HUMAN VACCINES & IMMUNOTHERAPEUTICS 2016, VOL. 12, NO. 12, 3097–3098 http://dx.doi.org/10.1080/21645515.2016.1210746
COMMENTARY
Immunotherapies against antibiotics-resistant Klebsiella pneumoniae Xiaodong Xiao, Herren Wu, and William F. Dall’Acqua Department of Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD, USA ARTICLE HISTORY Received 23 June 2016; Accepted 5 July 2016
News headlines reporting the emergence of a “superbug” in the US last May instilled a nationwide fear (e.g. http://www.cnn. com/2016/05/26/health/first-superbug-cre-case-in-us/). This fear is legitimate. The culprit is an E.coli strain that harbors plasmid-mediated colistin resistance mechanism MCR-1 and was first reported in China.1 It is resistant to essentially all available antibiotics and “…heralds the emergence of a truly pan-drug resistant bacteria.”2 Of special concern is that this bacterial strain carries transferrable plasmids that encode antibiotics resistance genes. These plasmids can be passed onto other enterobacteriaceae family members including Klebsiella pneumonia, which can become antibiotics-resistant. Indeed drug-resistant Klebsiella pneumoniae carbapenemase (KPC) also made headline when it spread through the NIH Clinical Center in Bethesda, MD, killing a dozen patients in one single outbreak in 2011.3 Klebsiella pneumoniae has emerged as an exceedingly problematic infectious agent in the past decades and this trend is not showing signs of slowing down.4 Against these KPC bacterial strains, clinicians are facing increasing challenges due to the dwindling options of new classes of antibiotics. New strategies are urgently needed to deal with this crisis and the help may come, at least partially, in the form of immunotherapy. Immunotherapy harnesses human body’s own immune system in either an active (vaccine) or passive (immunoglobulin) form. It uses different mechanism of protection and is not subjected to the resistance mechanism bacteria have adopted against antibiotics. It is also likely to have fewer side effects than antibiotics. Critical to both active and passive immunotherapies are the identifications of bacterial targets that are appropriate to serve as either vaccine immunogen for active immune-prevention or antibody targets for therapy. Much effort has been made in this regard in the anti-bacterial field and some progress seen with Klebsiella pneumoniae as well as Pseudomonas aeruginosa studies.5,6 Klebsiella pneumoniae is a gram negative bacterium and an etiological agent for nosocomial infections.7 Typical to a gram negative bacterium its surface is covered with polysaccharide including both capsules and LPS, which are the most accessible and immunodominant bacterial antigens. There are 77 different capsular and 9 different LPS serotypes.8,9 Without precise, longitudinal epidemiology data, it is difficult to preemptively develop an immunotherapy based on specific one or collections of capsule or LPS serotypes. Developing an all-inclusive vaccine CONTACT Xiaodong Xiao © 2016 Taylor & Francis
[email protected]
or antibody therapy covering all capsular or LPS serotypes is not possible in practice, even though a multivalent vaccine approach has been attempted.10 We and others have hypothesized that the solution may lie in the identification of antigens that are highly conserved among all or most Klebsiella pneumoniae serotypes so that vaccine antigens with broad coverage and antibodies that are broadly protective can be developed. Various strategies have been applied to look for such antigens with limited success. Our team used a target-agnostic approach, which started by looking for antibodies that possess protective activities against multiple strains of klebsiella pneumoniae in several in vitro and in vivo models without having actual knowledge of the antibody targets. The identifications of antibody targets are then resolved through immunological and mass spectrometry methods. Our target-agnostic approach aimed to avoid the generation of antibodies against the most immunodominant and serotype specific antigen instead of looking for antibodies targeting the otherwise hidden and conserved antigens. To achieve this we used a Klebsiella pneumoniae mutant devoid of capsule and LPS for the first round of either phage panning or immunization of animals for hybridoma generation. This was followed by additional rounds of panning or immunization with wild type Klebsiella pneumoniae strains of different serotypes in order to generate antibodies that 1) have in vivo relevance, 2) target non-polysaccharide and preferably protein antigens, and 3) target serotype independent antigens. As a result, MrkA was identified as the antigen recognized by a group of antibodies, which were discovered through both phage panning and hybridoma approaches.6 We expect that such a strategy should be of universal usage for neo-antigen/antibody discoveries in diverse therapeutic areas. MrkA is a major component of the Type III fimbrial complex. Its functions include being the principle building block of the fimbrial shaft, biofilm formation and establishment of infection.11 It is conserved among the majority of enterobacteriaceae and therefore an ideal candidate for vaccine and antibody development. Limited work has been done in this regard and no monoclonal antibodies have been tested for any protective effect until recently. We found in in vitro experiment that MrkA is important for biofilm formation in agreement with previous reports. Further we found that all the antibodies we identified mediated opsonophagocytic killing (OPK). Unexpectedly, all the antibodies identified targeted one overlapping
One MedImmune Way, Gaithersburg, MD 20878, USA.
3098
X. XIAO ET AL.
epitope.6 The precise protective mechanism and the relevance of epitopes to protective effect remain important topics for indepth investigations. Another potentially interesting antigen that may be useful in immunotherapy against Klebsiella pneumoniae is a conserved surface polysaccharide Poly-N-acetylglucosamine (PNAG). It was reported to be present on the surface of a surprisingly wide collection of bacterial and fungal strains including Klebsiella pneumoniae and one anti-PNAG antibody F598 is under clinical development.12 Its impact on Klebsiella pneumoniae remains to be evaluated. A note of caution is that with such a broad coverage, the impact on microbiota should be monitored carefully when immunotherapy is developed based on such a target. Although it is very challenging to identify highly conserved antigens among various Klebsiella pneumoniae strains, it is safe to predict that more and perhaps better antigens will be discovered as future researches are focused in this direction. As promising as these newly identified targets are, their identifications represent only the beginning of a long journey in developing immunotherapies against the antibiotics resistant bacteria such as Klebsiella pneumoniae. Each target antigen plays unique functional roles in supporting the life-cycle of the bacteria. They each also exhibit a different virulent mechanism against the host. The elucidation of their functional roles as well as their virulent mechanism will help to establish the candidate drug profiles and guide the development of the most efficacious immunotherapies. In this regard, the agnostic approach itself will need continuous modifications as we gain further insight into what mechanism of action (MOA) and what functional screening protocols are the most relevant ones in predicting the therapeutic outcomes of a specific immunotherapy. The critically important questions we need to address in future studies include: 1) Are there better MrkA epitopes to target? 2) Are there other in vitro assays that can better predict the in vivo activities than the OPK? 3) What are the major MOA of the current group of antibodies? and 4) how do we incorporate these information into future screening processes? Immunotherapy development is a field that sees its boundaries continuously being pushed as a result of better understanding of human immunology and advancing technologies. One future direction may consist of combining a small number of partially broadly protective antibodies that would help to achieve near complete coverage. Additionally, important effector functions including OPK can be enhanced universally through antibody engineering independent of antigens or epitopes. Finally, a “non-functional” antibody can become a functional one through conjugations to powerful anti-bacterial drugs. Realizing all these possibilities depends on our continued search of “conserved targets,” which may or may not show any value today.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016; 16:161-8; PMID:26603172; http://dx.doi.org/ 10.1016/S1473-3099(15)00424-7 [2] McGann P, Snesrud E, Maybank R, Corey B, Ong AC, Clifford R, Hinkle M, Whitman T, Lesho E, Schaecher KE. Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First report of mcr-1 in the USA. Antimicrob Agents Chemother 2016; PMID:27230792 [3] Snitkin ES, Zelazny AM, Thomas PJ, Stock F, Henderson DK, Palmore TN, Segre JA. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci Transl Med 2012; 4:148ra16; http://dx.doi.org/10.1126/ scitranslmed.3004129 [4] Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013; 13:785-96; PMID:23969216; http://dx.doi.org/10.1016/S1473-3099(13)70190-7 [5] DiGiandomenico A, Warrener P, Hamilton M, Guillard S, Ravn P, Minter R, Camara MM, Venkatraman V, Macgill RS, Lin J, et al. Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening. J Exp Med 2012; 209:1273-87; PMID:22734046; http://dx.doi.org/10.1084/ jem.20120033 [6] Wang Q, Chang CS, Pennini M, Pelletier M, Rajan S, Zha J, Chen Y, Cvitkovic R, Sadowska A, Heidbrink Thompson J, et al. Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit. J Infect Dis 2016; 213:1800-8; PMID:26768253; http://dx.doi.org/10.1093/infdis/jiw021 [7] Broberg CA, Palacios M, Miller VL. Klebsiella: a long way to go towards understanding this enigmatic jet-setter. F1000Prime Rep 2014; 6:64; PMID:25165563 [8] Hansen DS, Mestre F, Alberti S, Hernandez-Alles S, Alvarez D, Domenech-Sanchez A, Gil J, Merino S, Tomas JM, Benedı VJ. Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries. J Clin Microbiol 1999; 37:56-62; PMID:9854064 [9] Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, Decre D. wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol 2013; 51:4073-8; PMID:24088853; http://dx.doi.org/10.1128/JCM.01924-13 [10] Campbell WN, Hendrix E, Cryz S, Jr., Cross AS. Immunogenicity of a 24-valent Klebsiella capsular polysaccharide vaccine and an eightvalent Pseudomonas O-polysaccharide conjugate vaccine administered to victims of acute trauma. Clin Infect Dis 1996; 23:179-81; PMID:8816151; http://dx.doi.org/10.1093/clinids/23.1.179 [11] Allen BL, Gerlach GF, Clegg S. Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae. J Bacteriol 1991; 173:916-20; PMID:1670938 [12] Cywes-Bentley C, Skurnik D, Zaidi T, Roux D, Deoliveira RB, Garrett WS, Lu X, O’Malley J, Kinzel K, Zaidi T, et al. Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens. Proc Natl Acad Sci U S A 2013; 110:E2209-18.
COMMENTARY
Immunotherapies against antibiotics-resistant Klebsiella pneumoniae Xiaodong Xiao, Herren Wu, and William F. Dall’Acqua Department of Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD, USA ARTICLE HISTORY Received 23 June 2016; Accepted 5 July 2016
News headlines reporting the emergence of a “superbug” in the US last May instilled a nationwide fear (e.g. http://www.cnn. com/2016/05/26/health/first-superbug-cre-case-in-us/). This fear is legitimate. The culprit is an E.coli strain that harbors plasmid-mediated colistin resistance mechanism MCR-1 and was first reported in China.1 It is resistant to essentially all available antibiotics and “…heralds the emergence of a truly pan-drug resistant bacteria.”2 Of special concern is that this bacterial strain carries transferrable plasmids that encode antibiotics resistance genes. These plasmids can be passed onto other enterobacteriaceae family members including Klebsiella pneumonia, which can become antibiotics-resistant. Indeed drug-resistant Klebsiella pneumoniae carbapenemase (KPC) also made headline when it spread through the NIH Clinical Center in Bethesda, MD, killing a dozen patients in one single outbreak in 2011.3 Klebsiella pneumoniae has emerged as an exceedingly problematic infectious agent in the past decades and this trend is not showing signs of slowing down.4 Against these KPC bacterial strains, clinicians are facing increasing challenges due to the dwindling options of new classes of antibiotics. New strategies are urgently needed to deal with this crisis and the help may come, at least partially, in the form of immunotherapy. Immunotherapy harnesses human body’s own immune system in either an active (vaccine) or passive (immunoglobulin) form. It uses different mechanism of protection and is not subjected to the resistance mechanism bacteria have adopted against antibiotics. It is also likely to have fewer side effects than antibiotics. Critical to both active and passive immunotherapies are the identifications of bacterial targets that are appropriate to serve as either vaccine immunogen for active immune-prevention or antibody targets for therapy. Much effort has been made in this regard in the anti-bacterial field and some progress seen with Klebsiella pneumoniae as well as Pseudomonas aeruginosa studies.5,6 Klebsiella pneumoniae is a gram negative bacterium and an etiological agent for nosocomial infections.7 Typical to a gram negative bacterium its surface is covered with polysaccharide including both capsules and LPS, which are the most accessible and immunodominant bacterial antigens. There are 77 different capsular and 9 different LPS serotypes.8,9 Without precise, longitudinal epidemiology data, it is difficult to preemptively develop an immunotherapy based on specific one or collections of capsule or LPS serotypes. Developing an all-inclusive vaccine CONTACT Xiaodong Xiao © 2016 Taylor & Francis
[email protected]
or antibody therapy covering all capsular or LPS serotypes is not possible in practice, even though a multivalent vaccine approach has been attempted.10 We and others have hypothesized that the solution may lie in the identification of antigens that are highly conserved among all or most Klebsiella pneumoniae serotypes so that vaccine antigens with broad coverage and antibodies that are broadly protective can be developed. Various strategies have been applied to look for such antigens with limited success. Our team used a target-agnostic approach, which started by looking for antibodies that possess protective activities against multiple strains of klebsiella pneumoniae in several in vitro and in vivo models without having actual knowledge of the antibody targets. The identifications of antibody targets are then resolved through immunological and mass spectrometry methods. Our target-agnostic approach aimed to avoid the generation of antibodies against the most immunodominant and serotype specific antigen instead of looking for antibodies targeting the otherwise hidden and conserved antigens. To achieve this we used a Klebsiella pneumoniae mutant devoid of capsule and LPS for the first round of either phage panning or immunization of animals for hybridoma generation. This was followed by additional rounds of panning or immunization with wild type Klebsiella pneumoniae strains of different serotypes in order to generate antibodies that 1) have in vivo relevance, 2) target non-polysaccharide and preferably protein antigens, and 3) target serotype independent antigens. As a result, MrkA was identified as the antigen recognized by a group of antibodies, which were discovered through both phage panning and hybridoma approaches.6 We expect that such a strategy should be of universal usage for neo-antigen/antibody discoveries in diverse therapeutic areas. MrkA is a major component of the Type III fimbrial complex. Its functions include being the principle building block of the fimbrial shaft, biofilm formation and establishment of infection.11 It is conserved among the majority of enterobacteriaceae and therefore an ideal candidate for vaccine and antibody development. Limited work has been done in this regard and no monoclonal antibodies have been tested for any protective effect until recently. We found in in vitro experiment that MrkA is important for biofilm formation in agreement with previous reports. Further we found that all the antibodies we identified mediated opsonophagocytic killing (OPK). Unexpectedly, all the antibodies identified targeted one overlapping
One MedImmune Way, Gaithersburg, MD 20878, USA.
3098
X. XIAO ET AL.
epitope.6 The precise protective mechanism and the relevance of epitopes to protective effect remain important topics for indepth investigations. Another potentially interesting antigen that may be useful in immunotherapy against Klebsiella pneumoniae is a conserved surface polysaccharide Poly-N-acetylglucosamine (PNAG). It was reported to be present on the surface of a surprisingly wide collection of bacterial and fungal strains including Klebsiella pneumoniae and one anti-PNAG antibody F598 is under clinical development.12 Its impact on Klebsiella pneumoniae remains to be evaluated. A note of caution is that with such a broad coverage, the impact on microbiota should be monitored carefully when immunotherapy is developed based on such a target. Although it is very challenging to identify highly conserved antigens among various Klebsiella pneumoniae strains, it is safe to predict that more and perhaps better antigens will be discovered as future researches are focused in this direction. As promising as these newly identified targets are, their identifications represent only the beginning of a long journey in developing immunotherapies against the antibiotics resistant bacteria such as Klebsiella pneumoniae. Each target antigen plays unique functional roles in supporting the life-cycle of the bacteria. They each also exhibit a different virulent mechanism against the host. The elucidation of their functional roles as well as their virulent mechanism will help to establish the candidate drug profiles and guide the development of the most efficacious immunotherapies. In this regard, the agnostic approach itself will need continuous modifications as we gain further insight into what mechanism of action (MOA) and what functional screening protocols are the most relevant ones in predicting the therapeutic outcomes of a specific immunotherapy. The critically important questions we need to address in future studies include: 1) Are there better MrkA epitopes to target? 2) Are there other in vitro assays that can better predict the in vivo activities than the OPK? 3) What are the major MOA of the current group of antibodies? and 4) how do we incorporate these information into future screening processes? Immunotherapy development is a field that sees its boundaries continuously being pushed as a result of better understanding of human immunology and advancing technologies. One future direction may consist of combining a small number of partially broadly protective antibodies that would help to achieve near complete coverage. Additionally, important effector functions including OPK can be enhanced universally through antibody engineering independent of antigens or epitopes. Finally, a “non-functional” antibody can become a functional one through conjugations to powerful anti-bacterial drugs. Realizing all these possibilities depends on our continued search of “conserved targets,” which may or may not show any value today.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016; 16:161-8; PMID:26603172; http://dx.doi.org/ 10.1016/S1473-3099(15)00424-7 [2] McGann P, Snesrud E, Maybank R, Corey B, Ong AC, Clifford R, Hinkle M, Whitman T, Lesho E, Schaecher KE. Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First report of mcr-1 in the USA. Antimicrob Agents Chemother 2016; PMID:27230792 [3] Snitkin ES, Zelazny AM, Thomas PJ, Stock F, Henderson DK, Palmore TN, Segre JA. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci Transl Med 2012; 4:148ra16; http://dx.doi.org/10.1126/ scitranslmed.3004129 [4] Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013; 13:785-96; PMID:23969216; http://dx.doi.org/10.1016/S1473-3099(13)70190-7 [5] DiGiandomenico A, Warrener P, Hamilton M, Guillard S, Ravn P, Minter R, Camara MM, Venkatraman V, Macgill RS, Lin J, et al. Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening. J Exp Med 2012; 209:1273-87; PMID:22734046; http://dx.doi.org/10.1084/ jem.20120033 [6] Wang Q, Chang CS, Pennini M, Pelletier M, Rajan S, Zha J, Chen Y, Cvitkovic R, Sadowska A, Heidbrink Thompson J, et al. Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit. J Infect Dis 2016; 213:1800-8; PMID:26768253; http://dx.doi.org/10.1093/infdis/jiw021 [7] Broberg CA, Palacios M, Miller VL. Klebsiella: a long way to go towards understanding this enigmatic jet-setter. F1000Prime Rep 2014; 6:64; PMID:25165563 [8] Hansen DS, Mestre F, Alberti S, Hernandez-Alles S, Alvarez D, Domenech-Sanchez A, Gil J, Merino S, Tomas JM, Benedı VJ. Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries. J Clin Microbiol 1999; 37:56-62; PMID:9854064 [9] Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, Decre D. wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol 2013; 51:4073-8; PMID:24088853; http://dx.doi.org/10.1128/JCM.01924-13 [10] Campbell WN, Hendrix E, Cryz S, Jr., Cross AS. Immunogenicity of a 24-valent Klebsiella capsular polysaccharide vaccine and an eightvalent Pseudomonas O-polysaccharide conjugate vaccine administered to victims of acute trauma. Clin Infect Dis 1996; 23:179-81; PMID:8816151; http://dx.doi.org/10.1093/clinids/23.1.179 [11] Allen BL, Gerlach GF, Clegg S. Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae. J Bacteriol 1991; 173:916-20; PMID:1670938 [12] Cywes-Bentley C, Skurnik D, Zaidi T, Roux D, Deoliveira RB, Garrett WS, Lu X, O’Malley J, Kinzel K, Zaidi T, et al. Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens. Proc Natl Acad Sci U S A 2013; 110:E2209-18.
