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OPEN Outer membrane protein C is a

protective and unique vaccine
antigen against Shigella flexneri 3a
Anna Jarząb1, Anna Dąbrowska2, Piotr Naporowski1, Karina Krasna1, Agnieszka Szmyt2,
Michał Świat1, Krzysztof Pawlik1, Danuta Witkowska1, Edmund Ziomek1 & Andrzej Gamian1
The anti-Shigella vaccine is one of the WHO’s top priorities. Every year the disease kills more than
200,000 people worldwide and poses a serious threat to children under 5 years of age and the elderly.
Increasing antibiotic resistance and limitations in diagnostics emphasize the need to develop an
effective vaccine. Recent research and clinical trials report multiple approaches used in Shigella-vaccine
development. However, despite the efforts of researchers, pharmaceutical companies and health care
organizations, there is no licensed vaccine against shigellosis available to the community. Here, we
expressed, broadly characterized and demonstrated the protective properties of outer membrane
protein C as an effective molecule serving as a universal antigen for Shigella vaccine. Most of the
current approaches to the development of Shigella vaccine are based on the polysaccharide antigens,
which are serotype specific and have always been challenging in terms of their high specificity,
targeting the most exposed surface antigens identified for certain Shigella serotypes. Here, we confirm
immunogenic and protective properties of the recombinant OmpC protein, which protects mice against
a lethal dose of a virulent strain 2 weeks after active immunization.

Keywords Shigella, Vaccine, OmpC, Diarrhea, Infectious disease

Vaccinations save the lives of billions of people and protects public health against many microbial infections.
Even though, currently we have only few licensed vaccines protecting against several diseases and the need
to develop new, effective and highly protective vaccines remains unmet. This applies in particular to vaccines
protecting against diseases that kill people in low and middle-income countries (LMICs), and are therefore not
sufficiently profitable from an economic side.
Shigellosis (bacillary dysentery), a diarrheal disease caused by Gram-negative bacteria of the genus Shigella, is
a major health risk problem in poor, developing countries and natural disaster zones. Infection of the intestine’s
epithelial lining leads to acute inflammation of the digestive tract. Among the characteristic symptoms are
watery diarrhea with blood or mucous in the stool, nausea, vomiting, fever and abdominal pain1. According
to Global Burden of Diseases Study 1990–2016 it was estimated that shigellosis was responsible for 212,438
deaths and about 13.2% of all diarrhoeal deaths per year globally. Moreover, shigellosis was the second leading
cause of diarrhoeal mortality in 2016 among all ages and accounted for 63,713 deaths in children under 5 years
of age2. Increasing antibiotic resistance, even to the most advanced antibiotics and lack of reliable and quick
diagnostic tests, limit the shigellosis treatment options and makes calls for development of an effective vaccine
more urgent3,4. Unfortunately, no approved vaccine against shigellosis currently exist5. Anti-Shigella vaccine is
of particular use in LMICs countries, where sanitary hygiene is low, but it is also of high interest among tourists
and people traveling to developing countries: sailors, soldiers, medical personnel. For these reasons the World
Health Organization has given a high priority to the development of anti-Shigella vaccine6. Various approaches
have been explored over the past few decades, including whole-cell killed, live attenuated, and subunit vaccine
strategies. These vaccines, tested on humans, were characterized by poor immunogenicity, caused side effects or
were limited by specific recognition of the LPS O antigen characteristic of individual serotypes7,8. Approximately
50 serotypes belonging to 4 serogroups are associated with shigellosis in humans. These include S. dysenteriae
(15 serotypes), Shigella flexneri (around 18 serotypes), S. sonnei (one serotype), and S. boydii (20 serotypes)9.
Due to the wide range of Shigella serotypes and subtypes, there is a need for a broad range vaccine that is effective
against all of these species and serotypes. Our previous study showed that the 39-kDa outer membrane protein

1Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla Str. 12,
53-114 Wroclaw, Poland. 2Department of Animal Products Technology and Quality Management, Wroclaw
University of Environmental and Life Sciences, Chelmonskiego Str. 37/41, 51-630 Wroclaw, Poland. email:
anna.jarzab@hirszfeld.pl

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isolated from Shigella flexneri 3a plays a major protective role among other outer membrane proteins and is an
ideal vaccine candidate for the prevention of bacillary dysentery10.
Outer membrane protein C (OmpC) is located in the outer membrane of Shigella flexneri 3a and other Gram-
negative bacteria of the Enterobacteriaceae family10. This β-barrel protein with a molecular weight of 39.4 kDa
consists of 352 amino acids. The protein was isolated from a crude mixture of the other outer membrane proteins
and characterized11. In our previous studies, we have demonstrated that active immunization with the intact
protein protected mice against infection with live pathogen. In serological studies we observed that OmpC
reacted strongly with serum from immunized mice, normal human plasma and human umbilical cord serum.
Protection against S. flexneri 3a after OmpC vaccination performed in an animal model, and serological studies
indicate that cord plasma antibodies interacting with the protein may also have a protective effect in humans.
Interestingly, this protein is recognized by human umbilical cord serum IgG, which is transferred from mother
to fetus and may then play a key role in innate immunity. Such antibodies may serve as an early marker of
innate immunity, especially directed against enterobacteria and may protect newborns before their adaptive
immune response kicks in10,12. Furthermore, the OmpC epitope was observed to be surface exposed on intact
bacteria and could induce a protective immune response against the pathogen. Due to its embedding in the outer
membrane of the bacterial cell, OmpC is an excellent target for antibody recognition. This suggests that this
protein may become an ideal antigen for designing a vaccine protecting against shigellosis.
In our previous study11, we have extracted and purified the protein using size exclusion and ion exchange
chromatography. The methods used obtaining almost homogenous protein, but the procedure was time
consuming. Due to the embedding/anchoring in the lipid bilayer, membrane proteins pose a challenge for
purification and structural studies in a form of isolated molecules13,14. OMPs extracted from the lipid bilayer of the
outer membrane are often unstable, which often results in difficulties in their solubilization and purification11,15.
For this purpose, we turned to molecular biology techniques to obtain OmpC as a recombinant protein.
In this work, we cloned and overexpressed Shigella flexneri 3a OmpC in a prokaryotic expression system
using E. coli BL 21 as a host. Furthermore, we assessed the immunological and structural features of recombinant
OmpC and related its immunomodulatory properties to native, extracted and purified OmpC protein. Finally,
we confirmed that both proteins retain their protective immune properties against infection with pathogenic
bacteria and have great potential for use as an effective vaccine antigen.

Results
Immunological activity of recombinant protein
To investigate the immunomodulatory properties of rOmpC and compare its activity with the native protein,
we purified both proteins to homogeneity. We purified native OmpC (nOmpC) from Shigella flexneri 3a by
chromatographic methods using a Sephacryl S-200 molecular sieve and a DE-52 ion exchanger. Next, we
obtained a recombinant porin (rOmpC) of similar purity via one-step purification on Ni-NTA affinity
chromatography using a His-tag located at the N-terminus (Fig. 1A and Supplementary Fig. 4). To extract and
purify nOmpC from the outer membrane of S. flexneri, a detergent (Triton X-100) was used. On the other hand,

Fig. 1. OmpC immunoreactivity. (A) Coomassie stained SDS-PAGE profile of the purified nOmpC and
rOmpC proteins. (B) Immunoreactivity of the nOmpC and rOmpC with human umbilical cord serum
antibodies detected by Western-blot. Images from one out of 5 reproducible experiments are shown. Full
SDS-PAGE gels and Western-blot membranes were presented on Supplementary Fig. 4. (C) The nOmpC and
rOmpC immunoreactivity with 23 different human umbilical cord sera measured by ELISA. Data are presented
as the mean with range of two technical replicates for each of 23 individual human serum.

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recombinant rOmpC was expressed in the E. coli cytoplasm as inclusion bodies, which were then dissolved in
urea buffer, refolded, and purified without the addition of detergent. To check cell-derived conatminantions we
have detrmined the content of LPS and nucleic acids in a 1 µg of nOmpC and r OmpC samples (Supplementary
Fig. 6B). In LPS silver staining we have detected LPS in the native OmpC extracted from the S. flexneri 3a
membrane. Adversely we did not completely detect contaminating LPS in the recombinant form of OmpC,
which was purified from inclusion bodies expressed into E.coli cytoplasm (Supplementary Fig. 6A). We did not
reveal significant contamination (A260/A280 < 1 in both nOmpC and r OmpC samples) with the nucleic acids
(ds DNA, ssDNA, RNA) in the nOmpC and rOmpC samples, which were estimated on the level of 2–3% (nucleic
acid /protein ratio) (Supplementary Fig. 6C).
nOmpC and rOmpC immunoreactivity were assessed by Western blot. Both proteins showed immunoreactivity
towards human umbilical cord serum (Fig. 1B and Supplementary Fig. 4). Further ELISA assay using human
umbilical cord serum from 23 patients with both proteins (nOmpC and rOmpC) demonstrated similar
immunoreactivity with all the serum samples (Fig. 1C). To sum up, enzyme immunoassay studies have shown
that we have obtained fully antigenic rOmpC, capable of interacting with human antibodies, similarly to the
interaction with nOmpC. In addition, the rOmpC was recognized by the monoclonal antibody raised against
the nOmpC (Supplementary Fig. 5). The monoclonal antibody was broadly described in our previous studies26.

Molecular weight confirmation by MALDI-TOF-MS analysis

To test whether the obtained recombinant protein has the correct molecular weight the MALDI-TOF-MS
(Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry) analysis was performed.
Molecular masses of the intact native OmpC (39,314 Da) and recombinant OmpC (41,075 Da) have shown the
expected masses for both proteins (calculated from the amino acid sequences, Supplementary Fig. 1A,B). The
mass spectrum of rOmpC expressed in E. coli and isolated native S.flexneri OmpC shows homogeneous proteins
with single (41,075 Da; 39,314 Da), doubly (20,546 Da, 19,652 Da), triple (20,546 Da; 19,652 Da), quadruple
charged (10,277 ; 9826 Da), the peaks of rOmpC (red) and nOmpC (black) proteins, respectively, are shown in
Fig. 2.

Protein structure analysis using molecular modeling, DLS and CD

Next, we exploited the tertiary 3D model of the protein based on the amino acid sequence of nOmpC translated
directly from the ompC gene (Supplementary Fig. 1A). The structure analysis was performed using the
AlphaFold server (https://alphafold.ebi.ac.uk/), an integrated service for modeling of protein structure based on
structural homology16 and visualized by Chimera software. The antigenic epitope, which was already described
in our previous work as highly antigenic protein part, was highlighted on the exposed surface of the OmpC loop
(Fig. 3A)20,26.

Fig. 2. Molecular mass of the nOmpC and rOmpC proteins determination by MALDI-TOF-MS.

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Fig. 3. Structural analysis of the OmpC protein. (A) 3D structure of the OmpC obtained from the AlfaFold
server (https://alphafold.ebi.ac.uk/) and visualized in Chimera v. 1.16. (B) Molar ellipticity spectrum of the
rOmpC protein measured by circular dichroism. (C) Experimental content of secondary structure of the
rOmpC deconvoluted from circular dichroism measurement and calculated by Capito (CD Analysis and
Plotting Tool). (D) Theoretical secondary structure content predicted for the rOmpC based on the amino acid
sequence by the Capito server.

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The particle distribution measured by dynamic light scattering (DLS) shows that the native protein (nOmpC)
consist mostly (92,6%) of a trimer form with an averaged dimension of d = 99,23 nm (Supplementary Fig. 2),
which is the most common form of the OmpC in the majority of Enterobacteriaceae family17. Furthermore, we
detected a small amount (7.4%) of monomeric protein d = 31.65 nm. The recombinant protein that was purified
and stored without detergent, in addition to the monomeric (d = 38.44 nm, 11.2%) and trimeric (d = 130.5 nm,
67.1%), showed larger aggregates (21.6%) of 427.3 nm. Based on our observations and other studies, we explained
the aggregation of rOmpC into larger particles because the expressed protein was purified on a nickel column
without contact with detergents, which facilitates aggregates solubilization18.
The three-dimensional OmpC model was used to calculate the α- and β-fold contributions to the secondary
structure of this protein and to relate the theoretical structure content to experimental data acquired by circular
dichroism (CD) spectra. CD spectra for rOmpC were measured in the UV region 187–300 nm. The utility of
nOmpC was limited for the experiment due to contamination with trace amounts of Triton X-100. There are two
absorption bands in the CD spectrum responsible for the far-UV CD spectrum. The first, stronger π → π′ band at
approximately 190 nm and the weaker n → π′ transition between 210 and 220 nm. Molar ellipticity (Supplementary
Fig. 3) shows both transitions for the nOmpC and rOmpC proteins. Secondary structure analysis was performed
on a curve fitting procedure based on a set of reference spectra with known secondary structure components
and was used to estimate the nOmpC and rOmpC spectral components by regression analysis using the Capito
server19. Deconvoluted absorption bands of both proteins show that their structure content is slightly different
and accounts for 4% of α-helix structure for both proteins (Supplementary Fig. 3B, Supplementary Table 1). The
differences measured for both proteins concern the content of β-sheets (46% for nOmpC and 35% for rOmpC)
and the content of irregular structure (50% for nOmpC and 61% for rOmpC). Deconvolution analysis shows that
the recombinant protein consists of a more irregular structure than native OmpC, as measured by CD. This may
be influenced by changes caused by protein folding after rOmpC expression outside the natural environment of
the bacterial outer membrane. Comparing the content of the irregular CD structure measured by nOmpC (50%)
and the theoretical one (34%), it can be expected that the extraction of proteins from their natural environment
may change the secondary structure content (Fig. 3).

Protective activity of the recombinant protein

Nevertheless, to check if those slight structural changes between native and recombinant protein may influence
its immunogenicity we performed animal studies. A main objective of this work was to obtain recombinant
OmpC that would exhibit protective properties similarly to nOmpC against the lethal dose of Shigella flexneri
3a. Mice were immunized with 3 doses of the nOmpC or rOmpC on days 1, 3, 5 and challenged with the virulent
strain after 2 weeks post the last dose (Fig. 4A). Animals receiving 3 doses of 5 µg/mouse of the rOmpC, at 2-day
intervals remained fully protected against the LD100 = 2 × 108 bacteria/mouse of S. flexneri 3a. Similarly, mice
immunized with nOmpC according to the same immunization schedule and dose were protected at 80% when
challenged with LD100 of S. flexneri 3a (Fig. 4B). This shows that both nOmpC and rOmpC exhibit protective
properties against the virulent strain of S. flexneri 3a.
Compared to our previous work20, a significant improvement in survival rate was observed after immunization
with 3 doses of the OmpC and without an adjuvant. Additionally, we observed an increase in the level of IgG
and IgM antibodies in the blood of mice against the nOmpC. The IgM and IgG level increased significantly
(p < 0.001) in mice serum, immunized with both the native and recombinant proteins (Fig. 4C).

Discussion
Although several candidate vaccines against Shigella have been evaluated in pre- and clinical trials in recent
years, no licensed vaccine against the disease is currently available. The most common approaches to Shigella
vaccine development involve attenuated or inactivated whole bacterial cells. Alternatively, subunit vaccines based
on Shigella surface antigens have been proposed. The main problem with those aforementioned approaches
was either high reactogenicity of live attenuated bacterial cells or the insufficient immunogenicity of subunit
vaccines, mainly based on the serum-specific glycoconjugates3,5.
According to the latest clinical trials, the most advanced vaccine is the recombinant conjugate vaccine. It
is a monovalent synthetic vaccine SF2a-TT15 (NCT04602975) with S. flexneri 2a, currently being tested on a
group of volunteers and showing immunogenic properties21. Another advanced prototype is the multivalent
altSonflex1-2-3 vaccine (NCT05073003), whose monovalent precursor from S. sonnei was safe and immunogenic
in tests on volunteers, but turned out to be ineffective against infection22. One another vaccine tested already in
humans is the multivalent S4V conjugate (NCT04056117). It has been shown to be safe and immunogenic and
the research on its effectiveness is currently ongoing23. All three vaccines are currently being tested in pediatric
populations in Kenya. The main limitation of this approach to vaccine design is their high specificity directed to
certain serotypes and they do not protect against all Shigella strains.
Apart from the above listed trials, there is number of research groups focused on attenuated vaccines, some of
which are in the first phase of clinical trials. The weakened strain of Shigella flexneri 2a SC602 causes an increase
in the level of IgG antibodies directed against bacterial lipopolysaccharides in the serum24. However, in the case
of this type of vaccine, the risk of mutation and reversion of the attenuated strain to its virulent form is always
high. Another drawback of this form of vaccine is its difficult administration, quality control of the formulation,
its production and storage.
The development of subunit vaccines is an important branch of anti-Shigella vaccines, of which the most
advanced is a formulation called Invaplex. This vaccine is based on protein antigens (IpaB and IpaC proteins)
and lipopolysaccharides isolated from the Shigella flexneri 2a strain. Studies in the preclinical phase have shown
that the administration of this vaccine resulted in an increase of IgG and IgA antibodies levels in volunteers’
serum. Additionally, apart from short-term irritation of the nasal mucosa, no other side effects were reported

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Fig. 4. Protective efficacy of OmpC on animal model. (A) Immunization and challenge schedule. Mice
were immunized with 3 doses of vaccine (5 ug OmpC each) in 2-days intervals. After 2 weeks the mice were
challenged with S. flexneri (LD100 = 2 × 108 S. flexneri cells/mouse) and the mortality was recorded for the next
72 h. (B) Mice survival after immunization with the native (nOmpC) and recombinant (rOmpC) protein and
the challenge of LD100 = 2 × 108 bacteria/mouse. Survival distribution was performed by log-rank test/Mantel-
Cox test, p < 0.01. (C) IgM and IgG levels in mouse serum after immunization with nOmpC and rOmpC
measured by ELISA against the nOmpC protein. 2way ANOVA, followed by Bonferroni posttest, p < 0.001.

in vaccinated volunteers. Therefore, the preliminary results regarding the effectiveness of Invaplex 50 are
encouraging. However, the cost of producing the vaccine seems high, primarily due to the complex process of
obtaining individual ingredients and standardization of its composition. In addition, a serious problem is the
risk of contamination with endotoxin (lipopolysaccharide, LPS), which can cause poisoning, especially in the
event of an overdose. Probably due to high production costs, this group is currently working on a synthetic
equivalent containing antigens originally isolated from bacterial strains25.
In our previous studies, we have observed that native OmpC isolated from Shigella flexneri 3a induced a
protective immune response against a lethal dose of this pathogen20. Similarly, as with the Invaplex 50 vaccine,
the purification process of the native protein was time-consuming and tedious. Furthermore, the final product,
although it meets protein purity standards forms complexes with low-molecular-weight substances, i.e. Triton
X-100. Additionally, the presence of LPS was observed in the crystal structure of E.coli OmpC, which indicates
its incorporation also in the structure of the OmpC protein from S. flexneri17. Since the use of native OmpC has
some caveats, including LPS contamination our goal was to develop a synthetic vaccine. Therefore, we identified
an antigenic epitope presented on the external surface of bacteria, synthesized this epitope, and conjugated it
to carriers20, in the hope that such conjugates could induce similar protection to the native OmpC protein26.
These studies demonstrated that the OmpC epitope based on the RYDERY amino acid sequence is the minimal
sequence that interacts with protective antibodies, and all amino acids located in the sequence are essential.
Unfortunately, after immunization of mice with peptide-based conjugates, we observed only a weak immune
response and low production of antibodies directed against immunogenic epitopes26. Our research has shown
that the construction of peptide vaccines, despite enormous advantages in terms of their preparation, production
costs, high safety and lack of social controversy, is an extremely difficult process due to their poor immunogenicity.
Therefore, we decided to go back to the entire OmpC, but in a recombinant version. Even the expression and
purification of recombinant membrane proteins have always been challenging we have obtain an immunogenic
form of the rOmpC. The recombinant protein had the correct molecular weight (Fig. 2), was soluble, but tended
to form larger aggregates than the native counterpart (Supplementary Fig. 2). This phenomenon is characteristic
of membrane proteins whose hydrophobic core is exposed to the aqueous environment. Even the OmpC protein
showed aggregation and dissociation tendencies, and most importantly, it still presented an antigenic epitope
loop, which made it highly reactive with antibodies in enzyme-linked immunoassays. Aggregation of rOmpC

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into trimers and larger aggregates most likely leads to improved protein stability and resistance to lysis, allowing
for longer protein presentation to the immune system and improved immunogenicity. Nevertheless, the most
important observation was that this protein showed enormous protective properties against a lethal dose of
a virulent strain. Mice injected three times with the pure, recombinant protein survived challenge with the
live pathogen, demonstrating that the protein itself induces a protective immune response without the use of
an adjuvant. To avoid cross-reactions with mammalian His-rich proteins, the His-tag, which is still present at
the C-terminus of rOmpC, must be removed before preclinical testing. However, there is a major advantage of
the recombinant rOmpC protein, which can be obtained in a pure form and without LPS contamination. This
highlights its utility and allows to omit the LPS purification process before vaccine preparation. Our results
open the way to a genetically modified vaccine against shigellosis based on the recombinant OmpC. In addition,
we have achieved complete protection against S. flexneri after 2 weeks from the last immunization. It has been
already shown that multiple-dose immunization in short time period leads to strong immunity against the
pathogen even within 1–2 weeks post immunization. This was shown already for the Vivotif vaccine protecting
against typhoid and paratyphoid fever27. Similar effect was observed in terms of BNT162b2 mRNA COVID-19
vaccine, which increases IgGs after 11 days post first immunization28. Those vaccines may be given even a week
before exposure to the live pathogen.
High protection after immunization with pure OmpC antigen, which was shown in our studies is promising
and might highlight the OmpC as an vaccine antigen inducing protective immunity shortly after vaccination.
Our results show significant elevation of the IgM and IgG antibodies in mice serum 14 days after immunization.
This might be crucial in terms of constructing a vaccine for people living in shigellosis regions but also for people
visiting high risk zones, who can be vaccinated shortly before travel.

Materials and methods

Bacterial strain, culture condition
Shigella flexneri serotype 3a strain (PCM 1793) used in the study was obtained from the Polish Collection of
Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences
(Wroclaw, Poland). Bacteria were grown on enriched Bacto Agar plates (Difco) and cultured for 8 h with gentle
shaking in liquid Brain-Heart Infusion (BHI) medium (Difco).

Animals
The study was describe in accordance with ARRIVE guidelines. All experiments were performed on six to
seven-week-old female BALB/c mice (20–25 g) obtained from the Mossakowski Institute of Experimental and
Clinical Medicine of the Polish Academy of Sciences in Warsaw, Poland and randomly divided into 3 groups.
To assess statistical significance and limit the number of animals used in the study each of the group consisted
of n = 5 mice. All animals were placed in cages enriched with toys and nesting material, with constant access
to water and standard feed. The temperature in the animal rooms was: 22 ± 2 °C, humidity 55%±10%, lighting
12/12 h. Before the experiments, the animals were kept in quarantine for a week. Animal care staff responsible
for mice immunization were unaware of allocation group to ensure that all animals in the experiment were
handled monitored and treated in the same way. After immunization, 0.1 ml of blood was collected from 3
randomly selected animals from the tail vein to determine the level of antibodies. The ELISA test was run on the
100 µl of 100-times diluted mice serum. The primary endpoint of this study was defined as a number of animals
survived after LD100 of S. flexneri 3a 3 days after challenge. Survival distribution and statistical assessment was
performed by log-rank test/Mantel-Cox test, p < 0.01. The experiments were designed in accordance with the
guidelines of the National Ethics Committee and approved by the First Local Ethics Committee at the Institute
of Immunology and Experimental Therapy of the Polish Academy of Sciences (LKE 020/2021/P1, 21.04.2021).

Sera
Human umbilical cord sera from healthy women were obtained from the Obstetric Clinic of the Medical
University of Wroclaw. Written informed consent was signed by all umbilical cord blood donors. The study
was approved by the Medical Ethics Committee of the Medical University of Wroclaw (KB-882-2012) and all
experiments were performed in accordance with its guidelines and regulations.

ELISA assays
The immunoreactivity of both native and recombinant OmpC with human umbilical cord serum IgG was
measured by ELISA. To perform the assay, the surface in 96-well plates (Nunclon TM) was coated with 1 µg/well
of OmpC in 100 µl of 0.2 M carbonate buffer, pH 9.6. Unadsorbed antigen was removed by washing the wells
three times with 200 µl of TBS-T buffer. The plate was blocked with 1% BSA (Kierkegaard & Perry Laboratories)
in TBS-T buffer, pH 7.5 at room temperature for 1 h. After blocking, all wells were washed three times with
200 µl TBS-T. The wells were then filled with 100 µl of human umbilical cord serum diluted 1:500 in PBS and
the plates were incubated at room temperature for 1 h. After washing three times with TBS-T, 100 µl of an
alkaline phosphatase-labeled goat anti-human IgG diluted 1:10,000 in PBS was added to each well. Following 1 h
incubation at room temperature, the plates were washed with 200 µl of TBS-T and then incubated with 200 µl of
APYellow (pNPP) substrate (Sigma). After 30 min, the reaction was stopped by adding 50 µl of 3 M NaOH and
the optical densities were read at 405 nm using an automatic microplate reader (Biotek).

Purification of the native OmpC protein fromS. flexneri 3a

The crude outer membrane proteins (OMP) fraction was extracted from dry bacterial mass with valeric
acid according to Arcidiacono et al.29. Protein concentration was determined by the Lowry method, using
BSA as a standard30. OmpC was purified from crude membrane fraction by gel filtration and ion-exchange

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chromatography as previously described20. Briefly, the crude OMP fraction was dissolved in extraction buffer
(0.4% Triton X-100, 50 mM NaCl, 50 mM Tris-HCl, pH 8) and centrifuged at 14,000 × g for 30 min. The
supernatant containing solubilized OMP was loaded onto Sephacryl S-200 h (Pharmacia) column (1.6 cm ×
100 cm) equilibrated with the extraction buffer. Fractions containing OmpC were pooled, dialyzed against water
and concentrated by ultrafiltration (10 kDa cut-off membrane, Millipore). The concentrated sample was applied
to a DE-52 h (Whatman) column (1.6 cm × 10 cm) equilibrated with 50 mM Tris-HCl, pH 8 containing 10 mM
EDTA. The OmpC was eluted with a linear gradient of 0–0.5 M NaCl. The fractions containing OmpC were
collected, dialyzed against 0.1 M ammonium acetate, pH 9.00 concentrated and stored at − 20 °C.

Expression of the recombinant OmpC in E. coli BL 21

Oligonucleotide primers were designed based on the appropriate ompC gene sequence of S. flexneri 2a and E.
coli. Since the DNA sequence of the ompC genes from Shigella and Escherichia genus are highly homologues,
primers for S. flexneri 3a ompC gene amplification (ompC for: 5′-​G​C​T​G​A​A​G​T​G​T​A​C​A​A​C​A​A​A​G​A​C​G​G-3′;
ompC rev: 5′-​G​A​A​C​T​G​G​T​A​A​A​C​C​A​G​G​C​C​C-3′) were designed using ompC gene sequences of S. flexneri 2a
(NC_004741.1) and E. coli (NC_000913), both available at Gene Bank. The isolation of S.flexneri genomic DNA
was performed as described previously20. The reaction mixture contained: 1 µl of genomic DNA; 2 µl of 10µM
starters (each); 0.5 µl of 10 mM dNTP, 1.5 µl of 25mM MgCl2; 2.5 µl of 10× polymerase buffer, 1 µl of DNA
polymerase (10U). A final volume of 25 µl was fixed with sterile water. The cycle conditions were: 95°C for 3 min;
(94°C for 1 min; 54°C for 30 s; 72°C for 1.5 min) × 35 cycles followed by 72°C for 10 min. The gene encoding
the ompC was amplified by PCR, cloned in pQE 80 L vector (Qiagen, Germany), sequenced and expressed in
E. coli Novablue by isopropyl thiogalactoside (IPTG) induction in the presence of ampicillin to select positively
transformed clones. We have registered the ompC of Shigella flexneri 3a gene sequence in Gene Bank under
Accession No. KC865276. The protein was purified on the Ni-NTA affinity chromatography. Briefly, the protein
was dissolved in 8 M Urea buffer and applied to the Ni-NTA column. In the next step the His-tag bounded
protein was eluted with 150 mM imidazole and the final protein fraction (c = 25 µg/ml) was dialyzed against
0.1 M ammonium acetate, pH 9.00 and stored at -20 ºC.

Immunoreactivity of the nOmpC and rOmpC

The purified nOmpC and rOmpC fractions were characterized by sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS-PAGE) using 12,5% resolving gels31. The gels were stained with Coomassie Brilliant
Blue and visualized by Gel Doc imager (Vilber Lourmat). To perform immunoblotting, nOmpC and rOmpC
protein bands were electro-transferred at room temperature from SDS-PAGE gels to Immobilon P membrane
(Millipore) using transblot system (Bio-Rad). The gels were immersed in a buffer composed of 10 mM Tris, 150
mM glycine, 20% methanol and transfer was performed at 100 V for 1 h.
To perform the Western blot with human umbilical cord serum antibodies the membranes with transferred
OmpC were blocked with 1% BSA and subsequently incubated for 1 h at room temperature with human umbilical
cord serum diluted 1:1000 in 1% BSA in TBS buffer (TBS-T, 20 mM Tris–HCl, pH 7.00, 50 mM NaCl, 0.05%
Tween-20). To visualize OmpC-IgG complex membrane was first incubated with a 1:10,000 dilution in TBS-T
of alkaline phosphatase-labeled goat anti-human IgG (Sigma), followed by three wash steps in TBS, 10–15 min
each. Western-blots were developed using NBT/BCIP in 100 mM Tris–HCl, pH 9.5, with 100 mM NaCl and 50
mM MgCl2. All steps were performed at room temperature.
To perform the Western blot with mouse anti-OmpC monoclonal antibody the membranes with transferred
OmpC were blocked with 1% BSA and subsequently incubated for 1 h at room temperature with anti-OmpC
monoclonal antibody diluted 1:1000 in 1% BSA in TBS buffer (TBS-T, 20 mM Tris–HCl, pH 7.00, 50 mM NaCl,
0.05% Tween-20). To visualize OmpC-IgG complex membrane was first incubated with a 1:10,000 dilution in
TBS-T of alkaline phosphatase-labeled goat anti-mouse IgG (Promega), followed by three wash steps in TBS,
10–15 min each. Western-blots were developed using NBT/BCIP in 100 mM Tris–HCl, pH 9.5, with 100 mM
NaCl and 50 mM MgCl2. All steps were performed at room temperature.

Molecular mass of intact proteins by MALDI-TOF-MS

The MALDI-TOF-MS matrix solution was prepared just before the experiment. The matrix (HCCA, alpha-
cyano-4-hydroxycinnamic acid) was dissolved in an aqueous FA/H2O/isopropanol (3:1:2, v/v/v) to obtain a
supersaturated solution. The resulting solution was sonicated for 10 min and spun down by centrifugation.
Sample preparation was performed by mixing 1 µl of the sample solution with a protein content between 0.5
and 1.0 mg/ml and 9 µl of the matrix solution. For analysis, 1 µl of this solution was placed on customary
stainless steel MALDI target plates and dried. Molecular mass calibration was performed using Protein Standard
II from Brüker (Germany), containing mixture of standard proteins with mass range between 22.3 and 66.5 kDa.
The MALDI-TOF-MS experiment was performed on an ultrafleXtreme mass spectrometer (Brüker, Germany)
and recorded using Flex Control v. 3.2 software. The instrument was operated in the positive ion linear mode
applying an accelerating voltage of 25 kV. All mass spectra were acquired by averaging 100 laser shots. Data
evaluation and spectra smoothing were performed using the Flex Analysis v. 3.2 software.

Protein structure analysis by bioinformatics

The tertiary structure of the rOmpC was determined from the amino acid sequence using AlfaFold server
(https://alphafold.ebi.ac.uk/). AlpfaFold is an useful fully automated tool for homology modeling of proteins16.
The 3D structure of OmpC was visualized using Chimera v. 1.16 software32.

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Dynamic light scattering

OmpC, as a membrane protein, tends to form aggregates. The crystal structure of the OmpC revealed the
existence of a trimeric form17. Dynamic light scattering (DLS) was performed to determine the size and the
degree of aggregation of both native (nOmpC) and recombinant OmpC (rOmpC). The Zeta-sizer nano series
(Malvern Zetasizer Nano ZS) was used to characterize the particle size distribution. The measurement was run
with an acquisition time of 10 s and a scattering angle of 173º. Both proteins were dissolved in 0.1 M ammonium
acetate and the samples were shaken well before measurements. After preparation, each sample was measured at
23 ºC. Data analysis was performed in Zetasizer Software v. 6.2.

Secondary structure analysis by circular dichroism

Conventional CD measurements were made on the Jasko-715 instrument. Baseline was measured using 0.1 M
ammonium acetate in which the proteins were dissolved. The sample was dissolved in 0.1 M ammonium acetate
at the concentration of 25 µg/ml. Data were collected at 0.2-nm intervals. Quartz cuvettes (path length 1 mm)
were used as a cells for all the measurements. At least ten repeated scans were obtained for each sample and
baseline. The averaged baseline spectrum was subtracted from the averaged sample spectrum and the spectrum
was smoothed using the Savitsky-Golay, Adaptive-Smoothing, or Binominal filters that are available in the
Spectra Analysis Manager software for Jasco-715. Measurements were only performed down to wavelengths
where the HT (V) values were still in its linear range. For nOmpC, this was 197 nm and for rOmpC it was
195 nm.
Protein secondary structure analysis tools CDNN33, Capito19 and CDPro34 were used to analyze CD spectra.
The CDPro software package consists of three popular secondary structure fraction determination methods:
SELCON335, CONTIN/LL34 and CDSSTR36. To analyze the structure of OmpC, the SMP56 reference set was
used, which is based on 43 soluble proteins and 13 membrane proteins with known structures. CDPro tools
analyze the content of α-helix (regular and disordered) and β (regular and disordered) strands, turns and
disordered structures. Together we analyzed the content of regular and disordered α and β structures. CDNN
analyzes the data to determine helix, anti- and parallel β-structure, turns and irregular structure. For this
analysis, the anti and parallel β structure was summarized. The results were compared and presented using the
Capito tool available online. This method enables the analysis of α, β and irregular structures.

Protection studies on animal model of BALB/c mice

The immunological protective properties of recombinant OmpC, i.e. expressed in E. coli BL 21, were compared
with that of native OmpC isolated from the outer membrane of S. flexneri 3a. Each experiment group was
consisted of 5 mice. Animals (BALB/c mice, female, 6–8 week old) were immunized with 3 doses (5 µg/dose) of
nOmpC or rOmpC. The antigen was dissolved in PBS and injected intraperitoneally in a total volume of 200 µl.
To determine the level of IgM and IgG levels in animal serum, blood was collected from the animal’s tail vein
14 days after immunization. The level of antibody titer in 100-diluted mice serum was determined by ELISA.
The control group received only PBS and served as control. After 10 days of immunization, the animals were
challenged intraperitoneally with the lethal dose (LD100 = 2 × 108 bacteria/animal) of the virulent S. flexneri 3a
strain. Animals were observed for 72 h after bacterial challenge and their mortality was recorded. In order
to minimize pain, suffering, or stress of the animals participating in the experiment, cervical dislocation was
chosen as the method of euthanasia. Euthanasia by cervical dislocation is a quick and non-comfortable method
of killing mice, and therefore no analgesic or sedative drugs were used before the procedure.

Detection of contaminants
To detect the % of contaminating nucleic acids in the nOmpC and rOmpC samples we have measured the
concentration of dsDNA, ssDNA and RNA in a protein sample adjusted to 1 mg/ml by spectrophotometric
measurement. 1.5 µl of protein sample was measured on the Nano Drop Lite Plus (Thermo Scientific). The 1 mg/
ml commercial BSA (Fluka, 05488) sample was measured as a control.
To detect contamination with LPS (lipopolysaccharide) we have run 1 µg of nOmpC and rOmpC sample
into 15% SDS-PAGE and stained the gel with LPS detection silver staining. The gel was fixed overnight with 40%
methanol and 10% acetic acid. LPS was oxidized in the gel with 0.75% periodic acid at room temperature for
5 min. The gel was then washed four times with distilled water for 10 min. The gel was stained for 10 min with
freshly prepared staining solution which was prepared as follows. A 1-ml volume of 25% ammonium hydroxide
was added to 14 ml of 0.1 M sodium hydroxide. Next, 2.5 ml of 20% (wt/vol) silver nitrate was added in drops
with stirring. After staining, the gel was then washed four times with distilled water for 10 min. The color was
developed by reduction in 250 ml of water containing 12.5 mg of citric acid and 0.125 ml of 37% formaldehyde.
The gel was photographed immediately after washing with distilled water.

Statistical analyses
Statistical analyses of the data were performed using GraphPad Prism v. 5.01. The Mantel-Cox log-rank test was
performed to analyze mice survival curves. A P-value of < 0.01 was considered significant. Two-way ANOVA
with multiple comparison tests with Bonferroni correction was performed for comparison between more than
two groups, and a P-value < 0.001 was considered significant. Data presented as mean ± SD. Error bars represent
SD.

Data availability
Supplementary Information is available in the online version of this manuscript. Files containing data sources for
the main text and supplementary figures can be downloaded from https://figshare.com/account/items/25894939/
edit under DOI number: https://doi.org/10.6084/m9.figshare.25894939.

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www.nature.com/scientificreports/

Received: 6 June 2024; Accepted: 16 October 2024

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Acknowledgements
The authors gratefully acknowledge Bernadeta Szostko for laboratory assistance. Anna Jarzab acknowledges
Leader XIII Program (Grant No. 0093/L-13/2022) and the National Centre for Science and Development for
support.

Author contributions
AJ: conducted experiments: protein expression and purification, serological tests, MALDI-TOF MS analysis, CD
analysis, supervised bioinformatics analysis, analyzed data, wrote the manuscript; AD and KP: supervised pro-
tein expression, PN: conducted and designed animal studies, KK: performed bioinformatics analysis, analysis of
contaminants (LPS, nucleic acids) in protein sample, Western blot analysis, AS and MŚ: conducted experiments
on protein expression and purification; DW: supervised the study, EZ: supervised the study and revised the
manuscript, AG: supervised the study, provided resources.

Declarations

Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1038/s41598-024-76745-8.
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