Abstract
Bacteriophage therapy is currently experiencing a renaissance. Therapeutic efficacy of bacteriophages depends on phage-bacterial and phage-host interactions. The appearance of neutralizing anti-phage antibody has been speculated to be one of the few reasons for bacteriophage therapy's failure. This study aimed to know whether there is a rise in the neutralizing antibody on the parenteral injection of bacteriophages in an animal model. This study included bacteriophages against five different bacteria, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhi and Staphylococcus aureus. These bacteriophages were isolated, propagated and purified. Bacteriophage specificity was confirmed by spot testing on the respective bacterial lawn. Weekly subcutaneous injection of purified bacteriophages (109PFU) was given to five rabbits for six weeks. Blood samples were collected before administering the next dose every week. The antibody response was tested by phage neutralization followed by plaque assay by using double agar overlay method. The rise in anti-phage neutralizing antibodies was observed usually after the 3rd week after immunization. Complete neutralization of bacteriophages could be seen between 3 and 5 weeks after immunization. A further rise in bacteriophage counts (PFU), especially on 1:1000 and 1:2000 serum dilutions, could be noticed by the end of 6th week against most bacteriophages injected. Background anti-phage neutralizing antibodies were observed against bacteriophage specific to Escherichia coli. However, it was absent against bacteriophages specific to other four bacteria. Bacteriophage interacts with mammalian host and induces anti-phages neutralizing antibody production. However, neutralization of phage depends on repeated administration and duration of therapy. The significant rise in neutralizing antibody could be seen at the end of 3rd week. Therefore, bacteriophage can be effectively used in acute cases where therapy duration is less than 2 weeks. However, for prolonged therapy, bacteriophage cocktail of different antigenicity may be suggested.
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References
Belinda L, Sebastian L. A call for a multidisciplinary future of phage therapy to combat multi-drug resistant bacterial infections. Infect Microbe Dis. 2020;2:1–2. https://doi.org/10.1097/IM9.0000000000000018.
Zumla A, Rao M, Wallis RS, Kaufmann SH, Rustomjee R, Mwaba P, et al. Host-directed therapies for infectious diseases: current status, recent progress, and future prospects. Lancet Infect Dis. 2016;16(4):e47–63. https://doi.org/10.1016/S1473-3099(16)00078-5.
Sybesma W, Pimay JP. Expert round table on acceptance and re-implementation of bacteriophage therapy. Silk route to the acceptance and re-implementation of bacteriophage therapy. Biotechnol J. 2016;11(5): 595–600. https://doi.org/10.1002/biot.201600023
Bogovazova GG, Voroshilova NN, Bondarenko VM. The efficacy of Klebsiella pneumoniae bacteriophage in the therapy of experimental Klebsiella infection. Zh Mikrobiol Epidemiol Immunobiol. 1991;4:5–8.
Nakai T, Park SC. Bacteriophage therapy of infectious diseases in aquaculture. Res Microbiol. 2002;153(1):13–8 (Review. d’Herelle, F. (1992)).
Park K, Cha KE, Myung H. Observation of inflammatory responses in mice orally fed with bacteriophage T 7. J Appl Microbiol. 2014;117(3):627–33.
Smith HW, Huggins MB, Shaw KM. Factors influencing the survival and multiplication of bacteriophages in calves and their environment. J Gen Microbiol. 1987;133:1127–35.
Smith HW, Huggins MB. The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophage. J Gen Microbiol. 1987;133:1111–26.
Smith HW, Huggins MB. Effectiveness of phages in treating experimental E. coli diarrhoea in calves, piglets and lambs. J Gen Microbiol. 1983;129:2659–75.
Smith HW, Huggins MB. Successful treatment of experimental Escherichia coli infections in mice using phages: its general superiority over antibiotics. J Gen Microbiol. 1982;128:307–18.
Soothill JS, Lawrence JC, Ayliffe GAJ. The efficacy of phages in the prevention of the destruction of pig skin in vitro by Pseudomonas aeruginosa. Med Sci Res. 1988;16:1287–8.
Soothill JS. Treatment of experimental infections of mice by bacteriophage. J Med Microbiol. 1992;37:258–61.
Soothill JS. Bacteriophage prevents destruction of skin grafts by Pseudomonas aeruginosa. Burns. 1994;20:209–11.
Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. Phage treatment of human infections. Bacteriophage. 2011;1(2):66–85.
Górski A, Międzybrodzki R, Borysowski J, Dąbrowska K, Wierzbicki P, Ohams M, et al. Phage as a modulator of immune responses: practical implications for phage therapy. Adv Virus Res. 2012;83:41–71.
Kucharewicz-Krukowska A, Slopek S. Immunogenic effect of bacteriophage in patients subjected to phage therapy. Arch Immunol Ther Exp (Warsz). 1987;35(5):553–61.
Bruttin A, Brüssow H. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Antimicrob Agents Chemother. 2005;49(7):2874–8.
Hodyra-Stefaniak K, Miernikiewicz P, Drapała J, Drab M, Jończyk-Matysiak E, Lecion D, et al. Mammalian host-versus-phage immune response determines phage fate in vivo. Sci Rep. 2015;5:14802.
Furfaro LL, Payne MS, Chang BJ. Bacteriophage therapy: clinical trials and regulatory hurdles. Front Cell Infect Microbiol. 2018;8:376. https://doi.org/10.3389/fcimb.2018.00376.
Van Belleghem JD, Dąbrowska K, Vaneechoutte M, Barr JJ, Bollyky PL. Interactions between bacteriophage, bacteria, and the mammalian immune system. Viruses. 2019;11:10.
Krut O, Bekeredjian-Ding I. Contribution of the immune response to phage therapy. J Immunol. 2018;200(9):3037–44.
Uhr JW, Finkelstein MS. Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage phi-X 174. J Exp Med. 1963;117:457–77.
Uhr JW, Finkelstein MS, Baumann JB. Antibody formation. III. The primary and secondary antibody response to bacteriophage phi X 174 in guinea pigs. J Exp Med. 1962;115:655–70.
Ha’jek P. Neutralization of bacterial viruses by antibodies of young animals. The development of the avidity of 19S and 7S neutralizing antibodies in the course of primary and secondary response in young rabbits immunized with PhiX 174 bacteriophage. Folia Microbiol (Praha). 1970;15:9–16.
Stashak PW, Baker PJ, Roberso BS. The serum antibody response to bacteriophage phi chi 174 in germ-free and conventionally reared mice. I. Assay of neutralizing antibody by a 50 per cent neutralization method. Immunol. 1970;18:295–305.
Wang B, Hu B, Xu M, Yan Q, Liu S, Zhu X, Sun Z, Reed R, Ding L, Gong J, Li QQ, Hu J. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa. Int J Mol Med. 2006;17:309–17.
Wang B, Hu B, Xu M, Yan Q, Liu S, Zhu X, Sun Z, Reed R, Ding L, Gong J, Li QQ, Hu J. Therapeutic effectiveness of bacteriophages in the rescue of mice with extended-spectrum beta-lactamase-producing Escherichia coli bacteremia. Int J Mol Med. 2006;17:347–55.
Biswas B, Adhya S, Washart P, Paul B, Trostel AN, Powel B, Carlton R, Merril CR. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun. 2002;70:204–10.
Żaczek M, Łusiak-Szelachowska M, Jończyk-Matysiak E, Weber-Dąbrowska B, Międzybrodzki R, Owczarek B, Kopciuch A, Fortuna W, Rogóż P, Górski A. Antibody production in response to Staphylococcal MS-1 phage cocktail in patients undergoing phage therapy. Front Microbiol. 2016;7:1681. https://doi.org/10.3389/fmicb.2016.01681.
Singla S, Harjai K, Katare OP, Chhibber S. Encapsulation of bacteriophage in liposome accentuates its entry into macrophage and shields it from neutralizing antibodies. PLoS ONE. 2016;11:e0153777.
Adams MH. Bacteriophages. New York and London: Inter-science Publishers; 1959.
Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by double agar overlay plaque assay. In: Bacteriophages, 2009; pp. 69–76. Humana Press.
Łusiak-Szelachowska M, Żaczek M, Weber-Dąbrowska B, Miedzybrodzki R, Klak M, Fortuna W, et al. Phage neutralization by sera of patients receiving phage therapy. Viral immunol. 2014;27(6):295–304.
Majewska J, Beta W, Lecion D, Hodyra-Stefaniak K, Kłopot A, Kaźmierczak Z, et al. Oral application of T4 phage induces weak antibody production in the gut and in the blood. Viruses. 2015;7(8):4783–99. https://doi.org/10.3390/v7082845.
Dąbrowska K, Miernikiewicz P, Piotrowicz A, Hodyra K, Owczarek B, Lecion D, Mierczak ZK, Letarov A, Gorski A. Immunogenicity studies of proteins forming the T4 phage head surface. J Virol. 2014;88(21):12551–7.
Genovese MC, Covarrubias A, Leon G, Mysler E, Keiserman M, Valente R, et al. Subcutaneous abatacept versus intravenous abatacept: a phase IIIb noninferiority study in patients with an inadequate response to methotrexate. Arthritis Rheum. 2011;63(10):2854–64.
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Archana, A., Patel, P.S., Kumar, R. et al. Neutralizing antibody response against subcutaneously injected bacteriophages in rabbit model. VirusDis. 32, 38–45 (2021). https://doi.org/10.1007/s13337-021-00673-8
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DOI: https://doi.org/10.1007/s13337-021-00673-8