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Summary of Professional Accomplishments
Małgorzata Kęsik-Brodacka, Ph.D.
Institute of Biotechnology and Antibiotics
Department of Bioengineering
Warsaw, 2017
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Table of content
1. Name and surname3
2. Held diplomas and scientific degrees – with the name, place and the year of acquisition and
the title of the doctoral dissertation 3
3. Information on current and previous employment in scientific institutions3
4. Scientific contributions related to article 16, paragraph 2 of the act of 14 March 2003 on
academic degrees and title and on degrees and title in the art (Dz. U. 2016, item 882 with
amendments in Dz. U. 2016, item 1311)4
4.1) Title of scientific achievement4
4.2) Publications comprising scientific achievement4
4.3) Discussion of the scientific goals of the publications comprising the scientific
achievement and potential of its further use6
5. Presentation of other scientific and research accomplishments26
6. Summary list of publications35
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1. Name and surname
Małgorzata Kęsik-Brodacka
2. Held diplomas and scientific degrees – with the name, place and the year of acquisition
and the title of the doctoral dissertation
2012 – 2013 Postgraduate study "Research project management and
commercialization of research results”, Lodz University of
Technology.
2011 – 2012 Postgraduate study "Manager of innovation" at Warsaw School of
Economics.
2005 Doctor of Philosophy in Biology, specialization: biochemistry.
Institute of Biochemistry and Biophysics, Polish Academy of
Science.
Title of the doctoral thesis: “Inclusion bodies from recombinant bacteria as a novel system for delivery of vaccine antigen by the oral
route”. Supervisor: Prof. dr hab. Andrzej Płucienniczak.
1999 Master of Science degree in biology, specialization at Microbiology,
Faculty of Biology, Warsaw University.
M.Sc. project: “ The role of p60 protein in pathogenesis of Listeria
monocytogenes”. Supervisor: Prof. dr hab. Jacek Bielecki.
3. Information on current and previous employment in scientific institutions
January 2006 – present Associate professor in the Department of Bioengineering,
Institute of Biotechnology and Antibiotics. Head of
Biochemistry laboratory.
February 2010 – April 2011 Postdoctoral Scholar, Department of Molecular Biology and
Microbiology Case Western Reserve University, Cleveland
OH, USA.
January2005 – December 2005 Assistant professor in the Department of Bioengineering,
Institute of Biotechnology and Antibiotics.
May 2002– December 2004 Technical assistant in the Department of Bioengineering,
Institute of Biotechnology and Antibiotics.
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4. Scientific contributions related to article 16, paragraph 2 of the act of 14 March 2003
on academic degrees and title and on degrees and title in the art (Dz. U. 2016, item
882 with amendments in Dz. U. 2016, item 1311).
4.1) Title of scientific achievement:
Immune potential of antigens produced using novel expression
systems.
4.2) Publications comprising scientific achievement:
I. Kesik-Brodacka M.* (2017) Progress in Biopharmaceutical Development. Biotechnol Appl
Biochem. doi: 10.1002/bab.1617.
* Corresponding author.
IF2016 1.41;MNiSW2016 scoring:20, number of citations: 0.
II. Kesik-Brodacka M*, Lipiec A, Kozak Ljunggren M, Jedlina L, Miedzinska K, Mikolajczak
M, Plucienniczak A, Legocki AB, Wedrychowicz H. (2017) Immune response of rats vaccinated
orally with various plant-expressed recombinant cysteine proteinase constructs when challenged
with Fasciola hepatica metacercariae. PLoS Negl Trop Dis. 11:e0005451. doi:
10.1371/journal.pntd.0005451.
* Corresponding author.
IF2016 3.83; MNiSW2016 scoring: 45, number of citations: 0.
III. Kesik-Brodacka M*, Plucienniczak G. (2014) A universal flu vaccine.
Acta Biochim Pol. 61:523-30.
* Corresponding author.
IF2014 1.15; MNiSW2014 scoring: 15, number of citations: 2.
IV. Kesik-Brodacka M*, Romanik A, Mikiewicz-Sygula D, Plucienniczak G, Plucienniczak A.
(2012) A novel system for stable, high-level expression from the T7 promoter. Microb Cell Fact.
11:109.
* Corresponding author.
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IF2012 3.31; MNiSW2012 scoring: 40, number of citations: 3.
V. Wedrychowicz H, Kesik M, Kaliniak M, Kozak-Cieszczyk M, Jedlina-Panasiuk L, Jaros S,
Płucienniczak A. (2007) Vaccine potential of inclusion bodies containing cysteine proteinase of Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the fluke.
Vet Parasitol. 147:77-88.
IF2007 2.02; MNiSW2007 scoring: 24, number of citations: 14.
VI. Kesik M*, Jedlina-Panasiuk L, Kozak-Cieszczyk M, Płucienniczak A, Wedrychowicz H. (2007)
Enteral vaccination of rats against Fasciola hepatica using recombinant cysteine proteinase
(cathepsin L1). Vaccine 25:3619-28.
* Corresponding author.
IF2007 3.38; MNiSW2007 scoring: 24, number of citations: 26.
National and international patents that are part of scientific achievement:
1. Andrzej Płucienniczak, Małgorzata Kęsik, Grażyna Płucienniczak, Diana Mikiewicz-
Syguła. Patent granted:
11.06.2013 by the Patent Office of the Republic of Poland (patent number PL216037).
26.03.2014 by the European Patent Office (patent number EP 2109671).
14.01.2014 by the United States Patent Office (patent number US 8,628,954).
Title: „Expression cassette, use of the expression cassette, vector, host cell, a method for
producing a polypeptide”. Territorial protection: Poland, Europe (France, Germany, United Kingdom), United States.
2. Józef Kapusta, Małgorzata Kęsik-Brodacka, Violetta Cecuda-Adamczewska, Violetta
Sączyńska, Piotr Bociąg, Robert Brodzik, Bogdan Wolko, Janusz Kocik, Michał Bartoszcze, Grażyna Płucienniczak, Andrzej Płucienniczak. Patent granted 26.06.2013 by the Patent Office of the Republic of Poland (patent number PL
215808).
Title: “Universal carrier of bacterial antigen, antimicrobial vaccine, method of producing
a universal antigen carrier, use of a universal antigen carrier”. Territorial protection: Poland.
3. Małgorzata Kęsik, Agnieszka Romanik, Andrzej Płucienniczak, Grażyna Płucienniczak, Diana Mikiewicz-Syguła.
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Patent granted:
20.08.2015 by the Patent Office of the Republic of Poland (patent number PL222067).
16.11.2016 by the European Patent Office (patent number EP 2742140).
Title: „Prokaryotic host cell comprising expression vector”. Territorial protection: Poland, Europe (France, Germany, United Kingdom)
Total Impact Factor (IF) of the publications included in the reported scientific
achievement according to the year of publication (in the case of papers from 2017 the IF
is given as for 2016): 15.1.
Total points for a uniform cycle of publications according to the list of scientific journals
of Ministry of Science and Higher Education consistent with the year of publication: 168.
The total number of points for the granted patents forming part of the scientific
achievement (accepted rating category according to the Regulation of the Minister of
Science and Higher Education Dz. U. 2016 item 2154): 110.
4.3) Discussion of the scientific goals of the publications comprising the scientific achievement
and potential of its further use
Introduction
Introduction of vaccinations was one of greatest achievements of modern medicine. Vaccinations
are one of the most effective ways in the prevention of infectious diseases. One of most
spectacular successes of vaccination is eradication of smallpox, which in the first half of XX
century was causing 2 m human deaths yearly. Introduction of vaccinations reduced in the United
States and Western Europe the incidence of infectious diseases such as diphtheria, pertussis,
tetanus, yellow fever, polio, measles, mumps and rubella, by 95-99% compared to the pre-
vaccination period [1].
It is noteworthy, that vaccination successes are not limited to preventing human diseases only.
Vaccines are currently the most effective method of limiting animal suffering and preventing
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economic losses caused by infectious diseases in animals. They also have the potential to control
diseases, which develop drug resistance.
As the vaccination coverage grows, there also grow requirements and expectations toward
new pharmaceuticals. These requirements are formulated both by health authorities and by
societies. This is another force driving worldwide research on next generations of medicaments,
bringing us ever closer to the ideal of vaccines that are safe, effective and widely available due to
their low cost.
The search for possibly safest and side effects-free vaccines resulted in development of
subunit vaccines [2]. They have well-defined chemical composition, because they contain only
selected, highly immunogenic, pathogen components. This approach eliminates risk of
vaccination-induced infection, which risk in vaccines using whole pathogens, cannot be fully
avoided. However, use of only selected antigens results in a reduced effectiveness of subunit
vaccines when compared to the classical approach. This reduced effectiveness stemming from
weak or ineffective immune response, is one of challenges of subunit vaccines development [3].
What is required to overcome this issue is development of antigens, which will be effective and
induce lasting protection against given pathogen. Special challenge is here posed by highly
variable pathogens (e.g. flu virus). A perfect vaccine should be viable against possibly wide
spectrum of pathogen variations, protecting against multiple pathogen variants. I analysed the
current state of human knowledge in a review article discussing universal flu vaccine.
Kesik-Brodacka M, Plucienniczak G. (2014) „A universal flu vaccine”. Acta Biochim Pol.
61:523–530.
Another requirement is the widest possible availability of vaccines, which requires, among
other things, development of suitable production methods. The basis for subunit vaccines
development is production of given pathogen’s antigens in another organism (production
system), e.g. bacterial, plant or animal. Such a system should be sufficiently stable and efficient
to enable lowering vaccine production costs. Relatively new expectation is reduction of negative
environment impact incurred by industrial expression of antigens used in subunit vaccines. One
of possible responses to this expectation is development of systems with no need for the use of
antibiotics, which have the potential to reduce both cost of environmental protection (simpler,
and therefore less costly spent broth disposal) and direct expenses by avoiding the need to
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purchase antibiotics routinely used to exert selection pressure during cultivation. Additionally,
this approach also addresses antibiotic resistance – an issue recently attracting still more
attention. According to the data of European Society of Clinical Microbiology and Infectious
Diseases’ experts, antibiotic-resistant bacteria may cause death of over 1mln people by 2050.
Responding to this threat, European Commission prepared guidelines which propose
development and implementation of a complex system aimed at stopping the decline in antibiotic
efficiency in treatment of infections and infectious diseases, which is caused by development and
spread of antibiotic resistance among microorganisms. The system is also expected to rationalise
antibiotic use.
Additional advantage of antibiotic-free recombinant protein expression systems is
elimination of another medical risk – anaphylactic shock which may be induced in susceptible
individuals by remnants of the selection antibiotic in the preparation used for vaccination. This
risk has been addressed by, among others, European Medical Agency and US Food and Drug
Administration, which agree to register medical substances, whose production process uses as
antibiotic resistance genes selection markers, only in exceptional situations, and when presented
with extensive justification [5,6]
The aim of the work was researching immunisation potential of antigens produced using
novel recombinant protein expression systems.
My research of immunisation potential of antigens included use of plants as antigen
producing systems. Plant expression systems exhibit advantageous characteristics predisposing
them for applications in the generation of vaccine antigens. Antigens biosynthesized in edible
plants may be used in oral immunization with no need for costly and time-consuming purification
stages. We used the thus produced antigen in studies on subunit vaccine against the common liver
fluke.
The common liver fluke is a pathogen causing considerable economic losses, connected not
only with mortality of infected animals, but also their reduced productivity. To date no vaccine
has been generated against this pathogen. What is more, development of a vaccine against the
common liver fluke has proven to be exceptionally difficult. This results from the fact that the
common liver fluke presents the host organism with a great variety of antigen particles, of which
only a slight fraction induces protective immune response. The antigen used in our study was
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cysteine proteinase cloned from adult common liver flukes Fasciola hepatica (CPFhW). Cysteine
proteinases serve many important functions in the physiology of F. hepatica, e.g. they participate
in the decomposition of host tissues during their migration through tissues, parasite nutrition as
well as combat against immune response of the host. For this reason it is assumed that they may
be antigens inducing immune response and protection against liver fluke infection [7].
In the nucleotide proteinase sequence we distinguish three coding parts, i.e. the signaling,
leader and catalytic fragments. In the course of the studies I designed and obtained gene
constructs facilitating efficient biosynthesis of various variants of the CPFhW antigen in
transgenic lettuce. They comprised the leader region of the protein, its catalytic part and hybrid
proteins composed of the catalytic part of CPFhW and the core antigen of hepatitis B virus, as
well as the catalytic part of CPFhW and ubiquitin derived from Saccharomyces cerevisiae (Fig.
1). The hepatitis B virus antigen used in the gene constructs was to enhance immunogenicity of
the attached CPFhW fragment [8]. In turn, the fusion of CPFhW with ubiquitin was believed to
provide a solution to the common problem with obtaining efficient production of recombinant
proteins. Literature data indicate that the presence of the ubiquitin promoter or the ubiquitin gene
may considerably increase the expression of the gene attached to ubiquitin [9].
Fig. 1. A scheme of antigens produced in the plant expression system.
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We observed differences in the expression levels depending on the tested construct. The
highest expression level, as high as 20 μg per gram wet mass, was obtained for the hybrid protein
comprising the catalytic CPFhW fragment and ubiquitin derived from Saccharomyces cerevisiae.
In order to standardize the content of a given antigen in the material for immunization the plants
were freeze-dried and homogenized. Experimental animals were immunised twice with
individual antigens contained in freeze-dried transgenic lettuce. The antigen was administered by
the oral route. The objective of enteral antigen administration in those experiments was to induce
mucosal immune response, which is particularly important in the case of infections with F.
hepatica – a pathogen colonizing the host organism through the intestinal tract. After antigen
administration animals were infected with liver fluke metacercariae in order to evaluate
protective potential of administered antigens.
Studies showed that antigens produced and administered in lettuce induced immune
response and protection against infection. In animals both humoral and cellular immune response
was induced after the administration of the antigen. A distinct response in the IgG1 and IgM
isotypes was maintained for 4 weeks after immunization. Moreover, we obtained significant
protection of animals against liver fluke infection, manifested in a reduced number of flukes
colonizing the liver. The highest observed reduction in the number of flukes in the liver (65.4%)
was found after the administration of the hybrid antigens composed of the catalytic CPFhW
fragment and the hepatitis B virus core antigen. These results are satisfactory, since it is assumed
that providing a 50% protection against liver fluke infection could significantly reduce the scale
of infections with this pathogen [10].
Conducted studies indicate that in the adopted expression system we obtained functional
vaccine antigens. Natural protection for recombined protein, such as the plant cell wall,
predisposes this expression system for applications in the production of orally administered
antigens. It was particularly effective to use lettuce as the expression system, since lettuce is an
edible plant and its administration requires no preliminary thermal processing, which may
deactivate the vaccine antigen.
The accomplishment resulting from these studies was connected with the production of
recombinant CPFhW antigens generated in a plant expression system, which when used in
oral immunization effectively induce humoral and cellular immune response as well as
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protection against liver fluke infection, manifested in a significant reduction of the number
of flukes in the liver of immunized animals.
Research results were presented in the publication:
Kesik-Brodacka M, Lipiec A, Kozak Ljunggren M, Jedlina L, Miedzinska K, Mikolajczak M,
Plucienniczak A, Legocki AB, Wedrychowicz H. (2017) „Immune response of rats vaccinated
orally with various plant-expressed recombinant cysteine proteinase constructs when challenged
with Fasciola hepatica metacercariae“. PLoS Negl Trop Dis. 11:e0005451. doi:
10.1371/journal.pntd.0005451.
In our next experimental approaches we investigated potential antigens obtained from
a bacterial expression system to provide effective immunization against infection with the
common liver fluke. In my analyses I used gene constructs enabling in the E. coli strain
BL21(DE3) the production of various antigen variants, comprising the catalytic fragment or the
entire CPFhW antigen. The used expression vector (GenBank accession no. DQ485721) made it
possible to increase the amount of tRNA for codons rarely found in the bacterial genome (AGA
and AGG), which contributed to the abundant biosynthesis of recombinant proteins. In this study
rats were enterally immunized twice with individual CPFhW antigens, and next infected with
metacercariae in order to assess the protective potential of administered antigens. To identify the
mechanisms determining immunity of vaccinated animals in these experiments we analyzed the
humoral and cellular immune response. We showed that infection of immunized animals resulted
in a marked infiltration of eosinophils to the peritoneal cavity both in immunized and control rats.
However, the number of CD8 + and CD4 + lymphocytes in the peritoneal fluid was much higher
in vaccinated rats in comparison to the control rats. We obtained a very high reduction in the
number of flukes colonizing the liver. We recorded the highest decrease, as high as 80%,
following the administration of the catalytic fragment of CPFhW. Those studies indicate that
I obtained functional vaccine antigens, which provided a high reduction in the number of flukes
colonizing the liver, which was the primary objective of this immunization scheme.
The accomplishment resulting from these experiments was connected with the production
of CPFhW antigens in a bacterial expression system, which when administered in enteral
immunization of laboratory animals induce effective humoral and cellular immune
response, as well as provide a high level of fluke reduction.
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Research results were presented in the publication:
Kesik M, Jedlina-Panasiuk L, Kozak-Cieszczyk M, Płucienniczak A, Wedrychowicz H. „Enteral
vaccination of rats against Fasciola hepatica using recombinant cysteine proteinase (cathepsin
L1)“. Vaccine 2007;25:3619–3628.
Very promising results, which we obtained using the CPFhW antigen from the bacterial
expression system, including e.g. a very high level of resistance to liver fluke infection after
vaccination of laboratory animals, persuaded us to continue the initiated line of research. Our
initial question was whether the CPFhW antigens obtained in the bacterial expression system will
protect against liver fluke infection also these animals, which are natural hosts for this pathogen.
In our attempt to provide an answer to this question we conducted experiments on immunization
of calves and sheep. A significant part of this research was connected with the determination of
both the dose and the administration route of the CPFhW antigen obtained from a bacterial strain,
E. coli BL21(DE3). For the protection of animals against infection with such a pathogen as the
common liver fluke, it is essential to induce the above-mentioned mucosal immune response.
However, intragastric antigen administration in the case of ruminants due to their multi-
chambered stomach leads to the degradation of the antigen. For this reason a very interesting
accomplishment of this research was connected with the fact that the level of reduction in the
number of flukes colonizing the liver of immunized animals may be obtained after intranasal
administration of the CPFhW antigen. In female calves, which were administered two doses of
the CPFhW antigen onto the nasal mucous membrane and infected with metacercariae, the
number of flukes was reduced by 54.2% in comparison to the control. What is more, flukes found
in the bile ducts of immunized calves showed decreased reproduction rates. In the case of sheep
immunization the highest reduction in the population of flukes colonizing the liver (56.5%) was
obtained only after the administration of three intranasal doses of the CPFhW antigen. Moreover,
it was shown that vaccinated animals, both calves and sheep, had considerably reduced blood
eosinophil levels in comparison to the control animals.
In this study for the first time worldwide a significant degree of immunity against
F. hepatica infestation was obtained in ruminants as a result of mucosal immunization. What is
more, we found a correlation of great importance both from the scientific and practical point of
view between the provided level of protection against F. hepatica and the sex of immunized
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animals. So far literature sources have reported no data on this problem in the case of F. hepatica.
This issue is crucial in studies aiming at the development of a vaccine.
These studies showed that the application of the recombinant CPFhW antigen produced in
a bacterial expression system in the immunization of ruminants makes it possible to obtain
a statistically significant reduction in the number of flukes and alleviation of pathological
changes observed after infection with metacercariae.
The accomplishment resulting from these studies was connected with the production of
efficacious CPFhW antigens in a bacterial expression system, which when applied in the
immunization via the mucosal route in calves and sheep cause a significant reduction of the
number of flukes.
Research results were presented in the publication:
Wedrychowicz H, Kesik M, Kaliniak M, Kozak-Cieszczyk M, Jedlina-Panasiuk L, Jaros S,
Płucienniczak A. „Vaccine potential of inclusion bodies containing cysteine proteinase of
Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the
fluke“. Vet Parasitol. 2007, 147:77-88.
Very good results of immunization with antigens derived from the bacterial expression
system provided an inspiration for research on the development of an expression system, which
would be feasible in the commercial scale production of the antigens I obtained.
One of the most commonly used bacterial systems expressing recombinant proteins, including
vaccine antigens, is a system, in which the T7 RNA polymerase recognizes the T7 promoter. This
system is based on the application of a strong, inducible T7 phage promoter, present in the
expression vector, as well as an exceptionally selective and active T7 RNA polymerase found in
the bacterial chromosome. This expression system typically proves to be highly efficient;
nevertheless, its practical application is hindered by the fact that the biosynthesis of the
recombinant protein, which production is regulated by the T7 promoter, decreases with time. This
prevents the utilization of this system in the commercial scale production of proteins. Efforts
made to solve this problem since the very beginning of the application of the T7 promoter system
have been unsuccessful. It was shown that the primary cause for the amount of the biosynthesized
target polypeptide decreasing with time is connected with the appearing chromosomal mutations
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within the sequence coding the DNA-mediated T7 RNA polymerase, foreign for the bacterial
host cell, as a result of which it is deprived of its function [11]. Cells, in which such a mutation
has appeared, do not produce the target protein. My observations indicate that cells, which
metabolism is not burdened with the enforced production of the target protein, grow and divide
faster. This leads to the culture being overgrown with bacterial cells not producing the target
protein and it is the cause for the observed adverse phenomenon reducing the level of
biosynthesis of the recombinant protein. Thanks to this knowledge I may formulate the
hypothesis, according to which eliminating from the bacterial culture these cells, in which
a mutation appeared within the gene coding for the T7 RNA polymerase, makes it possible to
maintain a stable level of biosynthesis of recombinant proteins. In order to verify such
a hypothesis I adopted a pioneering approach consisting in the development of an expression
cassette, in which T7 RNA polymerase recognizes the T7 promoter regulating both the synthesis
of the target polypeptide and the selection factor, determining survival of the bacterial host cell in
culture with selection pressure (Fig. 2B). An appropriate selection factor determining survival of
the host cells is a protein factor, which needs to be produced in the cell, as otherwise this leads to
cell death. The factor determining cell survival, which I applied in my experiments, was
aminoglycoside-3’-phosphotransferase (APH(3')) (APH) encoded by aph gene (determines the
resistance to kanamycin). As the production of APH depends on the functioning of the T7 RNA
polymerase/promoter system in E. coli BL21(DE3) culture transformed with the vector with the
cassette, in the presence of kanamycin only these bacteria may develop which bear the vector and
at the same time in which the T7 RNA polymerase/promoter system works well, that is, only
those cells that produce the recombinant protein.
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Fig. 2 Scheme of the system for efficient production of recombinant proteins. 2A - E. coli strain BL21(DE3). 2B -
Expression cassette incorporated into the expression vector. Segments of the cassette: T7 promoter - T7 promoter
recognized by T7 RNA polymerase; recombinant protein - sequence encoding recombinant protein; stop - translation
stop codon; TT - transcription terminator for non phage promoters; aph gene - gene encoding aminoglycoside 3'-
phosphotransferase (kanamycin resistance). 2C - E. coli BL21(DE3) bacteria transformed with the expression
cassette in medium supplemented with kanamycin. IPTG - induction with isopropyl-β-D-1-thiogalactopyranoside;
APH - 3'-aminoglycoside phosphotransferase (kanamycin resistance).
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Additionally, the level of APH biosynthesis has been optimized experimentally so that it
is produced at the lowest level needed for bacterial cells to grow. The level of APH biosynthesis
is low enough to do not affect the reduction of recombinant protein biosynthesis. I achieved the
optimal level of biosynthesis by modifying the sequence of comprising Shine-Dalgarno non-
coding part of aph gene. Therefore, characteristic for T7 RNA polymerase/promoter high
biosynthesis is observed only for the recombinant protein. Thanks to this, the developed system
allow the biosynthesis of the recombinant protein at a level as high as that of the standard T7
RNA polymerase/promoter.
The cassette was included in the example expression plasmids. In the course of my studies
I observed that the obtained cassette does not serve its function, since the production of the
selection factor is not only regulated by the T7 phage promoter specifically recognized by the T7
phage polymerase, but also by other promoters found in the plasmid, into which the cassette was
cloned. I solved this problem by blocking the biosynthesis controlled by promoters found within
the plasmid outside the cassette. I obtained this by incorporating into the cassette the transcription
terminator for promoters other than phage promoters upstream of the 5' end of aph gene. This
prevents production of the factor determining survival of the bacterial host cell involving
promoters other than the T7 phage promoter. Additionally, between the sequence encoding the
recombinant protein and the sequence encoding the factor determining survival of the host cell,
the translation stop codon was incorporated. I showed experimentally that such a constructed
cassette (Fig. 2B) provides a stable and high expression of recombinant proteins. The application
of the cassette leads to the elimination from culture of these bacterial cells, in which chromosome
a mutation appeared preventing production of functional T7 phage polymerase and additionally
those, which as a result of mutation lost functionality of the T7 phage promoter. This means that
bacterial cells not producing the target polypeptide are eliminated from culture. The use of the
developed expression cassette prevents the culture from being overgrown with bacterial cells
unable to produce the target polypeptide and as a consequence - the decrease in biosynthesis of
the recombinant protein (Fig. 3). In this way I confirmed the hypothesis proposed at the
beginning of the research project.
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Fig. 3 Diagram of the division of bacteria and the appearance of mutants in the culture:
(top fig.) using an unchanged standard T7 RNA polymerase/promoter system. Bacteria that do not produce
recombinant proteins overgrow the culture;
(bottom fig.) developed novel system for stable expression of proteins based on the standard T7 RNA
polymerase/promoter system. Bacteria that do not produce recombinant proteins are eliminated from the culture.
What is more, the novel approach I applied was based on the phenomenon described in
literature as leaking, resulting in the non-specific expression of the gene encoding the
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recombinant protein. I showed that using this property of the system the selection factor may be
added to culture with no need to wait until the moment of induction. For this reason the selection
factor, i.e. the sequence encoding kanamycin resistance, serves a dual role: it facilitates selection
of host cells carrying the expression vector (the function of a selection marker) and those
producing the target polypeptide (the function of a selection factor), determining survival of host
cells. Thanks to the leaking phenomenon no additional selection factor is required at the stages
preceding induction, which reduces costs of culture and potential disposal of spent broth.
The functioning of the system in the presence of the cassette was verified for two different
target polypeptides (PA-4D from Bacillus anthracis and E2 from classical swine fever virus) and
two expression vectors. In all the experimental approaches the expression system using the
developed cassette provides a high and stable level of protein biosynthesis. I stated that the
generated expression system makes it possible to produce several times greater amounts of the
target polypeptide from the same culture volume in comparison to the non-modified system based
on T7 phage transcription. It reduces production costs, including recombinant protein purification
costs.
Two expression vectors with the embedded newly-developed cassette were provided by
IBA to KBI BioPharma Inc. (USA), where they are tested in the production of recombinant
proteins (copy of transfer agreement is in Attachment 5).
The accomplishment resulting from these studies was connected with the development of
a novel expression system based on a known system using T7 phage transcription. The
obtained system facilitates efficient commercial scale production of vaccine antigens.
However, it is not limited to applications connected solely with the generation of vaccine
antigens, but it also ensures stabilization of the production process for a wide range of
recombinant proteins. It is a universal system, which may be used extensively in the
production of recombinant proteins.
Research results were presented in the publication:
Kesik-Brodacka M, Romanik A, Mikiewicz-Sygula D, Plucienniczak G, Plucienniczak A. (2012)
„A novel system for stable, high-level expression from the T7 promoter”. Microb Cell Fact
11:109,
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and a patent:
Małgorzata Kęsik, Diana Mikiewicz-Syguła, Andrzej Płucienniczak, Grażyna Płucienniczak.
„Kaseta ekspresyjna, zastosowanie kasety ekspresyjnej, wektor, komórka gospodarza oraz sposób
otrzymywania polipeptydu”. Patent udzielony 11.06.2013 przez Urząd Patentowy
Rzeczpospolitej Polskiej; 26.03.2014 przez Europejski Urząd Patentowy; 14.01.2014 United
States Patent Office.
In my further research I investigated the vaccine potential of the antigen obtained in the
novel expression system described above, based on the antigen derived from the rod-shaped
bacterium of Bacillus anthracis. The antigen was the C-end domain of the protection antigen
(PA-4D) of B. anthracis. The applicability of the developed system for rapid production of large
amounts of the vaccine antigen to produce new-generation subunit vaccine against anthrax
became crucial in the context of bioterrorism threat. Additionally, the development of a new,
efficacious subunit vaccine against anthrax is essential in view of the fact that none of the
registered vaccines fully satisfies contemporary safety standards. Registered vaccines contain
a broad spectrum of undefined components, which are found in extracts from cultures of mutated
B. anthracis strains used in the production of these vaccines.
We showed in the course of this research that expression of PA-4D in the improved
expression system based on the transcription of the T7 phage is more efficient than using
conventional expression systems in E. coli. The obtained vaccine antigen was isolated from
bacterial cells and combined with an adjuvant – a saponin extract from Quillaja saponaria.
Analyses showed that the developed vaccine preparation induces immune response in mice both
after injection and mucosal administration. Results of these experiments also show that the
obtained antigen after its administration causes neutralization of anthrax toxins. The
neutralization effect is a characteristic of the protection level of the anti-anthrax response against
toxins as well as spore and vegetative forms of anthrax rods. Additionally, we showed that the
conducted immunization provides long-term response to the administered antigen. Obtained
results confirm that it is possible to apply the antigen obtained by our team to construct a subunit
recombinant vaccine against anthrax.
The accomplishment of these studies was connected with the confirmation of efficacy of the
PA-4D antigen derived using a novel system for stable recombinant protein biosynthesis in
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E. coli in the induction of specific, long-term immune response and causing neutralization
of anthrax toxins. The derived antigen may be used to construct a new-generation vaccine
against anthrax.
Research results were presented in the patent:
Michał Bartoszcze, Robert Brodzik, Violetta Cecuda-Adamczewska, Józef Kapusta, Janusz
Kocik, Małgorzata Kęsik-Brodacka, Grażyna Płucienniczak, Andrzej Płucienniczak, Violetta
Sączyńska, Bogdan Wolko. “Universal carrier of bacterial antigen, antimicrobial vaccine, method
of producing a universal antigen carrier, use of a universal antigen carrier”. Patent granted
26.06.2013 by the Patent Office of the Republic of Poland.
I confirmed the universal character of the novel expression system for antigens with
vaccine potential by deriving still another antigen. It was avian flu hemagglutinin (HA). Results
of this research were reported in a publication not included in the presented accomplishment:
Sączyńska V, Romanik A, Florys K, Cecuda-Adamczewska V, Kęsik-Brodacka M, mietanka K,
Olszewska M, Domańska-Blicharz K, Minta Z, Szewczyk B, Płucienniczak G, Płucienniczak A.
(2017) A novel hemagglutinin protein produced in bacteria protects chickens against H5N1
highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing
antibodies.
PLoS One. 12:e0172008. doi: 10.1371/journal.pone.0172008.
The accomplishment in this study was to confirm the efficacy of the HA antigen derived
using the novel system for stable recombinant protein biosynthesis in E. coli in the
induction of complete protection of poultry against infection by a homologous clade of
H5N1 virus including inhibition of excretion.
Results, which I obtained when running biosynthesis of various vaccine antigens in the
system I developed for stable production based on the T7 phage transcription, indicate that it is
a universal system and thus obtained antigens exhibit vaccine potential.
Apart of stability, another disadvantageous characteristic of the systems commonly used to
produce recombinant proteins is the use of antibiotic resistance genes and the application of
antibiotics during culture in order to maintain selection pressure. The need to apply antibiotics,
Attachment 2b
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particularly in large scale production, increases the risk of spreading antibiotic resistance in the
environment. Moreover, the use of antibiotics has a considerable effect on the increase in costs of
biosynthesis of target proteins, both connected with the cost of the reagent itself and the cost of
spent broth disposal. The above-mentioned adverse characteristics of commonly used systems
persuaded me to conduct studies on the development of an expression system, which would
provide stable and efficient expression with no need for the use of antibiotics. For this purpose, as
in the developed of stable biosynthesis recombinant proteins system, I used the system based on
the T7 RNA polymerase/promoter. In this system expression of the T7 RNA polymerase gene is
controlled by addition of IPTG. In addition to the need for selective antibiotics, this system is
characterized by a decreasing level of recombinant protein biosynthesis resulting from the
appearance of mutations within the gene encoding the T7 RNA polymerase [11]. In the course of
these studies we developed an appropriately modified bacterial strain E. coli BL21(DE3). This
strain was generated through targeted mutagenesis of the chromosomal aroA gene, which
product, 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), is required for survival
of E. coli cells (Fig. 4A). The obtained strain E. coli BL21(DE3)aroA is an auxotroph. This
strain is the host in which the biosynthesis of the recombinant protein is carried out with the use
of a vector comprising the cassette developed during the study. In this cassette, the T7 promoter
regulates both the synthesis of the recombinant protein and the selection factor determining
survival of the bacterial host cells. In this case, the EPSP synthase is the product of the aroA
gene, which functional copy is found in the cassette included in the expression plasmid (Fig. 4B).
The EPSP synthase is produced by the presence of the aroA gene in the plasmid, which
complements the deletion of the aroA gene in the host cell chromosome (Figure 4C). Since the
production of EPSP synthase depends on the functioning of the T7 RNA polymerase/promoter
system, in the E. coli BL21 (DE3)aroA culture only those cells may survive, which carry
a plasmid containing the designed cassette and at the same time have an effective polymerase/T7
phage promoter system that is, only those cells that produce the recombinant protein.
Additionally, the level of EPSP synthase biosynthesis was optimized experimentally so that
it is produced at the lowest level needed for bacterial cells to grow. The level of EPSP synthase
biosynthesis is low enough to do not affect the reduction of recombinant protein biosynthesis.
I achieved the optimal level of biosynthesis by modifying the sequence of comprising Shine-
Dalgarno non-coding part of aroA gene. Therefore, characteristic for T7 RNA
Attachment 2b
22
polymerase/promoter high biosynthesis is observed only for the recombinant protein. The
structure of the designed cassette provides biosynthesis of the target polypeptide in the
prokaryotic host cells at a level at least equivalent to that in the standard system containing the
same T7 phage promoter.
In addition, this system allows for stable production of the protein for a long time, thanks to
the fact that bacterial cells that have damaged the T7 RNA polymerase/promoter system are
eliminated from the culture. Therefore, bacterial cells that do not produce recombinant proteins
are eliminated from the culture. Thus the use of the developed expression cassette allow to avoid
the culture to be overgrowth with bacteria not producing the recombinant protein, and
consequently, to decrease the level of recombinant protein biosynthesis (Fig. 3).
The action of the cassette in the produced vectors was verified for the target polypeptide,
i.e. the PA-4D Bacillus anthracis antigen.
Attachment 2b
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Fig 4. Scheme of the system for efficient production of recombinant proteins without the use of antibiotics. 4A - E.
coli strain BL21(DE3) with deletion of aroA gene in chromosome (E. coli BL21(DE3)aroA). 4B - Expression
cassette incorporated into the expression vector. Segments of the cassette: T7 promoter - T7 promoter recognized by
T7 RNA polymerase; recombinant protein - sequence encoding recombinant protein; stop - translation stop codon;
TT - transcription terminator for non-phage promoters; aroA sequence - gene encoding 5-enolpyruvylshikimate-3-
phosphate (EPSP) synthase. 4C - E. coli BL21(DE3)aroA transformed with the expression cassette. EPSPS - 5-
enolpyruvylshikimate-3-phosphate synthase; IPTG - induction with isopropyl-β-D-1-thiogalactopyranoside.
Attachment 2b
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The developed system enable production of recombinant proteins with no need for the use
of antibiotics. Moreover, this system makes it possible to maintain high production of protein
over a long period, which is necessary for commercial scale systems.
The accomplishment resulting from this research was connected with the development of
a bacterial system for the biosynthesis of recombinant proteins, combining advantages of
the system for stable expression with elimination of the use of antibiotics during culture.
Research results were presented in the patent:
Małgorzata Kęsik, Agnieszka Romanik, Andrzej Płucienniczak, Grażyna Płucienniczak, Diana
Mikiewicz-Syguła. „Prokaryotic host cell comprising expression vector”. Patent granted
20.08.2015 by Patent Office of the Republic of Poland 16.11.2016 by the European Patent Office.
An important accomplishment resulting from the studies concerning the presented novel
expression systems is connected with their universal character, facilitating their application
in the production of a broad spectrum of antigens having immunization potential as well as
other recombinant proteins. The potential utilization of the research outcomes in specific
practical applications includes efficient large-scale production of vaccine antigens or other
recombinant proteins.
In view of the application potential of the presented results I prepared a review of the
latest literature concerning problems connected with the development of subunit vaccines and
feasibility of various production systems, as well as their marketability. I analyzed this potential
in relation to the latest accomplishments in the development of novel biopharmaceuticals.
A critical analysis of related problems is given in a review paper:
Kęsik-Brodacka M. (2017) Progress in Biopharmaceutical Development.
Biotechnol Appl Biochem. doi: 10.1002/bab.1617.
Summary of the results:
1. Recombinant CPFhW antigens produced in the plant expression system were obtained. The
antigens when used in oral immunization, effectively induce humoral and cellular immune
responses, and provide protection against liver fluke infection.
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2. Recombinant CPFhW antigens produced in the bacterial expression system that are used in
enteral immunization of laboratory animals induce an effective humoral and cellular immune
response and provide protection against F. hepatica infection.
3. For the first time worldwide, a significant degree of immunity against F. hepatica
infestination was obtained in ruminants as a result of mucosal immunisation. These results
were obtained with CPFhW antigens produced in the bacterial expression system.
4. An innovative expression system was developed based on a known system using T7 phage
transcription. The obtained system is universal and may be used extensively in the efficient
industrial-scale recombinant protein biosynthesis.
5. An innovative system for stable protein biosynthesis in E. coli was used to obtain the PA-4D
antigen. The efficacy of this antigen was demonstrated in the induction of specific, long-term
immune responses and neutralization of anthrax toxins. The obtained antigen can be used in
the construction of a new-generation vaccine against anthrax.
6. A novel bacterial system for expression of recombinant proteins was developed. It combines
advantages of the system for stable expression with the elimination of the use of antibiotics
during culture.
7. Analysis of issues related to the development of subunit vaccines, recombinant protein
expression systems as well as the market for recombinant proteins as active ingredients of
vaccines and biopharmaceuticals have been conducted.
Bibliography:
1. Sow SO, Tapia MD, Diallo S, Keita MM, Sylla M, Onwuchekwa U, Pasetti MF, Kotloff KL, Levine MM.
(2009) Haemophilus influenzae Type B conjugate vaccine introduction in Mali: impact on disease burden
and serologic correlate of protection. Am J Trop Med Hyg. 80:1033–1038.
2. Ada G. (2001) Vaccines and vaccination. N. Engl. J. Med. 345: 1042–1053.
3. Jarząb A, Skowicki M, Witkowska D. (2013) Szczepionki podjednostkowe – antygeny, no niki, metody koniugacji i rola adiuwantów. Postepy Hig Med Dosw. 67: 1128–1143.
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4. ESCMID. Antibiotic armageddon in UK and Europe by 2025. April 21, 2015.
https://www.escmid.org/fileadmin/src/media/PDFs/2News_Discussions/Press_activities/ECCMID_2015/ES
CMID_one_million_deaths_UK_consumer.pdf
5. EMA: The European Agency for the Evaluation of Medicinal Products. Note for Guidance on the Quality,
Preclinical and Clinical Aspects of Gene Transfer Medicinal Products
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/10/WC500003977.pdf
6. FDA: Guidance for human somatic cell therapy and gene therapy. (1998). Center for Biologics Evaluation
and Research, Food and Drug Administration.
7. Dąbrowska M, Kaliniak M, Wędrychowicz H. (2006) Wpływ szczepienia cieląt rekombinowaną proteazą cysteinową Fasciola hepatica na rozwój i inwazyjno c miracydiów. Wiadomo ci Parazytologiczne. 52: 305–309.
8. Ulrich R, Nassal M, Meisel H, Kruger DH. (1998) Core particles of hepatitis B virus as carrier for foreign
epitopes. Adv Virus Res. 50: 141–182.
9. Baker RT, Smith SA, Marano R, McKee J, Board PG. (1994) Protein expression using cotranslational
fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione
S- transferase. J Biol Chem. 269: 25381–25386.
10. Toet H, Piedrafita DM, Spithill TW. (2014) Liver fluke vaccines in ruminants: strategies, progress and
future opportunities. Int J Parasitol. 44: 915–927.
11. Vethanayagam JG, Flower AM. (2005) Decreased gene expression from T7 promoters may be due to
impaired production of active T7 RNA polymerase, Microb. Cell Fac. 7: 3.
5. Presentation of other scientific and research accomplishments
In the years 1994 - 1999 I studied at the Faculty of Biology, the University of Warsaw
(field of study biology, specialization microbiology). Thanks to my very high learning
achievements I was able to take an individualized program of study and undertake simultaneous
studies at the Faculty of Economic Studies, the University of Warsaw. I prepared my M.Sc. thesis
entitled "The role of 60 kDa protein in the pathogenesis of Listeria monocytogenes" at the
Department of Applied Microbiology, the University of Warsaw under the supervision of Prof. dr
hab. Jacek Bielecki. The aim of that thesis was to characterize the iap gene of L. monocytogenes
1043S. The objective of my research was to utilize the intracellular rod L. monocytogenes as
a live vector to construct a new generation vaccine, capable of inducing cell-mediated immunity
against various antigens. I continued research connected with the problems of vaccine
construction during my PhD studies.
Following the defense of my M.Sc. thesis I started my PhD studies at the Institute of
Biochemistry and Biophysics PAS. I conducted my experimental work under the supervision of
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Prof. dr hab. Andrzej Płucienniczak at the Institute of Biotechnology and Antibiotics (IBA). The
aim of my doctoral dissertation was to determine the applicability of inclusion bodies as a new
carrier of an orally administered vaccine antigen. I conducted this research within the framework
of project "Multivalent antigens based on modified nucleocapsids of hepatitis B virus (HBV)"
realized by a consortium comprising several leading scientific centers in Poland.
During my PhD studies I received a research grant of the School of Molecular Biology, the
Institute of Biochemistry and Biophysics PAS. The research conducted within the framework of
that grant concerned characteristics of active cysteine protease of Fasciola hepatica obtained
from inclusion bodies produced in Escherichia coli.
Already during my PhD studies, in 2002, I was employed at the Department of Bioengineering,
the Institute of Biotechnology and Antibiotics, at the position of biologist.
In 2005 at the Institute of Biochemistry and Biophysics PAS I defended my PhD
dissertation entitled "Inclusion bodies from recombinant bacteria as a novel system for delivery
of vaccine antigen by the oral route" and I received the PhD degree in biological sciences [doktor
nauk biologicznych] in the field of biochemistry.
The experience gained in the course of my research helped me to develop my research skills, gain
insight into problems connected with oral immunization and this resulted in the following
accomplishments:
Submission of a complete sequence of the cysteine protease coding gene of Fasciola
hepatica to the GenBank database (accession number GenBank AY277628).
Polish and international patents, of which I am a co-author (nos. 199190, 196114, 199189,
201419, 201420).
Publication:
Kęsik M, Sączynska V, Szewczyk B, Płucienniczak A. (2004) Inclusion bodies from
recombinant bacteria as a novel system for delivery of vaccine antigen by the oral route.
Immunol Lett. 91:197-204.
Publication:
Wędrychowicz H, Lamparska M, Kęsik M, Kotomski G, Mieszczanek J, Jedlina-Panasiuk
L, Płucienniczak A. (2003) The immune response of rats to vaccination with the cDNA or
protein forms of the cysteine proteinase of Fasciola hepatica. Vet Immunol
Immunopathol. 94:83-93.
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During the course of my research, which became the basis for my doctoral dissertation, I also
worked on the use of yeast Saccharomyces cerevisiae recombinant cells for the production of oral
vaccines. Yeast expression system exhibit advantageous characteristics predisposing them for
vaccine antigen production. They are considered safe for oral use. Foreign eukaryotic proteins
synthesized in recombinant yeast cells undergo co-translational or post-translational
modifications similar to those that occur in natural host cells. Also, the formation of a tertiary
structure of eukaryotic proteins in yeast cells takes place properly (native inter- and
intramolecular forms are formed). These features were an inspiration for the use of S. cerevisiae
as a host for the expression of heterologous proteins with medical and veterinary potential. The
research was aimed at obtaining vaccines against hepatitis B, classical swine fever and
F. hepatica. The results of the work were presented in Polish patent No. 199189, of which I am
co-author.
Results of my research conducted within the framework of my PhD studies contributed to the
IBA being awarded a specific targeted research project "Development and evaluation of
experimental edible vaccines against the common liver fluke F. hepatica and porcine
rotaviruses", which was financed by the State Committee for Scientific Research, and a grant
"Center of Biotechnology for medicinal products. A package of innovative biopharmaceuticals
for therapy and prophylaxis in humans and animals", co-financed from the funds of the
Operational Programme Innovative Economy (POIG). The POIG project was realized within
a consortium with the Institute of Biochemistry and Biophysics PAS and the University of
Gdańsk, and it was composed of 2 basic parts: generation of insulin analogues with modified
hypoglycemic effects and development of a vaccine against avian influenza. I participated in the
execution of both parts of this project. I was the head of two commissioned tasks within that
project: Production of expression plasmids for both virus proteins HA and NA in E. coli, and
Production of virus antigens in cells of E. coli. In addition, I was the contractor for the task:
Receiving analogues of human insulin in E. coli bacteria.
Within the framework of the first task I prepared around a dozen gene constructs and bacterial
strains facilitating efficient expression of the vaccine hemagglutinin antigen (HA). In the course
of this research I found solutions to several problems connected with the expression of
hemagglutinin in the bacterial system caused by the toxic effect of this protein on host cells.
I obtained antigens using an original system, which I developed for stable protein expression
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(Kesik-Brodacka M, Romanik A, Mikiewicz-Sygula D, Plucienniczak G, Plucienniczak A.
(2012) „A novel system for stable, high-level expression from the T7 promoter”. Microb Cell
Fact. 11:109. A publication that is part of the scientific achievement described in this summary of
Professional Accomplishments). I developed a method ensuring efficient production of this
protein, including culture of obtained strains and a method of antigen isolation from bacterial
cells.
Research results were presented in:
Sączyńska V, Romanik A, Florys K, Cecuda-Adamczewska V, Kęsik-Brodacka M, mietanka
K, Olszewska M, Domańska-Blicharz K, Minta Z, Szewczyk B, Płucienniczak G, Płucienniczak
A. (2017). A novel hemagglutinin protein produced in bacteria protects chickens against H5N1
highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing
antibodies.
PLoS One. 12:e0172008. doi: 10.1371/journal.pone.0172008.
That paper presented an effective vaccine based on the developed protein antigen and the
results of an experiment, in which the administered preparation provided chickens with complete
protection against infection with a homologous clade of the H5N1 virus along with the inhibition
of viral excretion. Moreover, the developed antigen and the resulting vaccine were the basis for
patent claims, of which I am a co-author: patent claim no. 408649 submitted at the Polish Patent
Office and international patent claim no. PCT/PL2015/050025.
The next objective of research conducted within the framework of the POIG project, in
which I was involved, was to develop a technology to produce recombinant human insulin
analogues of modified action. The production technology for insulin analogues was included in
the Polish patent and patent claim: 219335, 399287, as well as international patent claims:
WO2010002283, WO2013176560. I am a co-author of the above-mentioned patents and patent
claims. It needs to be stressed here that the patented production technology for the analogue of
recombinant human insulin of modified short-term hypoglycemic action (biosimilar to Lispro
insulin) was commercialized.
Another subject of research connected with the production of insulin, in which
I participated, was the production of a recombinant human enzyme: peptidylgycine α-amidating
monooxygenase (PAM). Within the framework of that research problem I participated in the
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experiments, which provided a stable eukaryotic cell line CHO/dhFr- secreting PAM to the
culture medium. This work resulted in the development of a two-stage PAM purification method
and verified the feasibility of the enzymatic reaction with a novel substrate - an analogue of
human insulin with single glycine attached at the C-end (GKR). The amidated form of GKR
insulin (GKR-NH2) was obtained. The biological activity of modified insulin was tested on rats
and its hypoglycemic effect was confirmed. It was shown that the GKR insulin analogue may be
a substrate for PAM. The produced recombinant PAM enzyme may be used to produce
recombinant peptide hormones, which require α-amidation to gain full biological activity.
Results of these research works were presented in:
Zieliński M, Wójtowicz-Krawiec A, Mikiewicz D, Kęsik-Brodacka M, Cecuda-Adamczewska V,
Marciniak-Rusek A, Sokołowska I, Łukasiewicz N, Gurba L, Odrowąż-Sypniewski M, Baran P,
Płucienniczak G, Płucienniczak A, Borowicz P, Szewczyk B. (2016) "Expression of recombinant
human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for
insulin analogue modification".
The next area of my scientific interests was connected with the applicability of the antigen
from inclusion bodies produced in Escherichia coli as a carrier for a bacterial immunogen to
obtain monoclonal antibodies of high affinity to the native antigen. In the conducted studies as
a result of the use of bacterial inclusion bodies containing the C-end domain of the protection
antigen of anthrax bacillus PA-4D we obtained a functional, recombinant monoclonal antibodies
(B10D2 oraz B8G2) of high affinity to the native antigen of anthrax bacillus. In our study, we
obtained transgenic plants (tobacco) producing recombinant B10D2 antibody variants containing
heavy and light chain antibody variable regions and IgG2a mouse monoclonal antibody. Plant-
expressed monoclonal antibody B10D2-P is an IgG2a isotype antibody and has an affinity for
PA-4D.
Research results were included in the patent:
Michał Bartoszcze, Robedrt Brodzik, Violetta Cecuda-Adamczewska, Józef Kapusta, Janusz
Kocik, Małgorzata Kęsik-Brodacka, Grażyna Płucienniczak, Andrzej Płucienniczak, Violetta
Sączyńska, Bogdan Wolko. Patent granted on 10.08.2015 by the Patent Office of the Republic of
Poland. Title: Recombinant monoclonal antibody of high affinity to a bacterial antigen of a native
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and functional structure, the manner to produce monoclonal antibodies and application of
recombinant monoclonal antibodies.
In my opinion a particularly important element for my scientific development was
connected with my Postdoc position at the Case Western Reserve University in Cleveland, USA.
In the course of that postdoc position I worked in a team investigating molecular mechanisms
mediating and controlling lentivirus-host cell interactions within the framework of project
"Molecular Biology of Lentiviral Vpr and Vpx Proteins", financed by the National Institutes of
Health. As a result of our study we discovered a long-sought cellular factor that works to inhibit
HIV infection of myeloid cells. These cells are a subset of white blood cells that display antigens
and hence are important for the body's immune response against viruses and other pathogens. The
factor is a protein called SAMHD1. This protein keeps cells from activating immune responses to
the cells own nucleic acids, thus preventing certain forms of autoimmunity from developing. We
observed that SAMHD1 prevents the replication of HIV in myeloid cells.
Our discovery provides a basis for conceiving of new therapies and treatment approaches,
which mimics this biological process. In consequence, it may contribute to prevent HIV infection
and/or its replication in myeloid cells and to stimulate body's own immune response to HIV.
Research in which I participated is crucial for the understanding of HIV pathogenesis and
it may be essential for the development of a vaccine against HIV.
Results of that research were presented in:
Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, Florens L,
Washburn MP, Skowronski J. "Vpx relieves inhibition of HIV-1 infection of macrophages
mediated by the SAMHD1 protein."
Nature. (2011) 474:658-661.
IF2011 36,28
Moreover, within the framework of that research we submitted a complete sequence of the
Homo sapiens SAMHD1 gene to the GenBank database.
I also participated in three other international scientific stays connected with the subject of
my research. Two of them were performed at the University of Lausanne in Switzerland. My
participation in those scientific stays was financed from the stipend I was granted from the funds
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of the 6th
and 7th
EU Framework Programmes. The third of my scientific stays was at the Uppsala
Universitet, Sweden. That stay was financed from a stipend of the 6th
Framework Programme of
the European Commission and the European Molecular Biology Organization. Participation in
those stays helped me to broaden my knowledge on the development of vaccines and vaccine
antigens as well as the application of adjuvants. I am using the knowledge and skills gained
thanks to those scientific stays in research on vaccines against influenza.
In 2014, I was granted a stipend funded by the European Patent Office, which made it
possible for me to participate in the European Patent Academy's Praktika Intern programme
Praktika Intern-Ship at the European Patent Office in Munich. My participation in that scientific
stay provided me with skills in the preparation of European patent claims and analyses of patent
applications and conducting opposition procedures. Thanks to my participation in that stay
I could realize objectives within the task of which I was the head in the project "Patent protection
of invention: Expression cassette, application of expression cassettes, expression vectors,
prokaryotic host cells containing expression vectors, bacterial strains and a method to produce
polypeptides" realized at IBA within the framework of the Operational Programme Innovative
Economy, priority axis Research and development of advanced technology. The aim of this
project was to ensure national and international legal protection, as well as transfer of research
and development results to the biotechnological and pharmaceutical sectors. Results of those
works covered inventions claimed in the patent, of which I am a co-author, which is described in
the section "Scientific accomplishments" of this self-presentation.
I am also using skills gained during my scientific stay at the European Patent Office to search for
novelties in current technologies, prepare descriptions of invention projects as well as prepare
specific responses to objections of experts of Polish and international patent offices in the course
of procedures to grant intellectual property protection for inventions resulting from my research
work.
In my opinion an element of paramount importance for my scientific development is
connected with my participation in the Top 500 Innovators: Science-Management-
Commercialization program, by the Ministry of Science and Higher Education within the
framework of the Operational Programme Human Capital. Within the framework of the program
I took a training stay at the Haas School of Business, University of California Berkeley, USA,
one of the best scientific centers worldwide according to the Academic Ranking of World
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33
Universities. Lectures given by outstanding academic teachers provided me with insight into
principles and methods to supervise the realization of research work and cooperate with
entrepreneurs. The scope of that program covered management of research activity, practical
aspects of research commercialization and cooperation of scientific and business communities, as
well as development of soft competences, i.e. team work and work in a multidisciplinary research
team, creative thinking, effective decision making, conflict resolution, etc. Moreover, within the
framework of that program I participated in study visits at the most innovative companies and
institutions worldwide (Google, NASA, Intel, etc.). They provided me with an opportunity to
observe work in leading companies, in which a significant role is played by commercialization of
research results, while I could also learn methods to conduct innovative research. I am using the
knowledge I gained when participating in the program in my activities as an expert evaluating
project proposals submitted at the National Centre for Research and Development, the Polish
Agency for Entrepreneurship and the European Commission and/or development works targeting
applications in economic activity. I also used this knowledge as a member of the Recruitment
Commission, appointed by the Minister of Science and Higher Education, during the
qualification of candidates for the following edition of the Top 500 program. I actively participate
in the initiatives of graduates of the Top 500 Innovators program. I am a founding member of the
Top 500 Innovators Association, a member of the Association Council and a member of the
Revision Committee.
Since 2012, I am a member of the Biotechnology Committee of the Polish Academy of
Sciences. In 2014 within the framework of works of the Committee I was an organizer and
chairman of a scientific conference "Red biotechnology as the basis for bioeconomy".
The list of my accomplishments following the defense of my PhD dissertation includes also
the presentation of research results during 44 scientific conferences (24 international and
20 national). After I was granted the scientific PhD degree I have delivered papers at 13 scientific
conferences, including 5 international conferences. I was a member of the Scientific Council of
one of the symposia.
I conducted research within the framework of 12 research projects financed from funds of
National Institutes of Health, National Centre for Research and Development, State Committee
for Scientific Research, the Ministry of Science and Higher Education, 10 of which after I
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34
received the PhD degree. I participated in 3 research projects, as the head for the task, in 6
research projects, as main contractor or contractor. In one project, I was acting on the basis of the
grant mandate of the grant manager in full scope resulting from the contract with the Scientific
Research Committee. I also participated in 2 projects financed or co-financed from EU funds,
serving the role of head of a task and a contractor. Two of the research tasks I realized were
conducted within the framework of R&D cooperation with an entrepreneur.
I am currently involved in the European Research and Technological Cooperation Program
funded by the European Commission under the Horizon 2020 program. I am a member of the
management committee and the leader of one of the teams.
After I was granted the scientific degree of doctor I participated in 6 international scientific
stays, including one within the framework of a Postdoc stipend at the Case Western Reserve
University, while another within the framework of a training program of the Ministry of Science
and Higher Education Top 500 Innovators Science-Management-Commercialization at the
University of California Berkeley.
My scientific experiments have been appreciated in Polish and international scientific
communities, as evidenced by invitations to review an application filed at the European
Commission within the framework of the Horizon 2020 program, 13 projects filed at the National
Centre for Research and Development as well as 17 projects submitted at the Polish Agency for
Entrepreneurship.
Moreover, I have reviewed manuscripts submitted at Polish and international scientific
journals.
I am the author of the chapter "Biopharmaceuticals and Pharmaceutical Biotechnology", in
"Biotechnology, biotechnology and new genetic engineering. Modern biotechnology based on
biotechnology " expertise prepared by the Biotechnology Committee of the Polish Academy of
Sciences. This expertise was submitted to the highest authorities of the state: the President of the
Republic of Poland, the President of the Council of Ministers, the Minister for Agriculture and
Rural Development, the Minister for the Environment, the Minister for Science and Higher
Education and Members of Parliament.
Due to the character of the unit, at which I am employed and where I conduct my research,
I have limited opportunities to be involved in teaching activity. Nevertheless, I have been
Attachment 2b
35
a scientific supervisor and scientific tutor for 7 M.Sc. theses and 2 Engineer's degree theses. In
this respect I cooperate with several universities in Warsaw: The Warsaw University of
Technology, the Warsaw University of Life Sciences and the University of Warsaw. I have also
been a supervisor for a scientific stay.
I was an independent supervisor and since 2014 I was a co-host for scientific stays
organized annually at IBA for a group of 10-12 students of biology and biotechnology.
I am a coordinator of activities popularizing science taught at IBA during the Science
Festival.
I organized the Biochemical laboratory at the Sub-department of Bioengineering at IBA
where the scope of research includes generation of gene constructs, new bacterial strains for the
production of proteins as well as isolation and purification of proteins of pharmaceutical
importance from prokaryotic systems. Since 2006 I have been the head of this laboratory. Since
2015, I am the Deputy Chair of the Commission for the Reception of Scientific Research and
since 2016 I have been a member of the animal welfare committee at IBA.
Summary list of publications. The Impact Factor for publications is given according to the year
of publication. In the case of papers from 2017 the IF is given as for 2016.
Total IF of the publications included in the reported scientific achievement–15.1
Total IF of all publications after receiving the PhD degree –55.54
Total IF of all publications–59.33
Total score of points for publications included in the reported scientific achievement
according to the classification of the Ministry of Science and Higher Education, according
to the year of publication–168
Total score of points for publications after receiving the PhD degree, according to the
classification of the Ministry of Science and Higher Education, according to the year of
publication–258
