thus be advantageously used in the treatment of the fungal infections mentioned above, whilst the expression vectors comprising the nucleotide sequence that encodes for the extracellular domain of the ZrfC protein of Aspergillus can be advantageously used as genetic vaccines, such as, for example, DNA vaccines.

The present invention will now be described, by an illustrative, but not limitative way, according to a preferred embodiment thereof, with particular reference to the figures of the appended drawings, wherein:

- Figure 1 shows a plasmid vector containing the cDNA of ZrfC, used for the immunisation of BALB/C mice (Envigo, the Netherlands) by in vivo electroporation on muscle tissue.

- Figure 2 shows a plasmid vector containing the cDNA of ZrfC, used for the transfection of HEK 293 cells (ATCC, #CRL-1573).

- Figure 3 shows an ELISA assay on immunised mice serum. An are the mice immunised by intramuscular injection of the vector pCMV6- AC-FC-S-ZRFC (pCMV6-AC-FC-S, ORIGENE, #PS100054) and subsequent electroporation. The immunisation was carried out 3 times: at time 0, on days 21 and 42. The various bars indicate the dilutions of the serum drawn from immunised mice 52 days after the first immunisation (see legend below the horizontal axis).

- Figure 4 shows a western blot analysis of protein extracts of A. fumigatus cultured at different Zinc concentrations (0 — 50 mM). A: pool of immunised BALB/c mice sera (Envigo, the Netherlands). B: pool of pre- immunisation sera.

- Figure 5 shows an ELISA assay on the hybridoma supernatant. The supernatant of the hybridoma 1316G8 and of a non-specific hybridoma (CTL-) were assessed by ELISA, whose result demonstrates the specificity of the hybridoma 1316G8.

- Figure 6 shows a cell proliferation test of conidia of Aspergillus fumigatus incubated at two different Zn concentrations (0.09; 50 mM) in the presence of the supernatants of two hybridomas. Only 1316G8, specific for the ZrfC protein, inhibits the growth of the fungus under limiting conditions of zinc concentration (0.09 mM), whereas the supernatants from hybridomas against non-correlated antigens (CTL-) do not show any inhibitory effect on fungal growth.

- Figure 7 shows an ELISA assay for assessing the specificity of 1316G8-lgG1. Specificity was assessed in terms of the antibody’s binding capacity towards the purified ZrfC-FC protein (0.075 pg/ml) and the non- purified ZrfC protein (full length). An antibody obtained by means of the same procedure was used as a negative control (lgG1 CTL-).

- Figure 8 shows a western blot analysis of the antibody 1316G8- lgG1.

- Figure 9 shows a western blot of the humanised antibodies. 1 Protein Dual Colour Standards; 2: hVH1/hVL1 ; 3: hVH2/hVL1 ; 4: hVH1/hVL2; 5: hVH2/hVL2.

- Figure 10 shows an ELISA assay for assessing the specificity of the humanised antibodies. Specificity was assessed in terms of the antibody’s binding capacity towards the purified ZrfC-FC protein (1 pg/ml). 1 : supernatant used as such; 2: supernatant diluted 1 :2; 3: supernatant diluted 1 :4.

- Figure 11 shows an ELISPOT assay for y-IFN, carried out on cells isolated from the spleen of Sprague-Dawley rats (Envigo, the Netherlands) immunised against ZrfC and stimulated with two pools of peptides representing the entire amino acid sequence of ZrfC.

EXAMPLE 1. Preparation of a humanised antibody against ZrfC according to the present invention.

With reference to Article 170bis of the Italian Industrial Property Code, it is hereby declared that the studies on genetically modified organisms cited below took place inside a facility with a biosafety level of BSL2, with notification ID RM/IC/lmp2/04/001 , Takis s.r.l. authorised on 09/04/2015.

Immunisation by electroporation of plasmid DNA expressing the target of interest With the aim of obtaining an antibody response against the target of interest, a gene therapy approach based on DNA electroporation in skeletal muscles was adopted. This technology allows the use of appropriately engineered cDNA variants of the protein of interest and enables endogenous expression in the muscle. The plasmid vector used encodes the extracellular domain of the ZrfC protein of Aspergillus fumigatus fused to the murine FC tag (mFC), in order to improve the presentation of the antigen (pCMV6-AC-FC-S-ZRFC-Fig.1).

The nucleotide sequence cloned in the vector is: extracellular domain of the ZrfC protein of Aspergillus fumigatus). Whereas the respective amino acid sequence of 190 aa is: ID NO:2, amino acid sequence of ZrfC of Aspergillus fumigatus).

The gene therapy protocol consisted of an injection of 50 pg of plasmid pCMV6-AC-FC-S-ZRFC (pCMV6-AC-FC-S, ORIGENE, #PS100054) into the quadriceps muscle of one leg of 6- to 7-week-old female BALB/c mice (Envigo, the Netherlands). The DNA was formulated in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml. The expression of the ZrfC protein was guided by the cytomegalovirus (CMV) promoter; the transcription was stopped thanks to the presence of a STOP codon at the end of the mFC tag. The DNA-EP was carried out with an electroporator of the IGEA Cliniporator type, using a plate electrode (electrode P-30-8-B). For the DNA-EP in the muscle, the following low- voltage electric conditions were applied: 8 pulses of 20 msec each at 110V, 8Hz, 120 msec interval between each pulse. The immunisation, in addition to the electroporation of plasmid DNA, provided for intraperitoneal injection of 100mI of the adjuvant CpG at a concentration of 2 mg/ml.

ELISA assay for verifying the specificity of the serum produced and the antibody concentration

With the aim of assessing the presence of anti-ZrfC antibodies in the plasma of mice immunised by DNA-EP (see example 1), an ELISA assay was set up.

In order to produce the recombinant protein of ZrfC as an antigen substrate, necessary for carrying out the ELISA assay, HEK 293 cells were transfected with the plasmid vector pCMV6-AC-FC-S-ZRFC (pCMV6-AC-FC-S, ORIGENE, #PS100054) containing the cDNA of ZrfC (Fig. 2). The supernatant of the transfected cells, containing the ZrfC protein, was diluted 1 :10 in a buffer at pH 9.6 (Carbonate-Bicarbonate Buffer, Sigma-Aldrich) and incubated on 96-well Nunc Maxisorp plates (50 pL/well) for 12 hours at 4°C. This procedure allows the absorption of the ZrfC protein on the inner surface of the wells. The day after the wells were saturated with 1% BSA in PBS, 0.05% Tween20 for 2 hours at 37°C. The serum of the electroporated animals was serially diluted from 1 :100 to 1 :12800 in the same buffer used for the saturation and added to the wells of the plate. The presence of anti-ZrfC IgG in the serum of the immunised mice was detected by incubation with an anti-murine IgG antibody conjugated with alkaline phosphatase (Sigma). As a chromogenic substrate for the catalytic activity of the alkaline phosphatase, Yellow pNPP (Sigma) was used. The reading of the assay was taken with an optical reader (BIORAD) at a wavelength of 405 nm. The polyclonal serum of all the immunised mice showed a capacity to bind to the antigen (Figure 3).

Furthermore, a pool of the polyclonal sera was prepared and used in a western blot on fungal lysates and on the full-length ZrfC protein in order to further validate its specificity. The pool of sera drawn from the mice before they were immunised (TØ) was used as a control for specificity.

In order to obtain the fungal lysates, the cultures of A. fumigatus cultured at different Zinc concentrations (0, 0.09, 0.18 and 50 mM) were pulverised with liquid nitrogen and then lysed with lithium acetate and sodium hydroxide. Finally, the fungal pellets were resuspended in Laemmli Buffer 4X. The loaded protein extracts were transferred onto nitrocellulose membranes blocked for one hour at room temperature with 0.05% PBST, 5% milk. In figure 4A the pools of anti-ZrfC sera diluted 1 :100 in blocking buffer are shown, whilst in figure 4B the pools of TØ sera diluted 1 :100 in blocking buffer are shown; the membranes were incubated at 4°C for 2 hours. After several washes, the membranes were incubated with a secondary anti-lgG antibody (whole molecule) conjugated with peroxidase at a 1 :2000 dilution in the saturation buffer for one hour at room temperature, washed and treated with ECL substrate (Amersham). The results confirm the presence of antibodies specific for the ZrfC protein in the pool of immunised mouse sera and not in the pre-immunisation mouse serum TØ. The absence of reactivity in the line loaded with fungal extracts cultured in 50 mM zinc (Figure 4A) indicates that the ZrfC protein is scarcely represented under these conditions, in accordance with what is reported in the literature [29]

Production of mAb/ hybridoma technology

The animals with the highest antibody concentration (1 :2700) were sacrificed; their spleens were removed and homogenised in order to recover the splenocytes. These cells were fused with murine myeloma cells (ATCC, #P3/X63Ag8653), following the classic procedure, in order to generate hybridomas and produce monoclonal antibodies. The hybridomas obtained were assessed by ELISA, as described, using 50 mL of the supernatant of each one of them. The hybridoma 1316G8 showed to be positive (Figure 5).

Proliferation assay

In order to verify the biological activity of the hybridoma 1316G8 in vitro, a proliferation assay was set up, in which 7500 conidia/well of A. fumigatus were cultured in the lack (0.09mM) or presence (50mM) of zinc to simultaneously test the conditions under which ZrfC is highly or poorly expressed. The conidia were incubated or not incubated with the hybridoma supernatant and the culture medium of the hybridomas and a hybridoma specific for a target other than ZrfC were used as negative controls. Readings at 630 nm were taken at 24, 48 and 63 hours. The hybridoma 1316G8 showed to be capable of inhibiting the proliferation of A. fumigatus when zinc was lacking (Figure 6).

CDR of 1316G8

Following RNA extraction from the culture of 1316G8 and reverse transcription, the cDNA obtained was amplified with different pairs of primers specific for light chains (K or l) and heavy chains of IgG and IgM antibodies. The PCR products were cloned and sequenced to verify which sequences were productive or unproductive. The productive sequences of the light chains were cloned in the vector pFUSE2-CLIg-mk (InvivoGen, #pfuse2-mclk), containing the constant region of murine Igk, whilst the productive sequences of the heavy chains were cloned in the vector pFUSE-CFIIg-mG1 (InvivoGen, #pfuse-mchg1), containing the constant regions of the heavy chain of murine lgG1. These constructs were transfected in pairs (one vector expressing a light chain and one expressing a heavy chain) in FIEK-293 cells (ATCC, #CRL-1573). For each pair, the formation of the recombinant lgG1 antibody was assessed by western blot and specificity was subsequently assessed by ELISA. Figs. 7 and 8 show the results of the respective experiments for the pair that gave positive results. The sequences that produced an lgG1 antibody capable of binding ZrfC are reported below. In particular, the sequences of the heavy and light chains of the murine immunoglobulins selected for the construction of the human recombinant chains are reported: nucleotide sequence of the light chain (murine) amino acid sequence of the light chain (murine) ( ) nucleotide sequence of the heavy chain (murine) amino acid sequence of the heavy chain (murine)

Humanisation of the 1316G8 antibody

With the aim of generating a humanised antibody, the super humanisation method described by Flwang et al. [30] was chosen. In accordance with the method, a determination was made of the nearest human germline sequence (NCBI IG-Blast), the structural models useful in the PDB, including the X-ray structure closest to the murine VFI and VL sequences and the X-ray structure most similar to the best human germline sequences. The murine sequences 40XT and 3J8W, for the heavy (VFI) chain, and 1 CBV, for the light (VL) chain, gave the best results in the overlay with the X-ray structure. Based on the comparison, the human germline VFI 5AZE (GeVH SEQ ID NO:15) was selected for the heavy chain as they appear to be the most similar to the CDR sequence of the murine antibody. The human germline VL 5DRX (hGeVL SEQ ID NO:16) was selected for the light chain.

The alignment of the sequences of the murine heavy (VFI) and light (VL) chains (mVH and mVL) with the respective human immunoglobin sequences (hVH1 SEQ ID:7, hVH2 SEQ ID NO:8; hVL1 SEQ ID No:9, hVL2 SEQ ID NO:10) is shown below.

The substitutions made in the human sequences for the humanisation of the murine monoclonal antibody produced by the hybridoma 1316G8 are indicated in bold.

Based on the comparison between the murine CDRs of 1316G8 and the CDRs of the human germline, the sequences SEQJD: 7 and 8 (hVH1 and hVH2, respectively) and SEQJD: 9 and 10 (hVL1 and hVL2, respectively) were designed.

The synthetic genes were generated by assembly of synthetic oligonucleotides and PCR, according to the method described by Stemmer et al. [31].

The synthetic genes were then cloned by recombination, using the Gateway system (ThermoFisherScientific) in plasmids containing, respectively, the synthetic genes coding for the human heavy chain type 1 and kappa light chain, respectively. The vectors thus obtained were designated pFlc1316G8 and pLc1316G8. For each pair, the formation of the humanised antibody was assessed by western blot and specificity was subsequently assessed by ELISA. Figures 9 and 10 show the results of the respective experiments.

The results in figure 10 show that the humanised antibody, derived from the murine one 1316G8, exhibits characteristics of specificity for the antigens comparable to that of the murine antibody. Both antibodies recognise the same recombinant protein containing the N-terminal domain of ZrfC fused to the FC domain of an immunoglobulin, used for immunisation. Therefore, on the basis of the principle of bioequivalence, the humanised antibodies according to the present invention show an antiproliferative activity comparable to that of the 1316G8 antibody, as shown in Figure 6.

EXAMPLE 2. Study of the cell-mediated response induced by an genetic vaccine against ZrfC according to the present invention

With the aim of verifying the possibility of inducing a cell-mediated immune response through the generation of T-lymphocytes specific against the zinc transporter zrfC, the same genetic vaccination procedure as used in BALB/C mice (Envigo, the Netherlands) to generate anti-ZrfC antibodies was used for the immunisation of Sprague-Dawley rats (Envigo, the Netherlands). The immunisation protocol consisted in 3 injections of 100mg of pCMV6-AC-FC-S-ZRFC (pCMV6-AC-FC-S, ORIGENE, #PS100054) in the quadriceps, 2 weeks apart. The plasmid was formulated in phosphate-buffered saline (PBS) at a concentration of

I mg/ml. DNA-EP was carried out with an electroporator of the IGEA Cliniporator type, using a needle electrode (electrode N-10-4-B). For the muscle DNA-EP, the following low-voltage electric conditions were applied: 8 pulses of 20 msec each at 110V, 8Flz, interval of 120msec between each of them. Two weeks after the last vaccination, the spleens of the immunised rats were removed and the T-lymphocytes were analysed by ELISPOT under conditions of stimulation (16 hours) with 2 different pools of 15 aa peptides, partially overlapped in the sequence for

I I aa, and which cover the entire ZrfC protein. As shown in figure 11, all of the rats responded to the vaccine, showing a cell-mediated immune response.

References

1. Verweij, P.E., et al., J Antimicrob Chemother, 2016. 71 : 2079.

2. Lazzarini, C., et al., Antimicrob Agents Chemother, 2015. 60: 682

3. Segal B. H. 2009. Aspergillosis. N. Engl. J. Med. 360:1870-1884

4. Luckay, A., J Virol, 2007. 81 : 5257

5. Reed, S.D. and S. Li, Curr Gene Ther, 2009. 9: 316

6. Fowler, V., et al., Antiviral Res, 2012. 94: 25

7. Aurisicchio, L., et al., Clin Cancer Res, 2009. 15: 1575

8. Peruzzi, D., et al., Vaccine, 2010. 28: 1201

9. Andre, F. and L.M. Mir, Gene Ther, 2004. 11 : 33

10. Gothelf, A. and J. Gehl, Flum Vaccin Immunother, 2012. 8: 1694

11. Neumann, E., S. Kakorin, and K. Toensing, Bioelectrochem Bioenerg, 1999. 48: 3

12. Lurquin, P.F., Mol Biotechnol, 1997. 7: 5 13. Mathiesen, I., Gene Ther, 1999. 6: 508

14. Mir, L.M., et al., Proc Natl Acad Sci U S A, 1999. 96: 4262

15. Rizzuto, G., et al., Hum Gene Ther, 2000. 11 : 1891

16. http://www.inovio.com.

17. http://www.ichorms.com.

18. http://www.igeamedical.com.

19. Fattori, E., et al., Somat Cell Mol Genet, 2002. 27: 75

20. Durieux, A.C., et al., J Gene Med, 2004. 6: 809

21. Diaz, C.M., et al., J Transl Med, 2013. 11 : 62

22. Sparrow, E., et al., Bull World Health Organ, 2017. 95: 235

23. Kousha, M., R. Tadi, and A.O. Soubani, Eur Respir Rev, 2011. 20: 156

24. Falvey, D.G. and A.J. Streifel, J Hosp Infect, 2007. 67: 35

25. Dagenais, T.R. and N.P. Keller, Clin Microbiol Rev, 2009. 22: 447

26. Mansour, M.K., J.M. Tam, and J.M. Vyas, Ann N Y Acad Sci, 2012. 1273: 78

27. Mehrad, B., R.M. Strieter, and T.J. Standiford, J Immunol, 1999. 162: 1633

28. Salvatori, G. and S. Campo, Med Mycol, 2012. 50: 225

29. Outten, C.E. and T.V. O'Halloran, Science, 2001. 292: 2488

30. Gaither, L.A. and D.J. Eide, Biometals, 2001. 14: 251

31. Amich, J., et al., Eukaryot Cell, 2010. 9: 424

32. Bignell, E., et al., Mol Microbiol, 2005. 55: 1072

33. Amich, J., et al., Cell Microbiol, 2014. 16: 548

34. Auld D., Biometals, 2001 14: 271 -313

35. Drozdetskiy A, Cole C, Procter J & Barton GJ. (2015), JPred4: a protein secondary structure prediction server, Nucleic Acids Research, Web Server issue (first published 15th April 2015); http://dx.doi.org/10.1093/nar/gkv332