Early experience with

Nina Chanishvili

The Eliava Institute of , and , ,

ESCMIDECCMID eLibrary- 2018 Madrid, Spain © by author The first evidences

“The first evidence for a viral-like agent with antibacterial properties was reported by M. E. Hankin in 1896. Found in the Ganges river in , it was temperature sensitive, capable of passing through a porcelain filter, and could reduce titres of the bacterium Vibrio cholerae in laboratory culture. Hankin suggested that it might help to decrease the incidence of cholera in people using water from the Ganges.” Sankar Adhya, Carl Merrill, The road to phage therapy, Nature, 2006, ESCMID 443:754-55. eLibrary © by author Discovery of

1898 1915 1917F 1918 ? Nikolai Gamalea Frederick Twort Felix D’Herelle George Eliava UK France Georgia Russ Arch Pathol An investigation on Sur un microbe invisible Clin Med Bacteriol 6 the nature of antagoniste des bacilles (1898),ESCMID pp. 607–613. ultramicroscopic eLibrary dysentériques (Comptes rendus de l'Académie des (1915), Sciences, Paris, 1917. ©Lancet by11: 1241 author165 :p.373-5. Phage Reproduction on the Bacterial Cell

ESCMID eLibrary © by author History of Phage Therapy

1919 – The first clinical trial - Hôpital des Enfants- Malades, Felix D’Herelle, Paris, France. 1921 – The first publication on phage therapy: Richard Bruynoghe and Joseph Maisin, Essais de thérapeutique au moyen du bacteriophage. C. R. Soc. Biol. 85:1120-1121. 1927 – The first mass application of phages, Campbell Hospital, Calcutta, ESCMIDIndia, Felix D’Herelle. eLibrary © by author G.Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia (1923-2010)

Eliava IBMV was founded in 1923 in Tbilisi, Georgia by Professor George Eliava.

George Eliava and a French-Canadian Professor Felix D’Herelle came across in 1920’s in the Pasteur Institute in Paris.

The main idea of G. Eliava and F. D’Herelle was creation of the World Centre of Phage Research. ESCMID eLibrary Professor Eliava was executed in 1937. © by author Realization ESCMID of the eLibrarydream © by author New Institute

ESCMID eLibrary © by author D’Herelle in Georgia (1933-1934)

ESCMID eLibrary © by author Traditional products of the Eliava Institute

 Mono-phages (staphylococcal, streptococcal, E.coli, Pseudomonas, Dysenterial, typhoid)  Poly-phages (Pyo, Intesti)  Standard (typical) phages  Indicatory phages  Sera to treat , tetanus, gangrene, , meningococcus  Sera for identification of Salmonella and Shigella The oldest phage preparations from  (anti-rabies, anthrax, the Eliava collection dated by 1930’s. ESCMID eLibrarybrucella, , intestinal, gono - ) © by author Past Experience in the World

 Commercial department at the Pasteur Institute (5 preps)  German Bacteriophage Society (dried phages in tablet forms)  German company Antipiol (Enterofagos)  Eli Lilly Company (USA) (7 products based on phages)  Swan-Myers of Abbot Laboratories  Squib and sons (now belongs to Brystol Myers Squibb)  Parke, Davis and Company (now part ESCMID eLibraryof Pfizer) © by author 1943

ESCMID eLibrary © by author “I am dying from the treatment of too many physicians.” Alexander the Great

ESCMID eLibrary © by author The miraculous development of exploded and drew much more attention than phage therapy ever did.

Following this, the outbreak of World War II broke lines of communication in both directions: the Western world turned to antibiotics, while many Soviet states used phage therapy instead.

ESCMID eLibrary © by author Bacteriophage production in the Former Soviet republics

In 1988 Scientific-Industrial Union “Bacteriophage” has been formed with the branches in:

Georgia, Tbilisi SIU “Bacteriophage” => Eliava IBMV, Ministry of Education of Georgia

After privatization in 1994-1995 10 small companies have been formed, two out of them with the phage profile:  ChemBioPharm Ltd.  BioPharm Ltd.

 Eliava BioPreparations Ltd. (formed in 2008)

Russia  Perm (Filial of “Microgen Ltd” => “Biomed Ltd”)  (Filial of “Microgen Ltd” => “Immunopreparat Ltd.”)  Nizhni Novgorod (GPU production “BactPreparations, Russia)  Khabarovsk (GPU production “BactPreparations”, Russia)  Saratov (Anti-plague Institute “Microbe” => Biofon Ltd.)  ObolenskESCMID (“Mikromir Ltd”) eLibrary © by author Traditional phage preparations presently in use in the former Soviet Republics Monovalent phage preparations:  Staphyloccocal  Streptococcal  E.coli  Proteus  Pseudomonas aeruginosa  Klebsiella (active against K.pneumoniae, K.ozena, K. rhinoscleroma)  Typhoid (active against Salmonella typhi A,B,C,D,E)  Dysenetrial  Salmonlla sp. Polyvalent (combined) phage preparations:

Intesti-bacteriophage Sh.flexneri serotypes 1,2,3,4,5, 5; Sh. sonnei; Salmonella paratyphi A & B; S. enteritidis, S.typhimurium.S. cholera- suis, S.oranienburg, enteropatogenic E.coli, Proteus vulgaris, P. mirabilis, Staphylococcus aureus, Streptococcus sp. pathogenic Enterococcus. Pio-bacteriophage Staphylococcus, Streptococcus, Ps. aeruginosa, E.coli, Proteus (in some caaes Klebsiella pneumoniae) Coli-Proteus

Diagnostic phages:  ESCMIDSaratov eLibrary 5 types of Cholera phages © by author The Eliava Phage Preparations Currently produced phage products:

Pyo-Phage for treatment of purulent infections caused by Saphylococcus, E.coli, Streptococcus, Pseudomonas, Proteus. ) Intesti-phage - a preparation for treatment and prophylaxis of intestinal infections (dysentery, salmonellosis, dyspepsia, colitis, enterocolitis caused by Shigella,Salmonella, entero- pathogenic 10 serotypes of E.coli, Proteus, Enterococci, Staphylococci and Pseudomonas aeruginosa. Enko-phage - against S. typhimurium, S. enteritidis, S. heidelberg, S. newport, S. cholerae suis, S. oranienburg S. dublin and S. anatum), Shigella flexneri, Shigella sonnei, enteropathogenic E.coli, Staphylococcus aureus, •Pharmaceutical forms: S. epidermidis, S. Saprophyticus) •Liquid SES phage - against S. aureus, S. epidermidis, S. •Dry (tablets) saprophyticus; , S. sanguis, S. salivarius and S. agalactiae), E.coli •Aerosol ESCMID eLibrary•Soft (suppositories, Fersis - against S. aureus, S. epidermidisS. Saprophyticus, Streptococcus pyogenes, S. salves) sanguis, S. Salivarius, © S. agalactiae. by author Historical experience of bacteriophage application in different fields: •Therapy  Medicine •Prophylaxis  Veterinary •Detection & Identification  Agriculture of the pathogen •Epidemiological aspects –  Environmental research phage-typing •Use of phages as tracers

ESCMID eLibrary © by author Comparison of bacteriophages vs. antibiotics

 Live bacterial viruses  Chemical substances  Viable  Standard  Specific action  General action  Self reproducible  N/A  No evidence  Side effects ( e.g. allergy)  Effective against drug- resistant  Spread of drug-resistance  N/A  Yeast, fungal infections  Non-hazardous industry  Hazardous industry  ESCMIDInexpensive eLibrary Expensive © by author ISTC Project G-1467 Preparation of a detailed review article/ monograph on the practical application of bacteriophages in medicine, veterinary, etc.

Screening results:  > 5000 volumes of scientific journals, selected articles, books and thesis of dissertations have been screened. About 10% of the total contain information on phages.  The gathered information was included into: “A Literature Review of the Practical Application of Bacteriophage Research”. ESCMID eLibrary Prof. Richard Sharp © by author “A Literature Review of the Practical Application of Bacteriophage Research” Content:

 Animal studies  Immune Response to Phage  Phage Therapy in Dermatology Therapy  Phage Therapy in Surgery and Wound Treatment  Phage Therapy against Septic Infections  Phage Therapy against Wound Infections  Phages in Veterinary  Phage Therapy in Urology  Use of Phages in Plant Protection  Phage Therapy in Gynecology  Environmental Role of  Phage Therapy in Ophthalmology Bacteriophages  Combined Phage- and Sero- Therapy  Phages Potentially Applicable  Combined use of phages and against Bioterrorism antibiotics  Methodology for Manufacturing of  Experience on “prophylactic Bacteriophages phaging”  Glossary  Immune Response to Phage ESCMIDTherapy eLibrary Addendum © by author Analysis of Old Scientific Literature

 Purtseladze, M.D. (1941) Treatment of purulent forms of mastititis. Obstetrics and Gynecology ("Akusherstvo i ginekologia"), 11-12, 22-24.  Rodigina, A.M. (1938) Pneumococcal bacteriophage: Its application for treatment of the ulcerous corneal serpens. Perm, USSR.  Ruchko, I. et al. (1936) Therapeutic effect of Staphylococcus phage for oral and dental infections. Soviet Stomatology (“Sovetskaya Stomatologia”), 1, 11-20.  Tsulukidze, A.P. (1938) Application of phages in urology. Urology (“Urologia”), XV, 10-13.  Vartapetov, L.I. (1947) Results of phage therapy of deep forms of piodermitis, Selected Articles, Institute of Dermatology and Veneric Diseases, Tbilisi, 205-207.  Vlasov, K.F. and Artemenko, E.A. (1946) Treatment of chronic dysentery, (1946) Soviet Medicine (“Sovetskaya medicina”), 10, 22- 28.  Ukelis, I.I. (1946) Treatment of furunculosis and other purulent skin infections with di-phage. Soviet Medicine (“Sovetskaya Medicina”), 3, ESCMID11-12. eLibrary © by author The First Mass Application of Phages - Field Trials

 Finnish Campaign 1938-1940

Krestnikova, 1947 Experience of phage prophylaxis based on the results of 3 mobile brigades.

I brigade applied phages for 2,500 solders, among them gangrene symptoms were revealed in 35 (1,4%) cases. In the control group of 7,918 solders gangrene was registered in 342 (4.3%) cases.

II brigade applied phage treatment for 941 solders, only 14 (1,5%) got sick. In the control group 6,8% were infected.

III brigade applied phage treatment for 2,584 ESCMIDsoders, gangrene eLibrary was developed in 18 (0,7%) cases, while in the control group incidence of infection© wasby 2,3% author Phages against Wound Infections

A.P. Tsulukidze (1940) Phage therapy in surgery, “Khirurgia”, 1940, 12, 132-133

Anaerobic phages produced by the Tbilisi ESCMID Institute were applied fro eLibrary prophylaxis of gangrene infection. © by author Stalingrad battle (1943)

Zinaida Ermolyeva

ESCMID eLibrary © by author Alexandrova M.B. et al., (1935) Phage therapy against dysentery. J. Microbiol. Epidemiol. Immunol. 11, 860-868

I Gr. - 86% cured in 3 days, no improvement - 14% II Gr.ESCMID– 49 %, cured in 3 days, no improvement eLibrary - 51% Phage therapy allows to achieve positive results in a short period of time!!! © by author Lurie, M.N. (1938) Treatment of dysentery and haemolytic intestinal diseases among children and adults. Selected articles of Azerbaijani Institute of Epidemiology and Microbiology, 6, 31-34

ESCMID eLibrary Children Adults I Gr – 67% cured in 5 days I Gr - 93% cured in 5 days II Gr – 69% cured in 5 days © byII Gr –author80% cured in 5 days Lurie, M.N. (1938) Treatment of dysentery and haemolytic intestinal diseases among children and adults, Selected articles of Azerbaijani Institute of Epidemiology and Microbiology, 6, 31-34

Due to phage therapy consequences of infection and mortality rate were ESCMIDminimized!!! eLibrary © by author Effect of Phage Therapy in Dermatology (K. Shvelidze, 1970)

ESCMID eLibrary Phage therapy decreases a number of relapsed cases, a stable result of treatment is achieved.!!! © by author Method of “Prophylactic Phaging” (E. Babalova et al, 1968)

 1963 - May 8 – October 25.  1964 – May 15 – October 31.  Two regions in the city Tbilisi with the similar sanitary conditions.  Children in the age of 6 months - 7 years.  A dose – 1-2 tablets/day (=10-20ml of liquid phage) according to the age. The dose was given once per 5-7 days.  The phaging of children was performed on one side of the street, while the children on the opposite site composed a control group.  The days of observation in these groups were almost equal 108,6 and 109,5 correspondingly.  ESCMID> 30 000 children were involved eLibrary into the study. © by author Characterization of the experimental and control groups (E. Babalova et al., 1968)

Number Group Number of children in each age group of children in the group 6 1-3 y 3-5 y 5-7 y Total months 1 y Exp. 2 467 5 595 4 883 4 099 17 044

Contr. 1 557 4 281 4 097 4 097 13 725

Effectiveness 5,7 3,7 3,4 - Average IR = 3,8 index (IR)

IR is the ratio between the expected and observed number of infected persons, calculated per 1,000 ESCMID persons. IR is used here as a tool to measureeLibrary the effectiveness of prophylactic measures . Conclusion: Due to prophylactic phaging the number of disease cases decreased in average 3.8 - times in comparison © withby the expected author outcome !!! “Prophylactic Phaging” – Mass Experiments Sayamov et al, 1963  Anti-cholera bacteriophage preparation was used by the Soviet doctors in East Pakistan in 1958 and in Afghanistan in 1960. Altogether approximately 270,000 persons underwent phage treatment Buryakovsky, 1974,  Experiment involved 49,523 children from kindergartens.  Exp. gr. - 30,023, contr. g. - 19,500  Incidence of disease decreased for 2,6-times in the exp.gr.

Ferdinand et al, 1978, Russia  Experiments were held in 93 towns, Experimental towns – 50, control - 43.  Food and dairy production personnel and kindergarten teachers were involved into the study. n=3000, kindergarten children n=20,000.

 In different towns frequency of Infection decreased for 30-60%. •BacteriophagesESCMID are persisting ineLibrary the human organism for 3- 5 days!!! © by author Mechanisms of phage therapeutic action in vivo  Lyses (kills) bacterial cells;  Triggers body immune response due to activation of T-lymphocytes, which start: a) production of against bacteriophages – a peak of production comes to the 14th-21st days, while the therapeutic effect usually is achieved within the first 5-7-10 days of treatment!!! b) production of antibodies against the cell surface antigens ESCMIDof the lysed bacterial cells eLibrary – simulation of vaccination!!! © by author Development of the therapeutic and prophylactic phages (The Soviet regulatory requirements)  I – Gathering of information about the circulating pathogens;  II - Construction of the host strain collection;  III - Selection of the phages active against the particular bacterial species (strain collection);  IV - Study of the clonal composition of phages (approval of their virulent nature);  V- Elaboration of the technology regulations for propagation of the phage clones taking into account possible interference of their action, especially in case of the polyclonal preparations;  V I- Manufacturing of the phage preparation and its control;  ESCMIDVII- Monitoring of the efficien ceLibraryy of the phage preparation s. © by author Main conclusions:

 Success outcome of phage therapy is up to 95%.  Rapid improvement and cure within 3-5-7 days.  Positive cosmetic effect (no scars left).  No relapsed cases.  No side effects.  Minimization of the mortality rate among children.  Due to prophylactic “phaging” number of actually registered disease cases in comparison with expected rates (i.e EI) is Publications dated by reduces 3-6 times. 1930s-1950s.  Mild disease cases in a “phaged” group.  Effect similar to vaccination.  Reduced hospital days. ESCMID eLibrary2009 2012 © by author Major errors found in the old publications

 Difference in the number of patients in experimental and control groups  The use of placebos and the coding of preparations is missing in the majority of the studies  Different methodological approaches and schemes, doses, titers of phage preparations, and the duration of treatment  No control groups  ESCMIDStatistically unreliable eLibrary © by author Results of phage therapy achieved by enthusiastic Georgian doctors

ESCMID eLibraryBesik Iashvili Guram Gvasalia © by author Intravenous Staphylococcal bacteriophage (1970-1990)

Prof. Vakhtang Bochorishvili Pediatrician – Galina Samsygina

Results of clinical studies Group Adults Children Fatal cases Fatal cases Number of Abs.no % Number Abs. no % cases of cases ESCMIDExp 215 eLibrary0 0 149 1 0.67 Control 208 11 3.6 98 8 8.2 Prof. Teimuraz Chanishvili © by author Overview of the recent bacteriophage therapy clinical studies (Total 14). Name Country Target Start/end dates Number of Clinical organisms recruited Trials.gov patients Identifier/ Reference Bacteriophages for Tsulukidze Urology E.coli, 2016-2018 81 NCT03140085 treating urinary tract Center & Eliava Enetrococcus,Strept infections in patients Institute of ococcus, undergoing Bacteriophage, P.aeruginosa, transurethral Microbiology & Proteus spp. resection of the Virology, Tbilisi, prostate: a Georgia randomized, placebo-controlled, double-blind clinical trial PhagoBurn. Phase 7 clinical sites in Escherichia coli & 2013-2017 Only 27 patients NCT02116010 I/II Clinical Trial. France, Belgiu and Pseudomonas between 11 centers This Project is a Switzerland aeruginosa were included, which European Research is far from the pre- & Development calculated 220 (R&D) Project patients Funded by the European Commission Under the 7FP Antibacterial Nestlé, Switzerland Escherichia. coli 2009-2013 Oral coliphages NCT00937274 treatment against in collaboration with (T4 bacteriophage) showed a safe gut diarrhea in oral Dhaka Hospital of transit in children, rehydration ESCMID solution the International eLibrarybut failed to achieve Centre for Diarrheal intestinal Disease Research, amplification and to Bangladesh improve diarrhea © by authoroutcome Open-label treatment of RCT with bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomized, placebo controlled, double blind clinical trial

Pre-treatment Post-treatment Prostat PSA Adverse value Case Age e size events/ Culture Titer Culture Titer (g) (ng/m L) remarks 1 62 E.coli 10^7 E.coli 10^5 70 1 No side effects

2 82 E.coli 10^7 Non N/a 140 2 No side effects pathogenic microflora 3 58 E.coli 10^4 E.coli 10^4 65 0.3 No side effects

4 85 E.coli 10^7 Non N/a 90 3.2 No side effects pathogenic microflora 5 56 Streptococ 10^7 Streptococcus 10^6 130 0.8 No side effects cus 6 85 Streptococ 10^5 sterile 0 29 2.3 No side effects cus 7 69 Enterococc 10^6 E.coli 10^7 65 1 Secondary us infection (?) 8 69 Enterococc 10^6 Non N/a 60 1.3 No side effects ESCMIDus pathogenic eLibrary microflora 9 58 Pseudomo 10^6 No data No data 60 1.3 Fever and chills nas started on the 3rd © by author day Thank you

ESCMID eLibrary © by author