PL201750B1 - Methods of polyvalent bacteriophage preparation for the treatment of bacterial infections - Google Patents

Abstract

1. The method of obtaining a multivalent strain of bacteriophage, characterized in that: (a) a sufficiently large collection of n different strains of bacterial pathogens of a certain type occurring at random is compiled in a given area, preferably the isolated bacterial strains are drug-resistant strains, (b) accumulating a sufficient quantity of different bacteriophage strains specific to at least one bacterial strain belonging to to a given type, (c) the lytic activity of the collected bacteriophage strain against each collected bacterial strain is determined and then for a bacteriophage strain, the p-value is calculated as the ratio of the number of strains lysed by a given phage to the total number of bacterial strains, d) for the obtained value of p, it is checked whether the numerical value of n meets the condition: PL 201750 B1 pq on a 2 1-α where: q = 1-p d is a constant not greater than 0.1, preferably not greater than 10%, p, Zia is a normally distributed random variable dependent on the 1-a confidence interval, which is not less than 0.95, e) a bacteriophage strain is selected which meets the criterion specified in d) and is characterized by a p value not lower than 2 / n, preferably not less than 0.5. 5. The method according to p. The process of any one of claims 1 to 4, characterized in that the multivalent bacteriophage strain is specific for bacteria belonging to the genus Staphylococcus found in Poland, isolated in step e) is a bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00005), S6 (PCM F / 00006) and S7 (PCM F / 00007). 7. The method according to p. The process of any one of claims 1 to 4, characterized in that the multivalent bacteriophage strain is specific for bacteria belonging to the genus Pseudomonas found in Poland, isolated in step e) is a bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027).

Description

Description of the invention

Field of the Invention

The invention relates to multivalent bacteriophage strains, methods for their preparation and use in the treatment of bacterial infections, especially with drug-resistant bacteria strains.

State of the art

Bacteriophages (phages) are a diverse group of viruses whose life cycle is associated only with the bacterial cell. Bacteriophages have a lytic or lysogenic life cycle. The most useful antibacterial agents are lytic bacteriophages, which, after infection of bacterial cells sensitive to them, multiply in them, causing their complete destruction (lysis), and the released new phage particles attack and destroy subsequent bacterial cells. This process can take place both in vitro and in vivo.

One of the important features of bacteriophages is the commonly known high specificity of their lytic activity. This feature is used, among others, by in typing different species of bacteria (see for example GB 2285684, US 5824468, SU 543260, and international patent applications WO 0100786, WO 0109370). Other known applications of bacteriophages include: the use of bacteriophages as tools in molecular biology useful, for example, for the expression and selection of desired proteins (e.g. US Patent 6,027,930), the use of phages in sterilization and cleaning agents (e.g. EP 0414304, EP 0290295, GB 2253859 and international patent applications WO 9808944, WO 9003122). Modified phages have been used for the production of vaccines (e.g. WO 9505454). Certain proteins of bacteriophage origin are also used (e.g. EP 0510907, US 5470573, WO 9607329). Methods for isolating bacteriophages and obtaining phage preparations are well known and are constantly being improved (e.g. GB 829266, CS 192212, RU 2109055).

Phage therapy has been used on a large scale since World War II at the Institute of Microbiology and Virology of Tbilisi (Georgia). Pools of various phage preparations are used there in the treatment of bacterial infections and in prophylaxis. The available data indicate the high effectiveness of phage therapy. Similar studies have been carried out for years in Poland, in the Bacteriophage Laboratory of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, where phage therapy for infections caused by drug-resistant forms of bacteria and not amenable to antibiotic therapy has been carried out for over 25 years (see: Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1981, 31, 293; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1983, 31, 267; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1984, 32, 317; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1985, 33, 219; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1985, 33, 241; Stefan Ś lopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1987, 35 , 569; Beata Weber-Dąbrowska et al., Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 31-37; Beata Weber-Dąbrowska et al., Archivum Immunologiae et Therapiae E xperimentalis, 2000, 48, 547-551). In the last 14 years, phage therapy has included 1473 patients with purulent infections of various tissues and organs, demonstrating the high effectiveness of phage therapy.

Complete disappearance of disease symptoms and recovery was recorded in 1,289 cases. It should be emphasized that in at least a dozen or so cases, specific phage therapy was the only way to eliminate the life-threatening infection. The therapy was carried out on individual patients. It consisted of:

a) cultivating and identifying a bacterial strain obtained from material obtained from the patient

b) determining the sensitivity of the strain to specific phages and selecting the phage showing the highest lytic activity towards the strain grown.

c) preparing a phage lysate with a high number of phage particles

d) preparing a sterile phage preparation for treatment

This procedure is quite costly, labor-intensive and time-consuming. Sometimes it takes 7-10 days from the moment of receiving the material for research to the delivery of ready-made sterile phage preparations for treatment. In certain disease states, such a waiting period is too long. Treatment with phages according to the procedure used so far is not feasible on a larger scale. Infections in cystic fibrosis pose particular problems. It is a genetically conditioned systemic disease consisting in a malfunction of the mucous glands, predisposing to chronic

Bronchial and pulmonary diseases. The secreted mucus is thick and sticky, which makes it difficult to remove it by natural means (expectoration). The accumulation and deposition of mucus in the bronchi, and the impaired clearance of bronchial tubes, creates a susceptible environment for bacterial infections leading to chronic bronchitis and pneumonia. The most dangerous pathogens causing infections in cystic fibrosis are Pseudomonas and Staphylococcus aureus. Permanently recurring infections with these microorganisms lead to serious pathological changes in the respiratory system and premature death. Only a few patients survive over 25 years. Treatment of such infections with available antibiotics, especially in recent years, creates serious therapeutic problems worldwide. The antibiotic therapy of these infections is becoming less and less effective because the vast majority of bacterial strains show resistance to all antibiotics, including the last-resort antibiotic - vancomycin. Therefore, there is an urgent need to introduce into medical practice an alternative method of treating persistent and very dangerous bacterial infections.

Object of the invention

In recent years, there has been a massive spread of bacterial strains resistant to all antibiotics, including the last-resort antibiotic vancomycin. As a result, treatment of bacterial infections caused by drug-resistant forms with antibiotics is ineffective. This condition creates drastic therapeutic problems. Therefore, there is an urgent need to introduce into medical practice an alternative method of treating persistent bacterial infections.

Therefore, the object of the invention is to develop a method for obtaining a multivalent phage strain that could be used to prepare preparations with guaranteed efficacy for the treatment of bacterial infections, without the necessity of individual selection of the therapeutic phage. In a particular embodiment, the object of the invention is to present a method that allows to obtain highly purified phage preparations, especially those devoid of endotoxins.

A particular object of the invention is to obtain multivalent phage preparations that could be effectively used for the treatment of bacterial infections in cystic fibrosis, without the need to individually select the phages to be treated.

The essence of the invention

The subject of the present invention is a method of obtaining a multivalent bacteriophage strain, characterized in that: a) a sufficiently large collection of n different strains of bacterial pathogens of a certain type occurring randomly in a given area is collected, preferably the isolated bacterial strains are drug-resistant strains, b) a sufficient number of different bacteriophage strains specific for at least one bacteriophage strain of a given genus, c) the lytic activity of the collected bacteriophage strain against each accumulated bacteriophage strain is determined, and then for the bacteriophage strain the p-value is calculated as the ratio of the number of strains lysed by the given phage to the number of all collected bacterial strains, d) for the obtained p value, it is checked whether the number of n meets the condition:

pq ^to

1-α where: q = 1-pd is a constant not greater than 0.1, preferably not greater than 10% of p, z _1-a is a normally distributed random variable dependent on the confidence interval 1-α, which is not less than 0.95,

e) a bacteriophage strain is selected which meets the criterion defined in d) and is characterized by a p value of not less than 2 / n, preferably not less than 0.5.

For each of the isolated phages, the range of its lytic activity against the population of pathogenic bacteria strains present in a given area is determined. For this purpose, a sufficiently large collection of randomly selected drug-resistant strains of bacteria should be collected. With regard to the established large sample of bacteria with the number n (in the described example n was 845 and 880, respectively for drug-resistant Pseudomonas and Staphylococcus strains isolated from Poland), we calculate the frequency of success, i.e. the ratio of the number of strains lysed by

The number of strains of a given phage to the number of all strains tested. We treat this frequency as the probability of success p. The probability of failure q is q = 1 - p.

It is crucial to establish the number n that the sample should be taken in order to be considered representative of the general population of the strains of a given genus (eg Staphylococcus, Pseudomonas).

For this purpose, the criterion of representativeness is established:

P (\ ν - p \ (d) = P (- d (v - pd) = 1 - α

The probability that the modulus of difference between the sample frequency ν and the success probability p is less than the predetermined value d is 1-α, which corresponds to the confidence level of 1-α. Typically, the value of 1-α is set to 0.95 or 0.98.

On the other hand, the sample size n should meet the condition:

pq n>! - 2z d ²

1-α where z _1-a is a normally distributed random variable. For 1-a = 0.95, _1-a = 1.96, and for 1-a = 0.98, z = 2.33 _s

On the basis of this criterion and the value of the frequency p obtained for the characterized phage, assuming reasonable d values of the 1-a confidence interval, it is possible to verify the number n used to determine the range of lytic activity of a given phage and determine whether the range of lytic activity obtained for this n , with the assumed values of di 1-a, can be considered true for the general population of bacterial strains of a given genus. Preferably in step b) the bacteriophage strain is isolated from the sample coming from the environment, by filtration through membrane filters with a pore size of 0.2-0.4 μm, a nutrient medium is added to the obtained filtrate and mixed with a broth culture of a specific type of bacteria, incubated at about 37 ° C for about 1 hour, an aliquot of the suspension is taken and rubbed on plates with a solid medium, incubated at about 37 ° C for about 2 to 24 hours, a sample of the medium including a single plaque is isolated, transferred it is for the culture of a broth of bacteria of a certain type and incubated until the culture lightens, and the obtained lysate is filtered through membrane filters with a pore size of 0.2-0.4 μm, obtaining a bacteriophage preparation, preferably sowing on a solid medium and re-isolation of the plaque is repeated 5 times. Also preferably, in stage c), the tested bacterial strain is sown on a suitable solid substrate, onto which a portion of the bacteriophage preparation obtained in stage b) is applied, incubated at approx. 37 ° C for approx. 4 hours and then left at approx. Approx. 4 ° C for about 2 to 24 hours, with the lytic activity of a bacteriophage strain being evidenced by the appearance of at least single plaques.

Preferably, the lysate is further purified, especially from endotoxins, by contacting the bacteriophage-containing mixture with a bed containing cellulose or a partially esterified derivative thereof, followed by washing with a solution to remove contaminants, especially endotoxins, and then washing of the purified bacteriophages. Preferably, the elution of endotoxins is carried out with water, a solution of a non-dissociating substance or a saline solution with a concentration of no more than 0.1 M, preferably buffered. Equally preferably, the elution of the bacteriophage fraction is carried out with a solution of the non-dissociating substance or any buffer or saline solution with a concentration greater than 0.05 M, preferably buffered. Equally preferably, the elution of endotoxins and bacteriophages is carried out at a temperature of -25 ° C to 100 ° C. Equally preferably, the elution of endotoxins and bacteriophages is carried out using an aqueous saline solution containing an organic solvent. Equally preferably, the organic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, isopropanol and acetone, and the resin used is cellulose partially esterified with an organic or inorganic acid. Preferably, cellulose partially esterified with acetic, nitric, sulfuric or phosphoric acid is used as the matrix, in particular cellulose is used as the matrix in which from 0.01% to 5% glucose molecules are esterified, preferably from 0.25% to 1% of the molecules glucose, most preferably from 0.5% to 1% glucose molecules.

Preferably, the pathogenic bacterial strains belong to the genus Staphylococcus or Pseudomonas.

A further object of the invention is an agent for the treatment or prevention of infections caused by bacterial pathogens, comprising an active agent and, optionally, a pharmaceutically acceptable carrier, characterized in that the active agent comprises a multivalent bacteriophage strain of this type obtained by the method of the invention.

Preferably, the agent according to the invention is characterized in that, as a multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus, it comprises at least one bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003) , S4 (PCM F / 00004), S5 (PCM F / 00006), S6 (PCM F / 00006) and S7 (PCM F / 00007), preferably the phages S1, S2, and S4 or S5. Preferably, the agent of the invention serves to treat or prevent infections occurring in patients with cystic fibrosis and as a multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus comprises at least one bacteriophage strain selected from F / 00002, F / 00004, F / 00007.

Preferably, the agent according to the invention is characterized in that as a multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas it comprises at least one bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010) , P4 (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), preferably P7, P20 and P6 phages. Preferably, the agent of the invention serves to treat or prevent infections occurring in patients with cystic fibrosis and as a multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas it comprises at least one bacteriophage strain selected from F / 00010, F / 00013, F / 00018.

Another object of the invention is the use of a multivalent bacteriophage strain obtained by the method of the invention for the preparation of a medicament for the treatment or prevention of infections caused by bacterial pathogens.

Preferably, at least one bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S3 (PCM F / 00003), S3 (PCM F / 00002), S3 (PCM F / 00003) is used for the preparation of a medicament for the treatment or prevention of infections caused by bacteria belonging to the genus Staphylococcus. PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00006), S6 (PCM F / 00006) and S7 (PCM F / 00007), preferably phages S1, S2, and S4 or S5. Preferably, at least one bacteriophage strain selected from F / 00002, F / 00004, F / 00007 is used for the preparation of a medicament for the treatment or prevention of infections caused in patients with cystic fibrosis by bacteria belonging to the genus Staphylococcus.

Equally preferably, at least one bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 is used for the preparation of a medicament for the treatment or prevention of infections caused by bacteria belonging to the genus Pseudomonas. (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), preferably P7, P20 and P6 phages. Preferably, at least one bacteriophage strain selected from F / 00010, F / 00013, F / 00018 is used for the preparation of a medicament for the treatment or prevention of infections caused in patients with cystic fibrosis by bacteria belonging to the genus Pseudomonas.

Another object of the invention is a multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus, selected from among S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S3 (PCM F / 00003), S4 ( PCM F / 00004), S5 (PCM F / 00006), S6 (PCM F / 00006) and S7 (PCM F / 00007), preferably of S1, S2, S4 and S5.

The subject of the invention is also a multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 (PCM F / 00011), P5 ( PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024 ), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), preferably from P7, P20 and P6. The study used its own collection of phages active against bacterial species that are the most common etiological factors of bacterial infections in humans. The disclosed embodiments include multivalent bacteriophages that are lytic against methicillin-resistant strains of Staphylococcus aureus and blue oil bacilli (Pseudomonas aeru6).

Ginosa), especially in patients with cystic fibrosis. These species are currently the cause of severe infections resulting in high mortality.

Advantages of the invention

The introduction to the treatment of polyvalent phage preparations containing multivalent phages obtained by the method according to the invention is a great advance in the fight against bacterial infections that cannot be treated with antibiotics. There are also some economic benefits associated with the new technology. It allows to significantly reduce material costs and shorten the time from collecting the material for testing, until obtaining a phage preparation for treatment. As a result, the phage preparation will wait for the patient and not the patient for the preparation of an individual phage preparation.

The wide range of lytic activity of phage preparations composed of several multivalent phages obtained by the method according to the invention is a very important feature from the point of view of their use in the treatment of bacterial infections. It is known that in the bacterial population, forms resistant to bacteriophages may appear with a frequency of about 1x10 ^-7 . In the presence of two phages, the frequency of such mutants is 1x10 ^-14 and with three phages only 1x10 ^-21 . In such a system, the problem of bacterial strain resistance to phage is almost non-existent.

The great applicability of the exemplary preparations composed of multivalent phages deserves special emphasis. They can be widely used in medical practice and will allow for effective combating of dangerous bacterial infections. It is also possible to use the phages according to the invention in the preparation of preparations for external use, used, for example, for the treatment and prevention of dermatological infections. It has also been found that oral administration of the phage lysates of the invention increases resistance to future bacterial infections. In some cases, phage therapy can be used together with antibiotic therapy.

Polyvalent phage preparations used in the treatment of bacterial infections have proved to be highly effective. Particular efficacy was observed in the treatment of severe sepsis (88% of cures) and in the treatment of infections in patients with cystic fibrosis.

List of figures

Figure 1 shows the results of the statistical analysis obtained for phages S1-S7.

Figure 2 shows the statistical analysis results obtained for the P1-P20 phages.

Figure 3 shows the results of the statistical analysis of efficacy against strains belonging to the genus Staphylococcus isolated from cystic fibrosis patients obtained for selected phage strains.

Figure 4 shows the results of the statistical analysis of efficacy against strains belonging to the genus Pseudomonas isolated from cystic fibrosis patients obtained for selected phage strains.

For a better understanding of the essence of the invention, it has been illustrated by the following examples.

Example 1. Isolation of bacteriophages

Staphylococcus and Pseudomonas bacteriophages have been isolated from municipal wastewater. The wastewater was filtered through membrane filters with a pore size of 0.2-0.4 μm, which retained bacteria and allowed bacteriophages to pass. Concentrated liquid nutrient medium was added to the obtained filtrate, and different dilutions of the filtrate were mixed with a young bacterial broth (Staphylococcus aureus or Pseudomonas aeruginosa). The samples were incubated at 37 ° C for 1 hour. After this time, 0.2 ml of suspension was pipetted from each sample and triturated on nutrient agar plates with a glass rod. After overnight incubation at 37 ° C, in the case of the presence of the desired phage on the plates inoculated with the appropriate dilution of the filtrate containing the young bacterial culture, uniform (nebular) bacterial growth and separately lying transparent small fields, the so-called plaques, which usually contained several million phage particles. A single plaque along with the agar were excised with an isolation loop, transferred to a fresh bacterial broth culture, and then incubated until the culture was light. After filtering through membrane filters with a pore size of 0.2-0.4 μm, which retained the bacteria still remaining in the lysate, only the bacteriophage was present in the filtrate. Plating on agar media of various dilutions of the obtained phage with appropriate bacteria and re-isolation of the plaques was carried out 5 times, which allowed to obtain a pure phage line.

Example 2. Determination of susceptibility of Staphylococcus aureus strains to bacteriophages specific for these microorganisms.

PL 201 750 B1

The sensitivity was determined on the medium described by Wahl. Tiles with this substrate dried at 37 ° C for 30 minutes. covered with young 4-hour shaking broth culture of Staphylococcus aureus, excess suspension was pipetted and dried again for 30 min. at 37 ° C. On one plate divided into 6 segments, the sensitivity of the tested strain to 6 different but specific bacteriophages was determined. 1 drop of 1/10 diluted bacteriophage was applied to the surface of each segment. The plates were incubated in an incubator at 37 ° C for about 4 hours and then left in the refrigerator until the next day. In the case of high sensitivity of the strain to a specific phage, on a medium covered with uniform bacterial growth at the site of phage application, bald transparent fields were observed as a result of complete destruction of bacterial cells (lysis). The lower sensitivity of the strain was manifested by the appearance of single plaques.

Example 3. Determination of the sensitivity of Pseudomonas aeruginosa strains to bacteriophages specific for these microorganisms.

The method of determining susceptibility was similar to that described in Example 2. In this case, however, the solid medium was nutrient agar supplemented with phosphate buffer and the incubation period of the plates with the phage applied was 5 hours.

Example 4. Selection of phages with the broadest spectrum of activity against Staphylococcus and Pseudomonas strains - obtaining multivalent phage strains.

The bacteriophages were isolated using the method described in Example 1. The sensitivity of 845 drug-resistant clinical strains of various Staphylococcus species isolated from all over Poland to phages from the Staphylococcus-specific bacteriophage collection and the sensitivity of 880 drug-resistant clinical strains of various Pseudomonas species to phages from the Pseudomonas-specific bacteriophage collection were determined.

The analysis of the sensitivity of both groups of strains to specific bacteriophages allowed for the selection of phages with unexpectedly high activity and a broad spectrum of lytic activity. These phages are shown in Tables 1 and 2.

T a b e l a 1. Staphylococcus phages with high lytic activity deposited at the Polish Collection of Microorganisms (PCM), Wrocław, Poland.

MARKING FAG POLISH MICRO-ORGANISM COLLECTION (PCM) DEPOSIT ACCESS NUMBER S1 F / 00001 S2 F / 00002 S3 F / 00003 S4 F / 00004 S5 F / 00005 S6 F / 00006 S7 F / 00007

T a b e l a 2. Pseudomonas phages with high lytic activity deposited at the Polish Collection of Microorganisms (PCM), Wrocław, Poland.

MARKING FAG POLISH MICRO-ORGANISM COLLECTION (PCM) DEPOSIT ACCESS NUMBER 1 2 P1 F / 00008 P2 F / 00009 P3 F / 00010 P4 F / 00011 P5 F / 00012 P6 F / 00013

PL 201 750 B1 cont. table 2

1 2 P7 F / 00014 P8 F / 00015 P9 F / 00016 P10 F / 00017 P11 F / 00018 P12 F / 00019 P13 F / 00020 P14 F / 00021 P15 F / 00022 P16 F / 00023 P17 F / 00024 P18 F / 00025 P19 F / 00026 P20 F / 00027

The results obtained for phages S1-S7 and P1-P20 were subjected to statistical analysis in order to determine whether the p values obtained for individual phages can be considered true for the general population of the respective types of bacteria. The results of these studies are presented in Figures 1 and 2. For most bacteriophages, the calculated values of n were lower than the numbers of the tested samples of bacterial strains, which suggests that the obtained lytic spectrum ranges are true at least for clinical strains occurring in Poland. In view of these results, the phages listed in Tables 1 and 2 can be considered multivalent phages according to the invention.

Surprisingly, in the case of Staphylococcus phages, 3 out of the 7 most active phages, ie S1, S2, and S4 or S5 phages, are lytic on 95% of the Staphylococcus strains tested. Also, 3 out of the 21 selected Pseudomonas phages, ie P7, P20 and P6 phages, have a lytic effect on 87% of the tested Pseudomonas strains.

Cystic fibrosis patients may also be considered as "defined area of bacterial pathogens" as defined in the essence of the invention. 137 bacterial strains were used to study the pathogens present in this ecological niche, including 84 identified as Pseudomonas and 53 as S. aureus. All strains were isolated from materials obtained from patients with cystic fibrosis from all over Poland. The analysis of the sensitivity of the studied strains to Pseudomonas and S. aureus phages allowed for the selection of phages with the highest lytic activity and the broadest spectrum of activity. Surprisingly, in the case of Pseudomonas phages, 3 out of 21 active phages, ie, F / 00010, F / 00013, F / 00018, are lytic on 75% of the tested Pseudomonas strains. (See figure 3 for probability of success with phage 3 = F / 00010, phage 6 = F / 00013, phage 11 = F / 00018). Among the selected Staphylococcus phages, 3 phages: F / 00002, F / 00004, F / 00007 are lytic on 98% of Staphylococcus strains. (See figure 4 for probability of success with phage 1 = F / 00002, phage 3 = F / 00004, phage 4 = F / 00007).

Example 5. Propagation of Staphylococcus phages and Gram-negative rod phages, obtaining phage lysates.

About 5% phage lysate was added to a young 4-hour culture on a broth with glucose containing Staphylococcus aureus bacteria (log phase), test tubes or bottles were placed on a shaker and incubated for 3 hours at 37 ° C. After the culture light broke (lysis), the samples were left in the cold store until the next day. In order to remove the remaining non-lysed bacterial cells, the obtained lysates were filtered through membrane filters with a size of 0.2-0.4 μm. The concentration of live phage particles was determined by the two-layer Gratii method. The control was an incubated culture without the addition of phage lysate.

PL 201 750 B1

Phage propagation of gram negative rods (Pseudomonas). Peptone water was used to amplify Pseudomonas phages. To a young 4-hour culture of Pseudomonas aeurginosa, 5% phage lysate was added and, after placing the tubes or bottles on a shaker, the samples were incubated for 5 hours at 37 ° C. After the culture lightened, the samples were left in the refrigerator overnight, and then filtered through membrane filters with the pore size as above. The number of viable phage particles in the lysate was determined by the Gratii method. Sterile broth phage lysates show long-lasting lytic activity. Stored in a cold room, they remain viable for several years, and in some cases even for several years.

Example 6. Preparation and use of polyvalent phage preparations

Sterile broth lysates obtained as described in Example 5, derived from selected broad-spectrum lytic phages from Example 4, or their concentrated or further purified (see another example) forms, were used to prepare polyvalent phage preparations.

An exemplary polyvalent preparation of Staphylococcus phages is a mixture containing equal volumes of phage lysates S1, S2, and S4 or S5, or derivatives of these lysates. In the case of infections caused by Staphylococcus in patients with cystic fibrosis, preparations of phages F / 00002, F / 00004, F / 00007 should be used.

An exemplary polyvalent Pseudomonas phage preparation is a mixture containing equal volumes of P7, P20 and P6 phage lysates, or derivatives of these lysates. In the case of infections caused by Pseudomonas in patients with cystic fibrosis, preparations of phages F / 00010, F / 00013, F / 00018 should be used. The preparations were administered orally 3 times a day, in an amount corresponding to 10 ml of lysate, 30 minutes before a meal, after the gastric juices had been neutralized (e.g. with Gell Alumini phosphorici, soda or Vichy water). When administered directly to the wound, the preparations were applied 3 times a day. In the case of topical administration, do not wash the wound with disinfectants that could inactivate phages. If necessary, the wounds were washed with sterile medium or physiological solution. In some cases, phage therapy may be used in conjunction with antibiotic therapy. Polyvalent phage preparations used in the treatment of bacterial infections have proved to be highly effective. Particular efficacy was observed in the treatment of severe sepsis (88% of cures) and infections in patients with cystic fibrosis.

Example 7. Further purification of bacteriophage preparations; removal of endotoxins from a mixture containing bacteriophages.

For some medical applications or routes of administration (e.g., intravenous administration), it is desirable to have highly purified bacteriophage preparations, in particular devoid of bacterial endotoxins. The method according to the invention uses the affinity of bacteriophages for a bed containing cellulose or preferably its esterified derivatives. In an exemplary embodiment, a commercially available sulfate derivative of cellulose was used, characterized by a low degree of esterification (8 micromol / ml of gel). This bed was used to remove endotoxins from a mixture containing bacteriophages. After rinsing the bed and removing endotoxins, the next purification step is elution of adsorbed bacteriophages.

The bed containing the sulphated cellulose derivative was filled with a sterile chromatographic column. The flow of the bed from the column was prevented by lining the lower part of the column with glass wool soaked in 70% ethanol.

The following elution buffers were prepared:

Buffer I: 0.01 M phosphate buffer pH 7.6;

Buffer II: 0.01 M phosphate buffer pH 7.6 containing 1 M sodium chloride.

The salts of the eluent were calcined for 1 hour at 145 ° C.

Solutions were prepared using apyrogenic distilled water. Prior to chromatography, 1 ml of the chromatographic column packing bed was washed with 5 ml of 0.01 M phosphate buffer, pH 7.6, followed by 5 ml of 0.01 M phosphate buffer, pH 7.6 containing 1 M sodium chloride.

The column was prepared for the actual chromatography by washing it with 10 ml of 0.01 M phosphate buffer pH 7.6.

Endotoxin removal was performed using the high molecular weight fraction obtained by the Sepharose 4B Sepharose 4B concentrate phage Ps F-8 lysate. 0.2 ml of the mixture of bacteriophages and endotoxins was applied to a chromatographic column containing 1 ml of a bed containing sulphated cellulose derivative. 0.2 ml fractions were collected.

PL 201 750 B1

Fraction 1 was eluted with 3 ml of buffer 1. Under these conditions, unbound endotoxins flowed out of the column. The second fraction was eluted with buffer II. Fraction II contained purified bacteriophages.

Balance of endotoxin units in fractions:

The material that was chromatographed on the sulphated cellulose bed contained 2500 endotoxin units / ml. The fraction eluted with buffer I contained 600-1000 endotoxin units / ml and the fraction eluted with buffer II contained endotoxin-free bacteriophages (approximately 1 unit / ml). Endotoxin content in bacteriophage preparations was determined using the gel method from Charles River Endosafe, Charleston, USA.

ΒΡ / Α / Π / Ϊ2 page 14

THE BUDAPEST TREATY OF ANIMALS ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, PAh ul, R Weigla 12

53-114 Wroclaw

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATION FOR INITIAL DEPOSIT · »πη4τΐΜ 1

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: SI Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00001 II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The following is attached to the mdcroorganizórz specified in point 1: Scientific description ΓχΙ - proposed taxonomic designation Siphoviridae SI19. (Mark the correct square with a cross) ΙΠ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism, specified in point I, (Date of initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY ' Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^Date >? /.

Where article 6.4 (d) of the treaty applies, Laka date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form EP / 4 (single page)

PL 201 750 B1

BP / A / H / 12 page 24

TREATY Hf mAPFSTTAKTSKT Π XjTTHnTVTJA> ONOWVM RECOGNITION OF MICRO-ORGAN DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences. n ui_ · ui.n.neigia 53-114 Wrocław

LIFE CERTIFICATE issued in accordance with 2 Article 10.2 by

POLISH COLLECTION OF MICRO-ORGANISMS

NAME 1 PAGE ADDRESS. FOR WHICH THE LIFE CERTIFICATE HAS BEEN ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: R. Weigla 12 53-114 Wroclaw Accession number given by: POLISH KOI ^ g ^ ICROORGANISM: Date of deposit or transfer ¹ : 07/10/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point II has been checked on 01/09/2001 ² . On that day, the tested microorganism was El · alive Π '· 1—1 me alive

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select a circle for the appropriate square

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / ΑΉ / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrodaw Signature of the person representing PCM or the authorized person (s): Date:

⁴ Fill ⁱⁿ if the information is required and if the test results negative entities.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSE OF PATENT PROCEDURES INTERNATIONAL QUANTITY

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: S2 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / OODG2 SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point I is accompanied by: □ scientific description ί ^ν 1 proposed taxonomic determination sleep lumiuac ou. (Mark the correct square with a cross) 4ΠΙ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism, specified in point L (Initial Datadeposit) ¹ 19.07,2001 IV, INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrociaw Signature of the person representing PCM or the authorized person (s) person (s):

¹ Where Article € .4 (d) of a treaty applies, this date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism,

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / ΑΖΠ / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ul. R Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

AND, SUBMITTED Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ^Address: ul.R.Weigla 12 53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / 00002 Date of deposit or transfer ¹ : 07/18/2001 ΠΙ. LIFE CERTIFICATE The viability of the micro-organism specified in point II, was checked on 1.09.2001 ^{2 on} days ^the tested microorganism was OD ³ alive Cl not alive

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases specified in an. 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

BP / AOT12 Page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: P olish Microorganisms Collection (PCM) A Tmiwiimnlrtrii ł TP ^ rannT ^ rtiwiJlHcTJllnfll a ~ aaaa *> i »<UteMJ SUS · · -» · »!«> · «r» »’ łw> »T ——’ J Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s) (vY 2cx? /

* Fill in when information is required and the test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

BP / A / H / 12 page 14

THE ARESHAN BUILDING TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSE OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunolonia and Experimental Therapy, Polish Academy of Sciences, ul. R. Weipla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by The DEPOSER; S3 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / OOD03 11. SCIENTIFIC DESCRIPTION OF FAVORITES PROPOSED TAXONOMIC MARKING The micro-organism specified in point 1 is accompanied by: □ scientific description - | _x and 1_1 proposed taxonomic designation Siphoviridae SI19. (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism, specified in point L Pata of the initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name; Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s) i Date: / {a, f 7 ^ '

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date of receipt of the micro-organism by the international depository orean.

Form BP / 4 (single page)

PL 201 750 B1 aP / A / n / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10U by

POLISH COLLECTION OF MICRO-ORGANISMS

NAME 1 ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

i. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: and _ns title of Immunology and Experimental Therapy of the Polish Academy of Sciences ^Address: ul.R.Weigla 12 53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / 00003 Date of deposit or transfer ¹ : 1S.07.2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point 11, was checked on 01/09/2001. on that day, the test microorganism was Ξ ³ alive, EU ³ dead

wanitz oaii nepozyiu puwąLKowego iuo, gay oo A new deposit has been made, and the latest appropriate date will be transferred.

In the cases specified in Article 10.2 (a) (u) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. RudoUh Weigla 12 53-114 Wroclaw Signature of the person representing PCM or the autotized person (s): Date: ot)

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

ΒΡ, Ά / Π / 12 page Η

FORM INTERNATIONAL Institute of Immunology and Experimental Era therapy of the Polish Academy of Sciences ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: 54 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 0D004 U. SCIENTIFIC DESCRIPTION. PLUB PROPOSED TAXONOMIC ASSIGNMENT □ o the micro-organism specified in point I is accompanied by: □ scientific description I χ I 1 1 proposed taxonomic designation Siphoyiridae SI10 (Mark the correct square with a cross) Ul. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism, specified in point !. (Initial deposit date) ¹ 19.D7.2QO1 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date:

¹ Where Article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such distance is still a further receipt of the micro-organism by the international depository authority.

Form BP / 4 (single page)

PL 201 750 B1

INTERNATIONAL BUDAPEST TREATY

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 24

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM ^Nazara: FAN Atłręs Institute of Immunology and Experimental Therapy: ul. R. Weigla 12 53-114 Wrocław Accession number given by; ΒΛΤ Cl / λ ΤΓΛΥΤ> ηντίΛΛηρ * αλ.ττ »τύ r. Date of deposit or transfer ¹ ; 10/07/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point IL was checked ^on 01.05.2001. on this day, the tested microorganism was 0 'im 1_1'

Please indicate the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

'In the cases specified in arL 10.2 (a) (ii) and (iii), specify the most recent lifetime test,' Mark the appropriate square with a cross.

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / ΑΛΙ / 12 page 15

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ui. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^Date:

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

INTERNATIONAL BUDAPEST TREATY

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

BP / A / H / 12 page 14

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy PA1 ul.R.Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM the identification capacity provided by The DEPOSER; S5 Accession number given by; POLISH COLLECTION OF MICRO-ORGANISMS: F / 00005 SCIENTIFIC DESCRIPTION MLUB PROPOSED TAXONOMIC MARKING It is attached to the micro-organism specified in point I L 1 scientific description Γχΐ. . 1—1 proposed taxonomic designation 5iphoviridae 5119 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point L (Date of initial deposit) ¹ IB. 07.21X11 IV. INTERNATIONAL DEPOSIT AUTHORITY .......... - Name: Pole's Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Under the person representing PCM or (·) person <y):

Where Article 6.4 (d) of the treaty applies, this is the date on which international depository authority status was acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

INTERNATIONAL BUDAPES2 TREATY

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 24

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10Λ by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: R. Weigla 12 53-114 Wroclaw Accession number issued by: POLISH COLLECTION OF MICRO-ORGANISM: F / DD005 Date of deposit or transfer ¹ : 07/18/2001 ΙΠ, LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was checked on 1.09.2001 L On this day, the tested microorganism wasl ί x | ^L 1 alive and dead ³

¹ Scroll to the date of the initial deposit or when made new deposit or transfer of appropriate _and most recent date.

In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

BP / A / W12 page 25

rv. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED * V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrodaw POdplS USftłtJj / ΓΟΜ Feb Matof ^ ttUk-nraAĄ person (s): Date:

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

PL 201 750 B1

BP / A / H / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology i

Experimental Therapy of the Polish Academy of Sciences ul. R. Weigla 12 53-114 Wrocław

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

Γ SUBSCRIBER- II. IDENTIFICATION OF MICRO-ORUANISM- Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: _u irWeigla 12 53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / OOOOS Date of deposit or transfer ¹ : 07/18/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was tested in ² · 01/09/2001 ^Ty ™ day of the test microorganism was alive U ^3. 1 l ³ is not alive

Please indicate the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / Ι2 page 25

rv. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy O / b1cVi> t Alrarfaimti A waeswnrj a λλλλλ ^^ τ ·· ~ ^ »- - ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): AND , fZ-l, Date; and in /

⁴ Fill in when information is required and the test results were negative

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Innunoloaii and Experimental Therapy of the Polish Humanitarian Organization ul. R. Weigla 12

55-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH MICRO-ORGANISM COLLECTION

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Therapy ΠαΧι.μ 1 on A PAKI υυθ) Ί1θυ ^ ώβ1Ι ISJ »ni i Address: R. Weigla 12 55-114 Wroclaw Accession number given by: dat cva νιΊτϊνητρ xfTVDnnbr.ńMT7MnW · F / 00007 _s Date of submission of the deposit: III. LIFE CERTIFICATE The viability of the micro-organism specified in point Π »was checked in 2001 ³ - On this day the tested microorganism was DD ³ alive. 1 and ³ dead

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date. .

² In the cases specified in ort. 10.2 (a) (u) orna (lii), he reduced the most recent teet of vitality, ³ Criticize the appropriate square

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴

V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ΰί. ^ Date: C * ?, Leo · /

⁴ Complete if information is required and test results were negative.

Form ΒΡΛ (page two and last)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

THE BUDAPEST TREATY OF ANIMALS ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy PA J ul. R. Weipla 12

53—114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT »« awt njuawtuw i> guuxuv <> ui ·

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER PI Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS; F / 00008 SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point I is accompanied by: 1 1 scientific description 1 χ | 1_1 proposed taxonomic designation Myoviridae MII. (Mark the correct square with a cross) ΙΠ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Initial deposit date) 'July 18, 2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date- "->

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; tara where, after acquiring this status, a deposit that does not comply with the Budapest Treaty is converted into a deposit in accordance with the treaty, such date is the date of receipt of the micro-organism by the international depository authority.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

ΤΙΙΑΚΤΑΤ STJDAJPSC2 * rJO’TGKX Ο

RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ul-R Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10 J by

POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE DEPOSER, U. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Therapy Π-Χ, .4 DALI UJOHJ.aUVABil ICJ 1 ΓΜΊ Address: ul. Weigl 12 53-114 Wroclaw Accession number given by: POLAND COLLECTION OF MICROORGANISM: F / GOOOB _Ł Date of submission of the lab transfer of the deposit: 07/18/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point U, was checked on 1.09.2001 - On this day the tested microorganism was Q ^and live Q was dead

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases specified in Art. 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / Π page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY ' Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy AV «rf4 * mii WanV f UlOlUtij * '« ł ** » ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: /? /, £ * ?, ^c

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. R. Weigla 12

53-114 Wroclaw

THE LIFE CERTIFICATE issued in accordance with Article 10.2 by

POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ^Address: R. Weigla 12 53-114 Wrocław Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00009, Date of deposit or transfer: July 18, 2001 m. LIFE CERTIFICATE The viability of the micro-organism specified in point U, was checked i Q9 2DD1 ² - On this day the tested microorganism was Θ ³ tZl not alive

¹ Indicate the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases specified in Article 10.2 (a) (ii) and ¢ 11), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immuologli i

Experimental Therapy of the Polish Academy of Sciences ul.R.Weiola 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATION FOR INITIAL DEPOSIT wvAmna 7 srt 7 * 1 rti-gąą

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P3 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 0001C II. SCIENTIFIC DESCRIPTION OF THE FAVORITES PROPOSED TAXONOMIC MARKING The micro-organism specified in point I is accompanied by: i- 1 scientific description LjJ proposed taxonomic designation Mvoviridas MI9. (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 10.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: z7 <f. i ??, l

Where Article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

BP / A / D712 page 24

INTERNATIONAL FORM FOR THE PURPOSE OF THE PATENT PROCEDURE

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED ^{1 * 3}

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: i _ns t _y t _u t Immunology and Experimental Therapy of the Polish Academy of Sciences Address: ul.R.Weigla 12 53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / 00010 Date of deposit or transfer ¹ : 07/18/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was checked on 01/09/2001 *. On that day, the microorganism was tested Β »' L l dead

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases specified in Article JO.2 (a) (ii) and (iii), determine the most recent viability test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: im> '

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (side drags and last)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy PAII R. Weigla 12

53-114 Wroclaw

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P4 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / oooir II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point I is accompanied by: 1_! scientific description 1 Y ί prop.nov.nne ο = λ ^ οκολΪο ttltoęnenuOTip MvnVT Γ1 riaP MT3 (Mark the correct square with a cross) III, CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 16.07.2001 IV, INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^Date: 7,

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / Π page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

1 SUBSCRIBER U. IDENTIFICATION OF THE MICRO-ORGANISM ' Name: FAN Institute of Immunology and Experimental Therapy ^Address: R. Weiola 12 53-114 Wrocław Accession number given to the owner: POLISH MICRO-ORGANISM COLLECTION: F / 00011 Date of deposit or transfer ¹ : 07/18/2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in R was checked on 01/09/2001. On that day, the test organism was θ '±> ΛΥ _Ύ □ ³ dead

Please indicate the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases specified in Article 10.2 (a) (ii) ora2 (iii). determine the latest life test

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED * V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM} Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^{Da, a:} / [r

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

AN INTERNATIONAL BUDAPEST TREATY

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

ΒΡ / Α / Β / 12 page 14

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P5 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00012 II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point I is accompanied by: 1 "J scientific description C3 proposed taxonomic designation Myoviridae MI9 (Mark the correct square with a cross) IU. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 1B. 07.2001 Tir W /ΓΡΤ\ΤΙΛ.Τ ΑΛΛΓιΛ, ΙΓΙ? Ζ \ Τ »isilt xv, i ri / usAJL / vvr i UEruii 1UVY ϊ Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrodaw Signature of the person representing PCM or the authorized person (s):

¹ Where Article 6.4 (d) of the treaty applies, that date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 24

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE LABEL issued in accordance with Article 10.2 by the POLISH MICRO-ORGANISM COLLECTION

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology i Experimental Therapy of the Polish Academy of Sciences Address: R. Weigla 12 53-114 Wroclaw Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00012, Date of deposit or transfer: July 18, 2001 IH. LIFE CERTIFICATE

in 1.09 H ³ alive □ ’ dead

^! . On this day, the tested microorganism was ¹ ΐιΛτ, & Jj dwKwiuuiw m, transfer of the most recent date.

² In the cases specified in ait 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross.

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

rv. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^Date; / t & iCfy.

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

BP / A.W12 page 14

INTERNATIONAL FORM

Institute of Immunology i

Experimental Therapy ΡΑΓν ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Art. 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: Sex Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00013 U, UPJS SCIENCE AND / OR PROPOSED TAXONOMIC MARKING The micro-organism specified in point I is accompanied by: 1 — J scientific description pq 1 — I proposed taxonomic designation Myaviridae MI3 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE '- The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ⁵ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY '..... ~ ........ Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s) person (s):

Where Article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Instytut Immunologii i Terapii Doświadczalne /! PAN ul R. Weigla 12 53-114 Wrocław

LIFE CERTIFICATE! issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED ^{1 * 3}

I. THE SUBSCRIBER H. IDENTIFICATION OF THE MICRO-ORGANISM Name; Institute of Immunology i Accession number given by; Experimental Therapy and the wound POLISH COLLECTION OF MICRO-ORGANISMS · '"7/00013 ^Address: R. Weiola 12 Date of deposit or transfer of the deposit ¹ : 53-114 Wroclaw 1Θ.07.2001 ΠΙ. LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was checked on 01/09/2001. On that day, the microorganism was tested 0 'tw Q 1 1 dead

¹ Specify the extent of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: i ^rx - \

Fill in when information is required and the test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / Π page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

Institute of Immunology i

Experimental Therapy PAb ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P7 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00014 Π. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT Attached to the microorganism identified in heel J is □ scientific description | χ i 1—1 proposed taxonomic designation Myoviridae MI 9. (Mark the correct square with a cross) III. CERTIFICATION 1 ACCEPTANCE The Polish Microorganism Collection accepts the ingested microorganism specified in point I, (Initial deposit date) ¹ ^ '2001 IV. MUSCLE MUCH OroAn deposit t-? cM.rra. PuliKa KuletaJafrlllUUUrgillllZinOW IPUMJ Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw FC List of the person representing PCM or authorized person (s):

Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER U. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: R. Weigla 12 53-114 Wroclaw Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / 00014 Date of deposit or transfer ¹ : 07/18/2001 III. LIFE CERTIFICATE The viability of the micro-organism specified in point Π. was checked on 01/09/2001. On that day, the tested microorganism was Γχ [3 alive □ ^{1 * 3} dead.

¹ Indicate the date of the initial deposit or, when a new deposit or transfer was made, the newest appropriate one

In the cases specified in Article 10.2 (a) (ii) and (iii), it will determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Imunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

1, IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P8 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00015 Π. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point I is accompanied by: CD scientific description K 1 proposed taxonomic designation Podoviridae PI3 (Mark the correct square with a cross) • UL CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point L (Date of initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY f Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s) person (s): Date:,?

¹ Where article 6.4 (d) of the treaty applies. such date is the date on which the status of international depositary authority is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER U. IDENTIFICATION OF THE MICRO-ORGANISM ΪΙοχμιι. ZnoLyLuL IlJhllUl IUluy ± ± X Therapy PAN Experimental Station Address: R, Weigla 12 53-114 Wroclaw autical manner naaudowane by: POLSKA KOL ^^ MICROORG ANIZMÓW; Date of deposit or transfer ¹ ; IB.07.2001 1Π. LIFE CERTIFICATE The viability of the micro-organism specified in point II was checked on 1-09.2001 and the eaten micro-organism was Q ³ alive Π 1 I not alive

¹ Enter the date of the initial deposit or, when a new deposit or transfer is made, to the latest appropriate date.

In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross.

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

iv. conditions under which the viability test was performed ⁴ V. international depository authority Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Μϊ "<!,

⁴ Fill in if information is required and if the test results were negative

Form BP / 9 (second and last page)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 24

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ^Address: ul.R.Weigla 12 53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM REGION: Date of deposit or transfer ¹ : 10.07.2001 m. LIFE CERTIFICATE The viability of the micro-organism specified in point Π was checked ^on 01/09/2001. On that day, the tested microorganism was B ³ alive n, · l — J dead

Specify the date of the initial deposit or if a new deposit or transfer was made to the latest applicable date.

In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

BP / A / H / ΙΪ page 25

⁴ Complete when information is required and test results were negative.

Form BP / 9 (side drags and last)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 14

Institute of Immunology and Experimental Therapy PA I R. Weigla 12

53-114 Wroclaw

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P1D Accession number given by; POLISH COLLECTION OF MICRO-ORGANISMS: F / 00017 u. SCIENTIFIC DESCRIPTION AND / OR PROPOSED MARKING TAKSON <Wię7NP ..... jud of the micro-organism specified in point_I is accompanied by: C-J Scientific Description [-3 proposed taxonomic designation Myoviridae M13 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE .................... ...... The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Society of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^Da “> .r.

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; tara where, after acquiring that status, a deposit not in accordance with the Eudapestan Treaty is converted into a deposit in accordance with that treaty, such date shall be the date on which the international depositary authority received the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

BUDAPEST TREATY ON INTERNATIONAL

RECOGNITION OF A MICRO-ORGANISM DEPOSIT

FOR PATENT PURPOSES

INTERNATIONAL FORM

ΒΡ / Α / Π / 12 page 24

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name; Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences Address; m, r. Weigl 12 53-114 Wroclaw Accession number given by; POLISH COLLECTION OF MICRO-ORGANISMS: F / 00017 Date of deposit or transfer: July 18, 2001 IH. LIFE CERTIFICATE Life of a microorganism referred to in point Π, was proven ⁱⁿ 1,09.2001 ™ you studied microorganism was a lively Q U ³ dead

³ Specify the starting date, or when a new deposit or transfer was made, to the newest one. the appropriate date.

³ In the cases specified in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM " Identification reference given to pizza The DEPOSER; Pil Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00018 II. SCIENTIFIC DESCRIPTION OF THE FAVORITES PROPOSED TAXONOMIC MARKING The micro-organism specified in point 1 shall be accompanied by: EU scientific description 01 proposed taxonomic designation Myaviridiae MI3 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE "' The Polish Collection of Microorganisms accepts the obtained microorganism, specified in point! (Initial deposit date) ¹ 07/19/2001 IV. INTERNATIONAL DEPOSIT AUTHORITY - ~ - Name? Pnfolrn KoTelceyn MilrmcrgnTUDniw (PCM) Address; Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, ul. Rudolfa Weigla 12 53-U4 Wroclaw Pędpb ojoby represent ^ this PCM or authorized {ej person (s): CX * Lc-J ’/ / £

Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM.

Institute of Immunology and Therapy Dodwiiąricząlnei PAS ul. R, Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with arL 10.2 by POLAND COLLECTIONS OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

L DEPOSER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: _U 1.R.Weigla 12 53-114 Wrocław Accession number given by: οητ cva rnizrrrp κπνρπηόΠΑνττΜΛνσ F / 00018 Date of deposit or transfer: July 18, 2001 III. LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was inspected on 1 09 2001. On that day, the microorganism was tested S alive Q dead

¹ Indicate the date of the initial deposit, or when a new deposit was made, or at the latest appropriate date.

² In the cases specified in Art. 10.2 (a) (ii) and (iii), determine the most recent freedom test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM insxyxux Immunologist and Experimental Therapy PAi R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P12 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00D19 Π, SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXSONOMIC MARKING The micro-organism specified in point J shall be accompanied by: □ scientific description ΓχΊ L_J proposed taxonomic designation SiDhoviricJae SI7. (Mark the correct square with a cross) ΙΠ, CERTIFICATION 1 ACCEPTANCE Polish Collection of Microoreanisms. The acclaimed mikmersnniTm nlrwilnny w pnnlrn »1 (Initial datadeposit) ια.υ / .zuui IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrodaw Signature of the person representing PCM or the autotized person (s) person (s): /! <$. T ^ a t Z

¹ Where Article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

THE AFHESIAN BUD TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: and _ns title of immunology and experimental therapy of the Polish Academy of Sciences Address: ul.R.Weigla 12 53-114 Wrocław Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00019 Date of deposit or transfer: 1Θ.07.2001 IU. LIFE CERTIFICATE The viability of the micro-organism specified in point Π was checked in 1.09.2DD1 ^{1 2} . On this day, the investigated microorganism was KI ³ alive Q nonworms

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases specified in Article 10.2 (a) (u) and (iii), determine the most recent viability test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, unit R. Weigla 12 53-112 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATION FOR INITIAL DEPOSIT łł n η Λ łrtn / ^ no · »* ιι4 1 fijuatrous · & gvurub s, tuk ι · λ

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P13 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / D0O2D. U. SCIENTIFIC DESCRIPTION OF FAVORITES PROPOSED TAXONOMIC MARKING The micro-organism specified in point I is accompanied by: Π scientific description H proposed taxonomic designation Podoviridae Pil. (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point L (Date of initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: / $ 1, O <?. Lol »/ / ^c

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴

V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s):

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE MICRO-ORGANISM DEPOSIT FOR THE PURPOSE OF THE PATENT PROCEDURE

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by The DEPOSER; P14 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00021 11. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT It is attached to the micro-organism specified in point I □ scientific description t3 proposed taxonomic designation Myoviridae MI3 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date pQcoqd <9Yvoge) l 18.0 * 7.3001 TV MIFn7VWADAnAU / VflDr, AXTTYCDriTVTiuiJV ............ ....... ............... - · yy Wliiwru. WW JU Π 1 Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy Polish Academy of Sciences ul. Rudolf Weigl 12 53-14 Wroclaw Signature of the person representing PCM or the authorized person (s) person (s): Date: z / dT, '' Loot '

Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

THE LIFE CERTIFICATE issued in accordance with Article 10.2 by

POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER U. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address - ul. R. Weigla 12 '53-114 Wrocław Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / D0021 Date of deposit or transfer ¹ : IB.07.2001 m. LIFE CERTIFICATE The viability of the micro-organism specified in U has been checked on 01/09/2001. On that day, the test microorganism was 0 ' _w □ ³ dead

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases referred to in Article 10.2 (a) (ii) and (iii), specify a more recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED

V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) a a in Tnrtvtiif imtmmnloeii and Terami Doświadczalna Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the autotized person (s): Date: £> rf ₍ ™ <-

⁴ Complete if information is required and if test results were negative,

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy and Polish Academy of Sciences H.R.Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P15 Pizza accession number: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00022 Π. SCIENTIFIC DESCRIPTION OF THE FAVORITES PROPOSED TAXONOMIC MARKING The micro-organism specified in point I is accompanied by: 1 1 scientific description □ proposed taxonomic designation Wyovirldae MI3 (Mark the correct square with a cross) III. CERTIFICATION AND ACCEPTANCE " The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 1B. 07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): "ZM, C7,

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which it acquires the status of international depository authority; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ul. R.WRłola i?

53-114 Wroclaw

LIFE CERTIFICATE

K Art. 10.2

POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

L DEPOSER U. IDENTIFICATION OF THE MICRO-ORGANISM Name: j _ns | y |. _u -t immunology and experimental therapy of the Polish Academy of Sciences Address: ul. R. Weigla 12 53-114 Wrocław Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00022 Date of deposit or transfer: 10.D7.2001 III. LIFE CERTIFICATE The viability of the micro-organism specified in point Π, was inspected on 1 09 2001. On that day, the tested microorganism was Γχ1 ₃ L_J alive Q dead

¹ Enter the date of the initial deposit or, when a new deposit or transfer is made, the latest applicable date.

³ In the cases referred to in Article 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS UNDER WHICH THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: (? · ?, LO ^ t

⁴ Fill in when information is required and test results were negative.

BP / 9 form (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Imnology and Experimental Therapy, Polish Academy of Sciences ul. * R.Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 10.2. POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Therapy DAM erase my and my mia Address: R. Weigla 12 53-114 Wroclaw Accession number given by: pn ^ gy a COLLECTION OF MICROORGANISMS: * ~ V / 00Q23 ** Date of submission of the deposit transfer ¹ : 13.07.2001 ΙΠ. LIFE CERTIFICATE The viability of the micro-organism specified in point E has been checked in and in 2001. On this day, the tested microorganism was 1_I ³ alive Q dead

¹ Indicate the date of the initial deposit or, where a new deposit was made, or a transfer to the most recent applicable date.

² In the cases specified in Art. 10.2 (a) (ii) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross.

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV. CONDITIONS. WHERE THE LIFE TEST WAS PERFORMED ⁴

V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date. ^^, ^ ioo l

⁴ Fill in when information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES JLARZ INTERNATIONAL

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

L IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P17 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00024 Π, SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The micro-organism specified in point 1 is accompanied by: L-J Scientific Description LxJ proposed taxonomic designation Siphoyiridae 5112. (Mark the correct square with a cross) ΙΠ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ 10.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date:

Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

THE LIFE CERTIFICATE issued in accordance with Art. 10.2 by the POLISH MICRO-ORGANISMS COLLECTION

L DEPOSER U. IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: R. Weigl 12 53-114 Wroclaw Accession number given by: POLISH MICRO-ORGANISM COLLECTION: F / OOD24 _χ Date of making or transferring the deposit ¹ : 07/18/2001 IU. LIFE CERTIFICATE The viability of the micro-organism specified in point Π was checked on 01/09/2001. On that day, the test micro-organism was LI ³ alive □ ³ dead

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

² In the cases specified in Article 10.2 (a) (u) and (iii), determine the most recent validity test.

Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 25

IV, CONDITIONS. WHERE THE LIFE TEST WAS PERFORMED ⁴ V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Λ Date: ^Date -Λ <i ?, ¢ 9 ^ 7. 'T - ** ⁷¹

^Α Fill in when information is required and the test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

PAEI Institute of Immunology and Experimental Therapy ul. R. Weigla 12

53-114 Wroclaw

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATION FOR INITIAL DEPOSIT • 'r Π 1 I \ rłP7 tołSEą ^ lekc ^ mkkiorganisms

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P18 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00025 Π. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The domicroorgnism specified in point 1 is attached with: CD scientific description jx 1 proposed taxonomic designation Myoyiridae MI3. (Mark the correct square with a cross) IU. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I. (Date of initial deposit) ¹ IB. 07.2001 M r% SM> 1 Λ ΛΓΐ / ikT ι · / kitnp IV. I υΐνυΛΙ ¹ * UCWil 1UYY I Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): ^{Da £ Ł} 'λ

¹ Where article 6.4 (d) of the treaty applies, that date is the date when international depository authority status was acquired; taro where, after acquiring this status, a deposit that does not comply with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R-Weigla 12

53-114 Wroclaw

SITE NAME AND ADDRESS.

FOR WHICH THE LIFE CERTIFICATE HAS BEEN ISSUED

LIFE CERTIFICATE issued in accordance with Article 10Z by the POLISH COLLECTION OF MICRO-ORGANISMS

I. THE SUBSCRIBER IL IDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address; _u L, 12 R. Weigla 53-114 Wroclaw Accession number given to pizza: _ cva νητίΠίΓΓΕ MICROORGANISMÓW: * - -------- F / 00025 _t Date of deposit or payment; 18> 07.2001 HI, LIFE CERTIFICATE The viability of the micro-organism specified in Π. was checked _on September 1, 2001 \ The tested microorganism was 0 ^{3 years old} Q dead _ ——

¹ Specify the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date. .

² In the cases specified in Article 10.2 (a) (ii) and fiu), determine the most recent viability test.

$ Mark the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

PL 201 750 B1

ΒΪ / ΑΛ3 / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy PAb ul. R. Weigla 12 53-114 Wrocław

NAME 1 ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued in accordance with Article 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER: P15 Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / aOOZ6 SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC ASSESSMENT The microorganism specified in plct.I is accompanied by: □ scientific description Γχ | L_J proposed taxonomic designation Mvoviridae MI3 (Mark the correct square with a cross) ΙΠ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the obtained microorganism specified in point I (Date of initial deposit) ¹ 18.07.2001 IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s): Date: _Λ

¹ Where article 6.4 (d) of the treaty applies, that date is the date on which international depository authority status was acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with joke 10.2 by the POLISH MICRO-ORGANISM COLLECTION

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. tDENTIFICATION OF THE MICRO-ORGANISM Name: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Address: R. Weigla 12 53-114 Wroclaw Accession number given by: POLISH MICRO-ORGANISM: Date of deposit or transfer ¹ : 07/18/2001 ΙΠ. LIFE CERTIFICATE The viability specified in point Π has been checked. in '. On this day the test microorganism was ³ El alive n> 1-b dead

Please indicate the date of the initial deposit or, when a new deposit or transfer was made, the latest applicable date.

In the cases of 10.2 (a) (Π) and (iii), determine the most recent viability test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

ΒΡ / Α / Π / 12 Page 25

IV. CONDITIONS. WHERE THE LIFE TEST WAS PERFORMED ⁴

V. INTERNATIONAL DEPOSIT AUTHORITY

Name: Polish Collection of Microorganisms (PCM)

Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolfa Weigl 12 53-114 Wrodaw

Signature of the person representing PCM or the authorized person (s):

Date: y, τχχ? /

⁴ Fill in when Information is required and test results were negative.

Form BP / 9 (second and last page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 14

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12 53-114 Wrocław

NAME AND ADDRESS OF THE DEPOSER

CERTIFICATE FOR INITIAL DEPOSIT issued pursuant to Art. 7.1 by

POLISH COLLECTION OF MICRO-ORGANISMS

I. IDENTIFICATION OF THE MICRO-ORGANISM Identification reference provided by OF THE DEPOSITER. P20 ' Accession number given by: POLISH COLLECTION OF MICRO-ORGANISMS: F / 00027 Π. SCIENTIFIC DESCRIPTION 1 / OR PROPOSED TAXONOMIC ASSESSMENT Attached to the micro-organism specified in point 1 is; And the first scientific description (x 1, t__1 proposed taxonomic designation Myoviridae MI? . (Mark the correct square with a cross) ΙΠ. CERTIFICATION AND ACCEPTANCE The Polish Collection of Microorganisms accepts the microorganism specified in point I. (Initial deposit date) ¹ ' IV. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wroclaw Signature of the person representing PCM or the authorized person (s) person (s):

¹ Where article 6.4 (d) of the treaty applies, such date is the date on which international depository authority status is acquired; where, after acquiring this status, a deposit not in accordance with the Budapest Treaty is converted into a deposit in accordance with that Treaty, such date shall be the date on which the international depositary authority receives the micro-organism.

Form BP / 4 (single page)

PL 201 750 B1

ΒΡ / Α / Π / 12 page 24

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF MICRO-ORGANISM DEPOSITS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12

53-114 Wroclaw

LIFE CERTIFICATE issued in accordance with Article 102 by the POLISH COLLECTION OF MICRO-ORGANISMS

NAME AND ADDRESS OF THE PARTY FOR WHICH THE LIFE CERTIFICATE IS ISSUED

I. THE SUBSCRIBER Π. IDENTIFICATION OF THE MICRO-ORGANISM Name, j _ns ^ y ^ of immunology and experimental therapy of the Polish Academy of Sciences Address * 'R. Weigla 12 ^ς 3-114 Wrocław Accession number given by: POLISH COLLECTION OF MICRO-ORGANTASMS: 'F / 00027 Date of deposit or transfer: 10/07/2001 HI. LIFE CERTIFICATE The viability of the microorganism specified in point Π was checked on 01/09/2001 - ^on this day the tested microorganism was S alive EJ ³ dead

¹ Indicate the date of the initial deposit, or, if a new deposit was made, or transferred to the most recent relevant date.

² In the cases referred to in Article 10.2 (a) (ii) and (iii), specify a fresh lifetime test.

³ Select the appropriate square with a cross

Form BP / 9 (first page)

PL 201 750 B1

BF / AZH / 12 page 25

IV. CONDITIONS. IN WHICH THE LIFE TEST WAS PERFORMED *

V. INTERNATIONAL DEPOSIT AUTHORITY Name: Polish Collection of Microorganisms (PCM) Address: Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences ul. Rudolf Weigl 12 53-114 Wrodaw Signature of the person representing PCM or the authorized person (s): ^Date: / t &, CQ,

* Fill in when information is required and the test results were negative.

Form BP / 9 (second and last page)

Claims (29)

1. The method of obtaining a multivalent strain of bacteriophage, characterized in that: (a) a sufficiently large collection of n different strains of bacterial pathogens of a certain type occurring randomly in the area is gathered, preferably the isolated bacterial strains are drug-resistant strains, (b) accumulating a sufficient number of different strains of bacteriophages specific to at least one strain of the bacteria belonging to the genus, c) the lytic activity of the collected bacteriophage strain against each collected bacterial strain is determined, and then the p-value for the bacteriophage strain is calculated as the ratio of the number of strains lysed by a given phage to the number of all bacterial strains collected, d) for the obtained value of p, it is checked whether the numerical value of n meets the condition: pq _z 2 d ^{2 ι-α} where: q = 1-pd is a constant not greater than 0.1, preferably not greater than 10% p, z _1-a is a normally distributed random variable depending on the confidence interval 1-α, which is not less than 0.95, e) a bacteriophage strain is selected which meets the criterion defined in d) and is characterized by a p value of not less than 2 / n, preferably not less than 0.5. 2. The method according to p. A method according to claim 1, characterized in that in step b) the bacteriophage strain is isolated from the sample coming from the environment by filtration through membrane filters with a pore size of 0.20.4 µm, a nutrient medium is added to the obtained filtrate and mixed with the broth culture of the specified bacteria. type, incubated at about 37 ° C for about 1 hour, an aliquot of the suspension is taken and rubbed on plates with a solid medium, incubated at about 37 ° C for about 2 to 24 hours, a sample of the medium is isolated including a single plaque, it is transferred to a broth culture of a certain type of bacteria and incubated until the culture lightens, and the obtained lysate is filtered through membrane filters with a pore size of 0.2-0.4 µm to obtain a bacteriophage preparation, preferably sowing on a solid medium and the plaque re-isolation is repeated 5 times. 3. The method according to p. 1 or 2, characterized in that in step c) the tested bacterial strain is sown on a suitable solid medium, onto which a portion of the bacteriophage preparation obtained in step b) is applied, and incubated at about 37 ° C for about 4 hours after which is left at the temperature of about 4 ° C for about 2 to 24 hours, the lytic activity of the bacteriophage strain is evidenced by the appearance of at least single plaques. 4. The method according to p. The method of claim 1, wherein the pathogenic bacterial strains belong to the genus Staphylococcus or Pseudomonas. 5. The method according to p. 1 or 4, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus occurring in Poland, isolated in step e) is a bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00005), S6 (PCM F / 00006) and S7 (PCM F / 00007). 6. The method according to p. 5, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus present in cystic fibrosis patients isolated in step e) is a bacteriophage strain selected from S2 (PCM F / 00002), S4 (PCM F / 00004) and S7 (PCM F / 00007). 7. The method according to p. 1 or 4, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas occurring in Poland, isolated in step e) is a bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022) , P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027). PL 201 750 B1 8. The method according to p. The method according to claim 7, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas present in cystic fibrosis patients isolated in step e) is a bacteriophage strain selected from P3 (PCM F / 00010), P6 (PCM F / 00013) and P11 (PCM F / 00018). 9. The method according to claim 1 or claim The process of claim 2, wherein the lysate is further purified, especially from endotoxins, wherein the mixture containing the bacteriophages is contacted with the bed containing cellulose or a partially esterified derivative thereof, then washed with a solution that removes contaminants, especially endotoxins, and then the purified bacteriophages are washed. 10. The method according to p. 9. The method of claim 9, characterized in that the elution of endotoxins is performed with water, a solution of a non-dissociating substance or a saline solution with a concentration not greater than 0.1 M, preferably buffered. 11. The method according to p. 9. A method according to claim 9, characterized in that the elution of the bacteriophage fraction is carried out with a solution of a non-dissociating substance or any buffer or salt solution with a concentration higher than 0.05 M, preferably buffered. 12. The method according to p. 9. The method of claim 9, characterized in that elution of endotoxins and bacteriophages is carried out at a temperature of -25 ° C to 100 ° C. 13. The method according to p. 9. The method of claim 9, wherein the elution of endotoxins and bacteriophages is performed using an aqueous salt solution containing an organic solvent. 14. The method according to p. The process of claim 13, wherein the organic solvent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, isopropanol, and acetone. 15. The method according to p. The process of claim 9, wherein the resin is cellulose partially esterified with an organic or inorganic acid. 16. The method according to p. The process of claim 9, wherein the resin is cellulose partially esterified with acetic, nitric, sulfuric or phosphoric acid. 17. The method according to p. The process of claim 9, characterized in that the matrix is cellulose in which from 0.01% to 5% of glucose molecules, preferably from 0.25% to 1% of glucose molecules, most preferably from 0.5% to 1% of glucose molecules are esterified. 18. An agent for the treatment or prevention of infections caused by bacterial pathogens, comprising an active agent and, optionally, a pharmaceutically acceptable carrier, characterized in that the active agent is a multivalent bacteriophage strain specific for bacteria of this type obtained by the method according to claim 1. 1-17. 19. The agent according to claim 1 The method according to claim 18, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus comprises at least one bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S3 ( PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00005), S6 (PCM F / 00006) and S7 (PCM F / 00007), preferably phages S1, S2, and S4 or S5. 20. The agent according to claim 1 The method of claim 18, which is for the treatment or prevention of infections in patients with cystic fibrosis, and as a multivalent bacteriophage strain specific for bacteria belonging to the genus Staphylococcus comprises at least one bacteriophage strain selected from S2 (PCM F / 00002), S4 (PCM F / 00004 ) and S7 (PCM F / 00007). 21. An agent according to claim 1, A method according to claim 18, characterized in that the multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas comprises at least one bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 ( PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023 ), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), preferably P7, P20 and P6 phages. 22. An agent according to claim 22 The method of claim 18, which is for the treatment or prevention of infections in patients with cystic fibrosis, and as a multivalent bacteriophage strain specific for bacteria belonging to the genus Pseudomonas comprises at least one bacteriophage strain selected from P3 (PCM F / 00010), P6 (PCM F / 00013 ) and P11 (PCM F / 00018). 23. The use of a multivalent bacteriophage strain obtained by the method of claim 1 1-17 for the manufacture of a medicament for treating or preventing infections caused by bacterial pathogens. PL 201 750 B1 24. Use according to claim 24 23. The method according to claim 23, characterized in that at least one bacteriophage strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00005), S6 (PCM F / 00006) and S7 (PCM F / 00007), preferably the phages S1, S2, and S4 or S5. 25. Use according to claim 1 23. The method according to claim 23, characterized in that at least one bacteriophage strain selected from S2 (PCM F / 00002), S4 (PCM F / 00004) and S7 is used for the preparation of a medicament for the treatment or prevention of infections caused in patients with cystic fibrosis by bacteria belonging to the genus Staphylococcus. (PCM F / 00007). 26. Use according to claim 1 23. The method according to claim 23, characterized in that at least one bacteriophage strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016) , P10 (PCM F / 00017), P11 (PCM F / 00018), P12 (PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), preferably P7, P20 and P6 phages. 27. The use of claim 27 23. The method according to claim 23, characterized in that at least one bacteriophage strain selected from P3 (PCM F / 00010), P6 (PCM F / 00013) and P11 is used for the preparation of a medicament for the treatment or prevention of infections caused in patients with cystic fibrosis by bacteria belonging to the genus Pseudomonas. (PCM F / 00018). 28. A multivalent bacteriophage strain having the characteristics of a strain selected from S1 (PCM F / 00001), S2 (PCM F / 00002), S3 (PCM F / 00003), S4 (PCM F / 00004), S5 (PCM F / 00005), S6 (PCM F / 00006) and S7 (PCM F / 00007), which strain is specific for bacteria belonging to the genus Staphylococcus. 29. A multivalent bacteriophage strain having the characteristics of a strain selected from P1 (PCM F / 00008), P2 (PCM F / 00009), P3 (PCM F / 00010), P4 (PCM F / 00011), P5 (PCM F / 00012), P6 (PCM F / 00013), P7 (PCM F / 00014), P8 (PCM F / 00015), P9 (PCM F / 00016), P10 (PCM F / 00017), P11 (PCM F / 00018), P12 ( PCM F / 00019), P13 (PCM F / 00020), P14 (PCM F / 00021), P15 (PCM F / 00022), P16 (PCM F / 00023), P17 (PCM F / 00024), P18 (PCM F / 00025), P19 (PCM F / 00026), and P20 (PCM F / 00027), which strain is specific for bacteria belonging to the genus Pseudomonas.