A Pentavalent Shigella flexneri LPS-Based Vaccine Candidate Is Safe and Immunogenic in Animal Models

A multivalent vaccine is much needed to achieve protection against predominant Shigella serotypes. Recently, we demonstrated the clinical applicability and immunogenic potential of tri-acylated S. flexneri 2a lipopolysaccharide (Ac3-S-LPS). Using a similar approach, we designed a pentavalent LPS candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). In this study, we performed molecular and antigenic characterization of the vaccine candidate and its preclinical evaluation. There were no signs of acute toxicity after subcutaneous administration of PLVF in rabbits at a proposed human dose of 125 μg. No pyrogenic reactions and adverse effects associated with chronic toxicity after repeated administration of PLVF were revealed either. The immunization of mice with PLVF led to ≥16-fold increase in S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies. In a serum bactericidal antibody (SBA) assay, we registered 54%, 66%, 35%, 60%, and 60% killing of S. flexneri 1b, 2a, 3a, 6, and Y, respectively. In the guinea pig keratoconjunctivitis model, the efficacy was 50% to 75% against challenge with all five S. flexneri serotypes. These studies demonstrate that PLVF is safe, immunogenic over a wide range of doses, and provides protection against challenge with homologous S. flexneri strains, thus confirming the validity of pentavalent design of the combined vaccine.


Introduction
Dysentery is an acute infectious colitis caused by an enteric bacterial pathogen known as Shigella. Dysentery is ubiquitous. About 188 million global cases of shigellosis are reported annually [1]. The main risk groups for shigellosis are composed of children under 5 years of age, underprivileged elderly people, and travelers in endemic areas [2,3]. Shigellosis caused by Shigella flexneri is predominantly found in low-and middleincome countries [1]. Despite the high epidemiological significance of Flexner's dysentery and progressive antibiotic resistance, a licensed vaccine against S. flexneri has not yet been developed.
Shigella vaccine development is being pursued in several directions: a live attenuated vaccine [4], a whole-cell inactivated vaccine [5], and a conjugated vaccine, including chemically conjugated or the most modern bioconjugate preparations (O-polysaccharide (O-PS) conjugated to the carrier protein, exotoxin A of P. aeruginosa, using gene engineering techniques) [6].
Live and inactivated whole-cell vaccines against S. flexneri have been immunogenic in clinical and preclinical studies [7][8][9]. However, a large number of observed adverse events and short-lived immune responses in clinical study subjects limits their regulatory approval and use in the clinic [10]. To reduce the adverse events of whole-cell vaccines, the

Bacterial Strains and Growth Conditions
The biomass of strains of S. flexneri 1b 1818, 2a 1605, 3a 2167, 6 281-55, and Y 2643, which represented the smooth (S) form of bacteria each serotyped with a homologous Shigella serum, was obtained through fermentation using Hottinger's broth (Nutrient Media, State Research Center for Applied Biotechnology and Microbiology, Obolensk Russia) in a 250 L fermenter (BioR 250, Prointech-bio, Pushchino, Russia) with stirring and forced aeration. Bacterial cells were separated from the liquid phase by flow centrifugation (Z-41, CEPA, Lahr, Germany). Wet cells were subsequently washed with sterile saline and water, and then lyophilized.
For serum bactericidal antibody (SBA) assay, S. flexneri 1b, 2a, 3a, 6, and Y were streaked on tryptone soy agar plates and incubated overnight at 37 • C. Then, bacterial cells were washed off plate surfaces using sterile 0.9% NaCl and further diluted in phosphatebuffered saline.

Isolation and Degradation of S-LPS
Freeze-dried bacterial cells were extracted with 45% aqueous phenol (Sigma-Aldrich, St. Louis, MO, USA) at 68-70 • C. The aqueous phase was separated, dialyzed, and lyophilized to give a crude LPS preparation. The preparation was dissolved in TRIS buffer containing 0.01% (w/w) CaCl 2  for 2 h at 55 • C and then dialyzed using ultrafiltration with a 50 kDa cut-off membrane (Vladisart, Vladimir, Russia), concentrated, and lyophilized to give a purified LPS preparation. S-LPS was obtained using gel-permeation chromatography on Sephadex G-150 (Sigma-Aldrich, St. Louis, MO, USA) in the presence of Na-deoxycholate (Sigma-Aldrich, St. Louis, MO, USA) as detergent. Fractions that contained S-LPS were combined and freeze-dried.
After the removal of lipid A (2% AcOH, 100 • C, 1 h), the carbohydrate portions of nLPS and S-LPS were profiled by an Agilent 1260 HPLC system with UV-and RI-detection on a TSK gel G3000PW (Toyopearl, Mainz, Germany) (7.8 mm I.D. 30 cm) column using 0.2 M NaCl elution solution and a flow rate of 0.5 mL min −1 . The content of low molecular mass compounds in S-LPS did not exceed 5-10%.
Partial de-acetylation of S-LPS was performed when S-LPS (300 mg) solution was heated with stirring in 8.3% aqueous ammonia and water (100 mL) containing 100 mg of Na-deoxycholate at 30 • C for 8 h, then cooled to 5-10 • C, diluted with 200 mL of water, neutralized with AcOH, and freeze-dried. The product was treated with 100% ethanol (200 mL), the precipitate was separated by centrifugation, washed with 100% ethanol (2 × 200 mL), vacuum-dried, dissolved in water, and freeze-dried to give Ac 3 -S-LPS (213 mg) [17].

Production of PLVF
The candidate vaccine product was manufactured based on Ac 3 -S-LPS compounds from S. flexneri 1b, 2a, 3a, 6, and Y (PLVF) as the active substances at a dose of 125 µg (25 µg of each antigen compound) and contained the following formulation excipients: phenol (preservative) 0.75 mg, NaCl 4.15 mg, Na 2 HPO 4 0.052 mg, and NaH 2 PO 4 0.017 mg (all Sigma-Aldrich, St. Louis, MO, USA), and 0.5 mL sterile pyrogen-free water for injection [16]. The final form of PLVF product was formulated and dispensed aseptically in ampoules at a GMP-compliant manufacturing suite of vaccine enterprise of the Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Sciences.

Chemical and Physical Analyses
SDS-PAGE was performed on a 12% acrylamide gel according to the Laemmli method using a Bio-Rad Mini-Protean electrophoresis system. Gels were stained with silver nitrate reagent (Sigma-Aldrich, St. Louis, MO, USA).
Electrospray ionization high-resolution mass spectra were recorded in the negative ion mode using a micrOTOF II instrument (Bruker Daltonics, Billerica, MA, USA). A sample (~50 ng µL −1 ) was dissolved in a 1:1 (v/v) water-acetonitrile mixture and sprayed at a flow rate of 3 µLmin −1 . End plate offset voltage was set to 0.5 kV and capillary voltage to 4.5 kV. Drying gas temperature was 180 • C. Mass range was from m/z 50 to 3000 Da. 1 H-and 13 C-NMR spectroscopy were performed for solutions in 99.95% D 2 O at 323 K on a Bruker DRX-500 spectrometer (Bruker Daltonics, Billerica, MA, USA) using sodium3-trimethylsilylpropanoate-2,2,3,3-d 4 (δ H 0) and acetone (δ C 31.45) as references for calibration. Prior to analysis, samples were freeze-dried from 99.5% D 2 O. The Bruker Topspin 2.1 program was employed to acquire and process the NMR data.

Animal Studies
The study on animal models was carried out in accordance with the ethical principles approved by the order of the Ministry of Health of the Russian Federation No. 199n from 4 January 2016. The study protocols were approved by the local ethics committees of the research organizations. Animals were housed in accredited animal facilities with free access to food and water. Before the start of a study, animals were placed in a separate room for a period of quarantine (14-21 days, depending on the animal species) and health-monitored.

Pyrogenicity
The standard method for determining the pyrogenicity is the rabbit pyrogen test (RPT). RPT was conducted on 21 adult Chinchilla rabbits (aged 3 months at the start of the experiment and weighing 2.8-3.05 g). Rabbits were randomized by weight into 7 groups. Three rabbits per group were intravenously injected with PLVF, each Ac 3 -S-LPS vaccine component or unmodified nLPS of S. flexneri 2a (as pyrogenicity control) at a dose of 0.025 µg kg −1 .
A substance was considered apyrogenic if the cumulative temperature rise of three rabbits did not exceed 1.15 • C in accordance with the European Pharmacopoeia requirements [18]. The PLVF doze used for RPT was chosen by analogy to the WHO-approved Vi-vaccine pyrogenicity test dose of 0.025 µg kg −1 .

Toxicology Study
A standard PLVF toxicity study was performed in rabbits. Chinchilla male and female rabbits were purchased from the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency", Russia. Acute toxicity studies were performed with a single subcutaneous injection of 125 µg/0.5 mL of PLVF (5 male and 5 female rabbits per group, aged 3 months at the start of experiment and weighing 2708 ± 71 g), followed by observation for 14 days. Animals in the control group were injected with 0.5 mL of PBS. Chronic toxicity studies were performed with daily subcutaneous immunization of rabbits with 125 µg/0.5 mL PLVF for a week (5 males and 5 females per group), followed by observation for 7 days. Animals in the control group were injected with 0.5 mL of PBS. Blood samples for hematology and biochemistry were collected before vaccination, and 7 and 14 days after vaccination. On the 14th day after vaccination, necropsy, histological examination of 16 internal organs and tissues of each rabbit, and the blood differential test were performed. The local irritation was studied by histopathological evaluation of the site of repeated subcutaneous injection of the vaccine preparation (macro-and microscopic description of the skin at the injection site).
Tissue samples for histological examination were fixed in 10% neutral buffered formalin, dehydrated in ascending concentrations of alcohol, and embedded in paraffin. Paraffin sections 5 µm thick were cut on a SM 2000R microtome (Leica, Wetzlar, Germany), stained with hematoxylin and eosin, and examined using a DM1000 microscope (Leica, Wetzlar, Germany).
The study of acute and chronic toxicity, as well as the study of the local tolerance of PLVF, was carried out in accordance with the recommendations and the requirements of local legislation (Federal Law of 12.04.10 N 61 "On the Circulation of Medicines").

Immunogenicity in Mice
Sixty (CBA × C57BL/6) F1 female mice (5 mice per group, 8-weeks-old at the start of the experiment and weighing 18 ± 0.3 g) were purchased from the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency", Russia and immunized intraperitoneally with 125 µg per mouse of PLVF or with two doses of 25 µg and 50 µg of Ac 3 -S-LPS 1b, 2a, 3a, 6, and Y. Two weeks after the primary injection, the mice were boosted with the same dose. At day 15 after the secondary immunization, serum samples were collected and levels of LPS-specific total IgG and IgM were evaluated by ELISA (using a standard protocol) with native S. flexneri LPS (nLPS) 1b, 2a, 3a, 6, and Y adsorbed on microplates (Greiner, Kremsmünster, Austria). Control animals were given saline.

SBA Assay
Heat-inactivated mouse serum samples were diluted 1:3000 in PBS and 100 µL were added into 96-well U-bottom plates (Medpolymer, Saint Petersburg, Russia). A 100 µL aliquot containing 10 4 CFU of each S. flexneri serotype 1b, 2a, 3a, 6, and Y separately and 25 µL of guinea pig complement (made in-house) were added into the wells. The final volume was 300 µL and serum dilution 1:9000. Plates were incubated for 1.5 h at 37 • C in a shaker at 200 rpm. Serum bactericidal activity was assessed individually for each mouse. The assay included complement control wells containing S. flexneri bacteria with guinea pig complement with no serum. This complement control was used to define 0% killing in the SBA killing rate calculation.
The percentage of bacteria killing (SBA rate) was determined by the equation [1-(colony forming units (CFU) of surviving bacteria/total CFU)] × 100.

Efficacy Evaluation of PLVF
Male (Agouti strain) guinea pigs were purchased from the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency", Russia. To examine the PLVF for eye protection against virulent S. flexneri stains, groups of 10 guinea pigs (aged 3 months at the start of the experiment and weighing 275 ± 3 g) were twice immunized subcutaneously dorsally with a dose of 125 µg of vaccine at an interval of 10 days. Control animals were given saline. Ten days after the last immunization, S. flexneri keratoconjunctivitis was induced in experimental and control groups of animals by inoculation into the mucosal surface of the conjunctiva of both eyes with a suspension of a virulent strain at a dose of 2 × 10 9 cells in 30 µL of sterile saline. Each group was inoculated with one of S. flexneri 1b, 2a, 3a, 6, and Y serotypes. Keratoconjunctivitis was assessed 7 days after challenge by visual inspection. The efficacy of PLVF was calculated by the formula: PLVF Efficacy = 100 × (Control attack rate -PLVF attack rate)/Control attack rate, where attack rate = number of infected eyes/total eyes [4].

Statistical Analysis
Statistical analyses of immunogenicity data were performed using GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA). For statistical significance while comparing groups of mice, the Student's t test was performed. Statistical significance was defined as p < 0.05.

Chemical Structure and Specific Epitope Identification of PLVF Compounds
PLVF contains modified long-chain S-LPS compounds with a length of O-PS chain of 15-25 repeating units ( Figure 1a). Mass spectrum analysis demonstrates that lipid A of 1b, 2a, 3a, 6, and Y represents highly homogenous Ac 3 -S-LPS and contains mainly three (the highest peak at m/z = 1053) residues of unsaturated fatty acids ( Figure 1b). It should be noted that no peaks for penta-and hexa-acyl derivatives were present in the mass spectra of the modified lipid A from S. flexneri serotypes 1b, 2a, 3a, 6, and Y.
noted that no peaks for penta-and hexa-acyl derivatives were present in the mass spectra of the modified lipid A from S. flexneri serotypes 1b, 2a, 3a, 6, and Y.  For immunochemical detection of PLVF components 1b, 2a, 3a, 6, and Y, we used monovalent Shigella antisera against type-specific antigens I, II, III, and VI, and groupspecific antigen 3,4, respectively. Each Ac3-S-LPS component in PLVF composition was antigenic (or serologically active) and reacted with the corresponding specific antisera, thus confirming the presence of type-or group-specific epitopes ( Figure 3). The serological activity of Ac3-S-LPS components of the vaccine was comparable to that of nLPS of the respective serotypes. For immunochemical detection of PLVF components 1b, 2a, 3a, 6, and Y, we used monovalent Shigella antisera against type-specific antigens I, II, III, and VI, and groupspecific antigen 3,4, respectively. Each Ac 3 -S-LPS component in PLVF composition was antigenic (or serologically active) and reacted with the corresponding specific antisera, thus confirming the presence of type-or group-specific epitopes (Figure 3). The serological activity of Ac 3 -S-LPS components of the vaccine was comparable to that of nLPS of the respective serotypes.

Immunogenicity in Mice
In all groups of mice immunized with Ac3-S-LPS 1b, 2a, 3a, 6, and Y, a significant increase in the titer of specific IgG and IgM was observed compared with the intact animals. The antibody response to five Ac3-S-LPS compounds did not change with an increase in immunization dose from 25 μg to 50 μg (Figure 5a,b). The immunogenicity in mice of low-endotoxic Ac3-S-LPS from S. flexneri 1b, 2a, 3a, and Y was almost equal. The level of serum antibody response in mice immunized with Ac3-S-LPS serotype 6 was higher.

Immunogenicity in Mice
In all groups of mice immunized with Ac3-S-LPS 1b, 2a, 3a, 6, and Y, a significant increase in the titer of specific IgG and IgM was observed compared with the intact animals. The antibody response to five Ac3-S-LPS compounds did not change with an increase in immunization dose from 25 μg to 50 μg (Figure 5a,b). The immunogenicity in mice of low-endotoxic Ac3-S-LPS from S. flexneri 1b, 2a, 3a, and Y was almost equal. The level of serum antibody response in mice immunized with Ac3-S-LPS serotype 6 was higher.

Immunogenicity in Mice
In all groups of mice immunized with Ac 3 -S-LPS 1b, 2a, 3a, 6, and Y, a significant increase in the titer of specific IgG and IgM was observed compared with the intact animals. The antibody response to five Ac 3 -S-LPS compounds did not change with an increase in immunization dose from 25 µg to 50 µg (Figure 5a,b). The immunogenicity in mice of low-endotoxic Ac 3 -S-LPS from S. flexneri 1b, 2a, 3a, and Y was almost equal. The level of serum antibody response in mice immunized with Ac 3 -S-LPS serotype 6 was higher. In the group of mice immunized with PLVF, an increase in the titer of specific I antibodies was also observed (p ≤ 0.05). Titers of IgG to LPS S. flexneri 1b, 2a, 3a, Y, an were 1213 ± 438, 1393± 980, 1056± 438, 2786 ± 2629, and 1600 ± 2360, respectively (Fig  6a).  In the group of mice immunized with PLVF, an increase in the titer of specific IgG antibodies was also observed (p ≤ 0.05). Titers of IgG to LPS S. flexneri 1b, 2a, 3a, Y, and 6 were 1213 ± 438, 1393± 980, 1056± 438, 2786 ± 2629, and 1600 ± 2360, respectively (Figure 6a). In the group of mice immunized with PLVF, an increase in the titer of specific IgG antibodies was also observed (p ≤ 0.05). Titers of IgG to LPS S. flexneri 1b, 2a, 3a, Y, and 6 were 1213 ± 438, 1393± 980, 1056± 438, 2786 ± 2629, and 1600 ± 2360, respectively ( Figure  6a). In the groups of mice immunized with only one component of PLVF, the titer of IgG In the groups of mice immunized with only one component of PLVF, the titer of IgG was 1393 ± 980 for 1b, 1213 ± 1403 for 2a, 1213 ± 438 for 3a, 2425 ± 2147 for 6, and 1213 ± 1043 for Y. These results were not significantly different from those in the group of mice immunized with PLVF. At the same time, titers in both experimental groups were at least 16 times higher than in the control group (Figure 6b). We did not register the occurrence of antigenic competition and suppression of the response when closely related polysaccharide antigens were combined into a single preparation.
Next, we evaluated the functional activity of S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies in sera from mice immunized with PLVF at a dose of 125 µg using the SBA assay. Insignificant killing (<15%) of S. flexneri of different serotypes was observed using nonimmune sera (Figure 7). After PLVF immunization, we registered significant rises in mouse immune sera bactericidal activity against virulent S. flexneri strains. The estimated killing rate was 54%, 66%, 35%, 60%, and 60% for S. flexneri 1b, 2a, 3a, 6, and Y serotype/serogroup, respectively. These data directly demonstrate the ability of modified S-LPS from different strains of S. flexneri to act as an effective protective antigen.
accines 2023, 11, x FOR PEER REVIEW assay. Insignificant killing (<15%) of S. flexneri of different serotypes was o non-immune sera (Figure 7). After PLVF immunization, we registered sign mouse immune sera bactericidal activity against virulent S. flexneri strains. killing rate was 54%, 66%, 35%, 60%, and 60% for S. flexneri 1b, 2a, 3a, 6 type/serogroup, respectively. These data directly demonstrate the ability LPS from different strains of S. flexneri to act as an effective protective antig Figure 7. Serum bactericidal antibody assay on immune sera from mice immunized dose of 125 μg against virulent S. flexneri strains, serotypes 1b, 2a, 3a, 6, and Y. We flexneri bacteria with guinea pig complement with no serum were used as compleme immune wells contained sera from naïve mice. Data represent mean SBA rate for SEM.

PLVF Protects against Keratoconjunctivitis in a Guinea Pig Model after Sepa with Each of S. flexneri 1b, 2a, 3a, 6, and Y Serotypes
To protect against conjunctivitis, guinea pigs were immunized with PL cutaneously with an interval of 10 days. On day 10 after the last immunizat animals were challenged with ID80 of virulent strains of S. flexneri 1b, 2a, 3a day seven after infection, visual assessment showed that in groups of immu the efficacy was 69%, 75%, 50%, 50%, and 69% against S. flexneri serotype and Y, respectively (  Figure 7. Serum bactericidal antibody assay on immune sera from mice immunized with PLVF at a dose of 125 µg against virulent S. flexneri strains, serotypes 1b, 2a, 3a, 6, and Y. Wells containing S. flexneri bacteria with guinea pig complement with no serum were used as complement control. Non-immune wells contained sera from naïve mice. Data represent mean SBA rate for each serotype ± SEM.

PLVF Protects against Keratoconjunctivitis in a Guinea Pig Model after Separate Infection with Each of S. flexneri 1b, 2a, 3a, 6, and Y Serotypes
To protect against conjunctivitis, guinea pigs were immunized with PLVF twice subcutaneously with an interval of 10 days. On day 10 after the last immunization, groups of animals were challenged with ID 80 of virulent strains of S. flexneri 1b, 2a, 3a, 6, and Y. On day seven after infection, visual assessment showed that in groups of immunized animals the efficacy was 69%, 75%, 50%, 50%, and 69% against S. flexneri serotypes 1b, 2a, 3a, 6, and Y, respectively (Figure 8). In unimmunized control animals, at least 80% of the eyes were infected depending on the serotype of the virulent strain of S. flexneri. Vaccines 2023, 11, x FOR PEER REVIEW 12 of 15

Toxicology Study
To assess the general toxicity, the effect of the vaccine preparation was studied in rabbits using single and repeated subcutaneous administration. The results of the acute toxicity study showed that PLVF was non-toxic at a dose of 125 μg. Animal mortality was not observed, there were no signs of toxicity, and there were no changes in body weight or hematological and biochemical tests. No pathological changes in bone marrow hematopoiesis were observed in all groups. There were no significant histological differences found in organs from PLVF and control group (Table S1, Figure S1).
The results of the chronic toxicity study showed that daily subcutaneous application of PLVF to rabbits did not cause disturbances in the functional state of the main organs and systems of the body.
There was no local irritating effect in the area of PLVF administration after repeated subcutaneous application to rabbits.

Discussion
Flexner's shigellosis represents a longstanding, difficult public health problem in many countries around the world. The achievement of field protection after vaccination against S. flexneri is an extremely difficult task.
Shigella-specific antibodies play an important role in promoting host defense against shigellosis. Children and adults living in areas endemic for shigellosis develop circulating antibody secreting cells and serum antibodies specific for Shigella LPS and invasion plasmid antigen. Sera of volunteers infected with S. flexneri 2a have a pronounced bactericidal efficacy [19].
Recent studies of functional antibodies after immunization with protein-lipopolysaccharide outer membrane vesicles (OMV) S. sonnei vaccine based on generalized modules for membrane antigen (GMMA) have shown that anti-LPS antibodies are the main drivers

Toxicology Study
To assess the general toxicity, the effect of the vaccine preparation was studied in rabbits using single and repeated subcutaneous administration. The results of the acute toxicity study showed that PLVF was non-toxic at a dose of 125 µg. Animal mortality was not observed, there were no signs of toxicity, and there were no changes in body weight or hematological and biochemical tests. No pathological changes in bone marrow hematopoiesis were observed in all groups. There were no significant histological differences found in organs from PLVF and control group (Table S1, Figure S1).
The results of the chronic toxicity study showed that daily subcutaneous application of PLVF to rabbits did not cause disturbances in the functional state of the main organs and systems of the body.
There was no local irritating effect in the area of PLVF administration after repeated subcutaneous application to rabbits.

Discussion
Flexner's shigellosis represents a longstanding, difficult public health problem in many countries around the world. The achievement of field protection after vaccination against S. flexneri is an extremely difficult task.
Shigella-specific antibodies play an important role in promoting host defense against shigellosis. Children and adults living in areas endemic for shigellosis develop circulating antibody secreting cells and serum antibodies specific for Shigella LPS and invasion plasmid antigen. Sera of volunteers infected with S. flexneri 2a have a pronounced bactericidal efficacy [19].
Recent studies of functional antibodies after immunization with protein-lipopolysaccharide outer membrane vesicles (OMV) S. sonnei vaccine based on generalized modules for membrane antigen (GMMA) have shown that anti-LPS antibodies are the main drivers of bactericidal activity. On the contrary, anti-protein antibodies had limited ability to either bind to Shigella cells or kill them in the presence of complement [20].
In modern LPS-enriched Shigella vaccines (Invaplex AR-Detox , GMMA) genetically modified Shigella strains (∆msbB or∆htrB) are being used to prevent production of the most endotoxic form of LPS with hexa-acylated lipid A domain [21,22]. However, in these vaccines, LPS remains heterogeneous like classical endotoxin and contains penta-and tetraacylated lipid A and, therefore, is still relatively endotoxic and pyrogenic. As a result, the doses of preparations containing genetically modified LPS proposed for the safe parenteral administration by the authors of the abovementioned studies are relatively low (in the range of 0.1-15 µg). Additional fractions of protein antigens, Ipa molecules (Invaplex AR-Detox ) or OMV proteins (GMMA), were also present in these vaccine preparations. Further studies aiming to increase the content of LPS, and thus change the design of GMMA-based vaccines to enhance their immunogenicity and efficacy, have been announced [20].
Our previous studies have shown that Ac 3 -S-LPS is apyrogenic and is the most immunogenic form of S. flexneri 2a S-LPS [16]. As we have already demonstrated the possibility of safe clinical use of S. flexneri 2a Ac 3 -S-LPS, it was reasonable to use this approach to obtain Ac 3 -S-LPS components of other serotypes/serogroups for the development of PLVF. Therefore, PLVF was designed to include in the vaccine dose the maximum number of S. flexneri antigens. Thus, PLVF contains five Ac 3 -S-LPS of S. flexneri, namely 1b, 2a, 3a, 6, and Y.
Each Ac 3 -S-LPS is a macromolecule, and at the same time is a full-fledged bioconjugate vaccine. In this respect, the O-PS conjugate is naturally produced by the bacterial cell and contains a built-in adjuvant-lipid A domain, the structure of which is deacylated to an apyrogenic form. The low content of pyrogens in PLVF preparation was confirmed using the rabbit pyrogenicity test. Preclinical toxicology studies in rabbits demonstrated the complete absence of signs of acute and chronic toxicity, local irritant action, damage to internal organs, and changes in biochemical and hematological parameters when PLVF was administered at a dose of 125 µg in 0.5 mL intended for human administration.
Thus, we achieved a significant extension of the dose range for a single parenteral administration to animals of Ac 3 -S-LPS as an active substance of PLVF to over 100 µg. Under the chronic toxicity regimen, multiple administration up to 875 µg of Ac 3 -S-LPS did not cause any endotoxic effects. These data once again demonstrate the critical difference between Ac 3 -S-LPS and natural endotoxin, the safe parenteral dose of which is sharply lower-just a few nanograms.
We used SDS-PAGE and mass spectrometry to determine the structure of Ac 3 -S-LPS of S. flexneri 1b, 2a, 3a, 6, and Y. Ac 3 -S-LPS from PLVF contains the long-chain S-LPS (O-PS chain length is about 20 repeating units) and has a triacylated lipid A. It should be emphasized that such vaccine grade Ac 3 -S-LPS is completely free from the most endotoxic highly acylated molecules of LPS-its hexa-and penta-acylated derivatives. The structure of the polysaccharide component of Ac 3 -S-LPS was determined using 13 C NMR and was distinct for each serotype.
Each Ac 3 -S-LPS of PLVF was identified using a homologous monovalent antiserum. The serological properties of PLVF components and nLPS of the same S. flexneri serotypes were similar. Thus, the methods used to obtain Ac 3 -S-LPS did not cause the alteration of antigenic determinants.
In double immunized mice, we have registered high IgG levels (16-28-fold rise) to all components of PLVF without an aluminum hydroxide adjuvant. High immune response in mice correlated with protection against dysentery eye infection in guinea pigs. We have registered 50-75% efficacy against dysenteric keratoconjunctivitis after double immunization of guinea pigs. In a recent paper [21], a 75% protection rate in the S. flexneri 2a Sereny test after triple subcutaneous immunization with Invaplex AR-Detox vaccine was reported. Thus, in addition to the 2a component, the simultaneous induction of a systemic immune response in mice and mucosal protection in guinea pigs has now been confirmed for four more S. flexneri O-antigens.
The correctness of the choice of Ac 3 -S-LPS with long-chain OPS as a protective antigen of PLVF was confirmed by the presence in the sera of mice immunized with the vaccine of significant amounts of bactericidal antibodies to each of the homologous virulent strains of S. flexneri, serotypes 1b, 2a, 3a, 6, and Y.
The results of this preclinical study substantiated a wide dose interval for the safe administration of the candidate vaccine product and, thus, successfully validated the design of a pentavalent vaccine combination. If the goal of protection in the field against Flexner's shigellosis using Ac 3 -S-LPS vaccine is achieved, the problem of comprehensive protection against shigellosis may be solved by the combined use of PLVF and the first available vaccine against Sonne's shigellosis. This vaccine is based on S. sonnei exopolysaccharide, which elicits protective antibodies, and has been successfully used for prophylactic immunization for 15 years. In addition, the comprehensive data on the safety of PLVF offers the possibility of extending our vaccine construction strategy to various serotypes of Enterobacteriaceae.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines11020345/s1, Figure S1: Hematoxylin and eosin stained sections of the internal organs of rabbits from the control group (a) and 7 days after the last injection of PLVF (b). PLVF was administered subcutaneously at a dose of 125 µg daily for a week. Representative images from 5 groups; Figure S2: Chemical structure of the repeating unit of the O-specific polysaccharide chain of S. flexneri serotypes 1b, 2a, 3a, 6, and Y; Table S1: Histopathological changes of internal organs after single or multiple subcutaneous injections with 125 µg dose of PLVF.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.