Generation of empty cell envelopes of Streptococcus pyogenes using biosurfactants

Background Bacterial ghost cells (BGCs) are cell envelopes that devoid of cytoplasmic and genetic contents in purpose of variable applications, including their great potential as vaccine candidates and their effectiveness as delivery systems for drugs and proteins. To our knowledge, this is the first study to produce Gram-positive BGCs by treating Streptococcus pyogenes ( S. pyogenes ) ATCC 19615 with Tween80 (TW80) or TritonX-100 (TX100), followed by prelimi-nary testing of their antigenicity and safety in NIH/Ola-Hsd mice. The produced BGCs were confirmed by the presence of intact cells under a light microscope, the absence of growth signs upon re-cultivation. The transmembrane tunnels were visualized using a scanning electron microscope, and subsequently, considerable quantities of released DNA and protein were detected in the culture supernatant of the BGCs. The antigenicity of the produced BGCs was tested through three intra-nasal immunization doses followed by infection. Afterward, the opsonic activity and the IgG levels were measured, followed by a comprehensive histopathological examination for selected tissues and organs. Results The sera of immunized mice exhibited a significant rise in both opsonic activity (TW80 produced BGC = 68% and TX100 produced BGC = 75%) and IgG levels (TW80 produced BGC = a threefold increase and TX100 produced BGC = a fourfold increase) when compared to the positive control group "non-immunized challenged with ATCC 19615." Histopathological analysis revealed that the BGCs produced by TW80 are relatively safer and have a less severe impact than those produced by TX100.


Background
Bacterial ghost cells (BGCs) are bacterial compartments that are depleted from all contents but retain most of their antigenic epitopes after production [1].The applications of BGCs are variable and widely studied, as they are used as bacterial vaccines, drug delivery, and antigen bacterial carrier systems [2][3][4].Those cells offer an excellent alternative to traditional bacterial vaccines, as they are cost-effective, easily stored, and have a longer shelf-life [5].The bacterial ghost (BG) system can stimulate innate and adaptive immune systems without using foreign adjuvants [4].It also carries pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS), which are detected by Toll-like receptor 4 (TLR4) and trigger the innate immune response [5].Recent studies have shown how powerful BGC is as adjuvants and how they can cause a variety of immunological and non-immune cell types to produce proinflammatory cytokines.These cytokines cause T and B cells to be widely recruited to lymph nodes, increasing the likelihood that they will come into contact with their cognate antigen, resulting in powerful immune responses.
The production of BGC relies on creating voids and preserving them as hollow structures, with the removal of all genetic and cytoplasmic contents [6].The regulated expression of the E-lysis gene was the initial approach employed in the generation of BGCs, although it is plagued by several limitations.It is limited to Gramnegative bacteria and highly expensive [7,8].Alternatively, chemical treatment using a sponge-like approach (chemical treatment) is another way to produce BGCs.That method involves determining the minimum inhibitory concentration (MIC) of different reagents such as NaOH, SDS, and H 2 O 2 , which can create small openings (transmembrane tunnels) in the bacterial cell wall [9].This method opened the door toward the production of Gram-positive BGCs, as Staphylococcus aureus was the first Gram-positive BGC to be produced using that method [10].However, using such chemicals has a destructive and corrosive effect on the antigenicity of the cells.A new chemical treatment utilizing a 7% of (TW80) was developed for the production of Gram-negative bacteria as Salmonella enterica-serovar Typhimurium ghosts.The TW80 approach successfully secured the integrity of the cells while also eliciting safe and effective immunological response in Gram-negative cells [11].Moreover, (TX100) was successfully used by our team as novel chemical treating agent to develop Shigella flexneri ghost cells, as those cells served as effective and safe vaccine candidates in vivo [12].
In the current study, Streptococcus pyogenes (S. pyogenes) was chosen to be the first Gram-positive candidate for bacterial ghost production using the chemical treatment of TW80 or TX100.
(S. pyogenes) is a Gram-positive cocci, responsible for causing a wide range of diseases related to the respiratory system and autoimmune disorders [13].The World Health Organization (WHO) stated in 2022 that there are a minimum of 500,000 fatalities annually caused by diseases related to Group A Streptococcal (GAS) infections, such as rheumatic heart disease, glomerulonephritis, and many invasive infections such as necrotizing fasciitis.Approximately 18.1 million individuals are afflicted with severe GAS sickness, and there are approximately 1.78 million new cases identified each year [14,15].The present work aimed to produce several batches of S. pyogenes ghost cells and evaluate their humoral immune response and opsonic activity in a in vivo model of immunization and challenge.Ultimately, the impact of these ghosts on various organs was examined using histopathology analysis.This study is a continuation of our prior work on the unique strategy for ghost cell production, as in this study we provided an early characterization and evaluation of the antigenicity of a newly developed Gram-positive BGCs generated using that approach.

Minimum inhibitory concentration determination of Triton X-100 (TX100)
A culture of S. pyogenes ATCC 19615 (KWIK-STIK, Microbiologics) was cultivated overnight in 5 mL of Trypticase Soy Broth (TSB) at 37 °C.Next, 150 µL of this culture was transferred to 15 mL of (TSB) and placed in an incubator at a temperature of 37 ℃ for 6 h.The concentration of the inoculum was adjusted to (1 × 10 6 CFU/mL) using an optical density (OD 600 :0.2).Multiple concentrations of v/v TX100 were created by diluting 10 mL of the solution at different percentages (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%).Subsequently, a volume of 100 µL of bacterial inoculum was introduced into the mixture, which was then subjected to incubation at 37 °C for a 24 h.Ultimately, an assessment of turbidity was conducted by measuring the optical density (OD) at 600 nm for all tubes that were incubated, and (MIC) was recorded.It was determined as the lowest concentration of TX100 that caused no visible turbidity, suggesting full bacterial eradication.Sub-MIC quantities of TX100, which are concentrations below the (MIC), were used for the generation of BGC.The sub-MIC concentration was evaluated at various incubation periods (1,3,6,12, and 24 h) to identify the most favorable harmless incubation time for producing BGCs [16,17].

Production of Streptococcus pyogenes bacterial ghost cells (Sp-BGCs) using TW80 (Sp-BGCs/TW80) and TX100 (Sp-BGCs/TX100)
Two sets of Sp-BGCs were generated deploying TW80 or TX100 individually following the chemical treatment protocol previously described [12].A 100 µL of bacterial inoculum of ATCC 19615 (1 × 10 10 CFU/mL, OD 600 :0.5) was added to 10 mL of v/v (7%) TW80/TSB and (5%) TX100/TSB (Alpha Chemika); those mixtures were incubated at 37 °C for 24 and 12h, respectively.A 180 µL of lactic acid was added to each of the treated set of cells in order to get the pH down to 2.9; afterwards, a 1 h of incubation was performed.The supernatant obtained after 10-min centrifugation at 4000 rpm was kept for further use.The produced ghost cells were washed and centrifuged twice at 4000 rpm with (0.45%) NaCl.Lastly, a (0.45%) NaCl was used for suspending the BGC pellets and kept at -20 °C for further use.

In vitro evaluation of the quality of BGCs
The produced sets of BGCs underwent various tests to assess and evaluate their quality.

Microscopic examination of BGCs
The BGCs pellets were subjected to Gram staining and observed under a light microscope (at a magnification of × 1000) in comparison with untreated ATCC 19615 cells.This was done to assess the dimensional stability and intactness of the treated cells [18].

Re-cultivation of BGCs
This procedure was conducted to guarantee the thorough eradication of all cellular activity and the lack of viable cells in the S. pyogenes BGC pellets.The pellets and untreated ATCC 19615 were cultivated separately on Todd-Hewitt agar supplemented with 0.5% yeast extract (THY) and TSB broth for 24-48 h at 37 °C.Any indications of the growth (colonies or turbidity (OD 600 ) have been detected and documented.

Measurement of protein and DNA
The supernatant from centrifuged BGCs was utilized to quantify the released protein and DNA Using Nanodrop (Jenway-7415 NANO) using Quartz cuvettes for both measurements at 280 nm and 260 nm, respectively [17,19].For a DNA measurements an extinction E260 = 1 that corresponds to 50 μg dsDNAmL − 1 was used, while measuring the released proteins was conducted through using Bovine Serum Albumin (BSA) standard [20].To sum it up, the supernatants of centrifuged, native, untreated S. pyogenes ATCC 19615 (1 × 10 10 CFU/mL, OD 600 :0.5) cultured in TSB for 24 h and 12 h at 37 °C served as controls for TW80 and TX100 preparations, respectively [19].That experiment was conducted in triplicates, where the average, standard deviation, and percentage of increase compared to native untreated cells were calculated for each set.

Scanning electron microscope (SEM) imaging of "Sp-BGC"
The morphological features of ghost cells have been investigated using Scanning Electron Microscopy (SEM) Quanta FEG [10].The cells had been fixed, dehydrated, and examined using the same protocol followed in [12].

In vivo models In vivo model of screening for the most sensitive mouse strain to S. pyogenes ATCC 19615 infection
Three mouse strains (BALB/C, B6, and NIH/Ola-Hsd) were used to assess the most sensitive strain toward S. pyogenes ATCC 19615 intra-nasal infection.Nine female mice, three from each strain, were obtained from Animal House.The mice were six weeks old at the time of infection.They were housed at a temperature of 25 °C and subjected to a 12-h light/12-h dark cycle.The mice had unrestricted access to a normal pellet meal and water.The mice were housed for one week to acclimatize them before beginning the screening process.Prior to initiating S. pyogenes ATCC 19615 infection, throat swab samples were obtained from each mouse and cultured on THY media to confirm the absence of S. pyogenes in the mouse throat.The collection of such samples was performed as described in Kurl et al. [21].Concisely, a sterile nasopharyngeal swab was introduced into the mouse's throat and thereafter placed in 1 mL of sterile, cold PBS for 5 min.Each sample was diluted in 180 µl of sterile, cold PBS in a 96-well plate, with 20 µl of each sample being used for the dilution.Ultimately, 10 µl of each dilution was spread onto THY and subjected to a 24-h incubation at 37 °C in a 5% CO 2 incubator.The number of viable, pale-white colonies of S. pyogenes was enumerated, and (CFU/mL) was calculated for each plate using the following equation: The infection was transmitted by administering 20 µl of ATCC 19615 (1 × 10 10 CFU/dose) through the nose.The dose was supplied once using a sterile micropipette (sized 20-200 µl) inserted into the nostril, allowing the mouse to inhale the bacterial inoculum, following the method described by Olive et al. [22].The evaluation of the infection's progression was performed using the same technique mentioned above.The number of viable (CFUs) of S. pyogenes was determined after infection on days one, three, six, and nine.The mouse strain with the highest number (CFU/mL) of S. pyogenes achieved during the infection period was selected as the most sensitive.

In vivo model of antigenicity assessment of BGCs
Twenty-eight adult female NIH/Ola-Hsd outbred mice, aged six weeks, were divided into four groups of equal size.Each group consisted of seven mice.The groups were as follows: negative control (mice not immunized and received (PBS)), positive control (mice not immunized but infected with ATCC 19615), Sp-BGCs/TW80 (mice immunized with bacterial ghost cells prepared with TW80 and then challenged with ATCC 19615), and Sp-BGC/TX100 (mice immunized with bacterial ghost cells prepared with TX100 and then challenged with ATCC 19615).The same housing and feeding conditions mentioned under (2.4.1) were followed.Before evaluating the antigenicity of the produced BGCs, a in vivo quality evaluation was conducted to check the viability of Sp-BGCs by cultivating throat swaps obtained from experimental animals immunized by the ghost cells on appropriate media.Throat swaps were collected before immunization with BGCs, after each immunization dose (1 × 10 8 cells/mL, OD 600 :0.4), and pre-and post-challenge with ATCC 19615 (1 × 10 10 CFU/dose, OD 600 :0.5).
After verifying the quality of the BGCs, the antigenicity evaluation was carried out by immunizing the mice with BGCs and then challenging them through intra-nasal administration of ATCC 19615 infection.
Immunization and challenge Immunization was carried out three times at two-week intervals through the intra-nasal (IN) route [23,24].The negative control group received 20 µl of PBS, whereas the TW80 and TX100 BGC groups were administered 20 µl (1 × 10 8 cells/mL, OD 600 :0.4) from each preparation of ghost cells.Throat swab samples were acquired and placed on THY media to culture viable S. pyogenes CFUs after each immunization dose with BGCs on days one, three, six, and nine.The procedure was essential in guaranteeing that there were no alive S. pyogenes cells present in any of the preparations.This served as an in vivo quality control for the created BGCs, confirming the lack of live cells among the ghost ones.
The challenge experiment commenced at week seven, two weeks following the final immunization dosage.The inoculum of ATCC 19615 was adjusted to a concentration of (1 × 10 10 CFU/dose, OD 600 :0.5).A single dose of 20 µl was delivered intranasally (IN) to all groups and 20 µl of PBS for negative control group.Throat swab samples were obtained from all groups after exposure (on days one, three, six, and nine) and cultured on THY medium to determine the number of (CFUs).

Protective efficacy and safety of Sp-BGC
The protective efficacy and safety of Sp-BGCs were assessed by analyzing the chosen organs for bacterial presence, comparing them to a group that was deliberately exposed to ATCC 19615 (positive control), as outlined in [10].All mice involved in the experiment were euthanized 10 days after being exposed to the challenge dose.At first, blood samples were collected from all tested groups, where serum was isolated and stored at − 20 °C in order to investigate the bactericidal activity and immunological response of the generated ghost cells, as described in [12,25,26].Afterwards, as the immunization and challenge experiment targeted respiratory infection; the superior and interior left lung, in addition to 0.25 cm of trachea, were collected, and the remaining organs were retained for histopathological study.The sample was homogenized manually in 1 mL of cold, sterile PBS using aseptic techniques.The homogenized tissues were diluted in a tenfold manner and then placed on THY agar.The agar was incubated for 24 h at 37 °C in a 5% CO 2 incubator and (CFU/mL) calculated for each plate.

Opsonization assay "indirect bactericidal activity" of Sp-BGCs
Opsonization assay was performed to detect the possible opsonic activity of the newly developed S. pyogenes ghost cells following the work performed by Brandt and his colleagues with some modifications [27].The experiment was conducted utilizing S. pyogenes ATCC 19615.The bacterium was inoculated onto a (THY) at 37 °C with 5% CO 2 for 24 h.One colony was transferred to 5 mL of (THY) broth and incubated at 37 °C with 5% CO 2 for 24 h, resulting in roughly 1 × 10 6 CFU/ml.The culture was progressively diluted to a concentration of 10 2 in (PBS).The modified amount of inoculum (10 µl) was mixed with heat-inactivated sera (20 µl) and horse blood (80 µl).The inactivated sera were generated by subjecting the tested blood samples to a temperature of 50 °C for a duration of 30 min using a heat block (Acculab DBH150-2, USA).A negative control was performed by combining a bacterial inoculum (10 µl) with PBS (20 µl) and horse blood (80 µl) in culture.Bacterial cells were incubated with sera and horse blood on a 96-well plate at a temperature of 37 °C for a duration of 4 h.A (10 µl) of the mixture was spread over (THY) agar containing 5% yeast extract in order to assess the survival of bacteria.The plates were placed in a 37 °C incubator with 5% CO 2 for 24 h, and the number of colonies was quantified in terms of (CFU).The opsonic activity of the antipeptide sera was assessed using the subsequent equation: CFU in the presence of antipeptide sera Mean CFU in the presence of sera from negative control × 100% The experiment was carried out in triplicates.

Determination of immune response
The immune response was determined in the sera of all mouse groups using enzyme-linked immunosorbent assay (ELISA) that performed in [12].A slight modification was made for negative control wells, as Shigella flexneri ATCC 12022 (Microbiologics) cultivated in Luria Bertani (LB) broth (Lab M) was used as irrelevant polypeptide in order to evaluate antibody specificity in a form of "mock vaccine" [11,28].

Histopathology analysis of pulmonary and tracheal tissues
The superior and interior right lung and the rest of the tracheal tissue samples were collected from all tested groups after animal termination.All tissues were flushed, fixed, and processed using the same protocol followed in [12].

Statistical analysis
GraphPad Prism 8 software (La Jolla, CA, USA) was used to examine statistical differences using one-way statistical analysis of variance (ANOVA) and two-way analysis of variance (ANOVA-mixed models).Significance was evaluated by p-values < 0.05 and 0.0001, indicating regular and high significance.Tukey's multiple comparison test was used to analyze data from the opsonization assay.

In vitro quality check for BGCs Microscopic examination of BGCs
Gram staining of both preparations showed the presence of Gram-positive cocci arranged in chains, as the intactness and cellular integrity were maintained.The same consistent result was observed when staining untreated ATCC 19615 cells (Supplementary Fig. 1).

Recultivation of BGCs
There was no detectable bacterial growth when Sp-BGC/TW80 or Sp-BGC/TX100 pellets were cultured in TSB and on TSA for 24-48 h at 37 °C (Supplementary Tables 1 and 2).In contrast, untreated ATCC 19615 exhibited positive bacterial growth, indicated by turbidity or the presence of colonies.

Quantification of released protein and DNA
Sp-BGC/TW80 and Sp-BGC/TX100 showed significant increases in the released protein and DNA when compared to native untreated ATCC 19615 cells.That confirms the successful generation of transmembrane tunnels which were responsible for evacuating the cellular content (Supplementary Tables 5, 6, 7, and 8) and (Supplementary Fig. 1).

Viable counts of throat swab cultures Screening model
No S. pyogenes colonies were observed from the throat swabs obtained before inducing intra-nasal infection with ATCC 19615 in all tested groups.Viable CFUs of S. pyogenes were obtained post-intra-nasal infection with ATCC 19615.NIH/Ola-Hsd outbred mice were the most sensitive, as they gave the highest CFU/mL at day six

Immunization and challenge experiment
No S. pyogenes colonies were observed in the throat swab samples obtained before and after any of the immunization doses with Sp-BGC/TW80 or Sp-BGC/TX100.Furthermore, no S. pyogenes was found in the throat swabs of immunized mice with BGCs from both groups after challenging them with ATCC 19615 starting from day one to day nine.Also, the negative control group gave no viable CFUs throughout the whole stage of the experiment.Consistent results were obtained from homogenized lungs and tracheas of immunized challenged groups with BGCs and the negative control group, where no bacterial colonies were found.Only the positive control group had viable S. pyogenes colonies in throat swabs after 24 h post-challenge in all mice until day nine postinfection.The highest number of viable ATCC 19615 colonies was observed on day six (1 × 10 6 CFU/mL) (Fig. 3).Additionally, viable colonies of S. pyogenes were found in the homogenized pulmonary tissues and tracheas of the positive control group at concentrations of 2 × 10 3 CFU/ mL and 1 × 10 2 CFU/mL, respectively.

Determination of opsonic activity of Sp-BGCs
Opsonic antibodies against ATCC 19615 increased in the sera of mice that were immunized with Sp-BGC/TW80 and Sp-BGC/TX100.Their opsonic activity increased significantly to 68% and 75%, respectively, compared to the positive control group's activity of 18% (Fig. 4).

Antibody response (serum IgG)
Both the Sp-BGC/TW80 and Sp-BGC/TX100 groups had significantly elevated serum IgG levels (p < 0.05) compared to the positive control and negative control groups at a dilution of 12.5 µl, with increases of threefold and fourfold, respectively.There was a notable difference between the immunized challenged groups and the negative control (p < 0.05), although there was no noticeable difference in the IgG serum levels of the two BGCs (p > 0.05) (Fig. 5).

Histopathology analysis of pulmonary and tracheal tissues
The pulmonary tissues of negative control group demonstrated normal morphological features in addition to intact vasculatures and minimal inflammatory infiltrates.Also, normal histological features were found in the tracheal wall with intact respiratory ciliated epithelium.The propria submucosa showed loose, delicate connective tissue in addition to intact vasculatures and minimal inflammatory infiltrates (Fig. 6).The non-immunized challenged (positive control) group demonstrated severe hemorrhagic pneumonia with diffuse records of extravasation of blood into the alveolar lumen as well as intra-bronchiolar lumen hyperplasia of the bronchial epithelium.There is a moderate increase of peribronchiolar mononuclear inflammatory cells with higher figures of interalveolar walls thickening by cellular infiltrates and congested blood vessels.Tracheal tissues showed marked diffuse necrotized respiratory epithelium with severe mucosal and submucosal inflammatory cell infiltrates.Some sections showed bloody luminal discharges mixed with necrotic tissue depressions (Fig. 7).Sp-BGC/TX100 showed minimal hemorrhage in the lung lobes as well as intact bronchiolar lumens with moderate hyperplasia of the bronchiolar epithelium.However, moderate thickening of interalveolar walls and a peribronchiolar infiltrate with inflammatory cells were observed, while tracheal tissues showed alternate areas of intact luminal epithelium and necrotic patches with mild records of mononuclear inflammatory cell infiltrates (Fig. 8).Sp-BGC/ TW80 showed a significant pulmonary protective efficacy with normal morphological features (as a negative control), and tracheal tissues showed intraluminal bloody discharges; however, scanty records of submucosal inflammatory cell infiltrates with apparent intact luminal respiratory epithelium were observed (Fig. 9).Results from the scoring system of pulmonary and tracheal tissue examination for all groups are displayed in (Supplementary Tables 3 and 4), respectively.

Discussion
Antigenicity is the ability to be recognized specifically by antibodies produced in response to a given pathogen or compound.Therefore, the first phase in producing antigenic candidates against a certain pathogen is to induce antibody production (humeral immune response) upon re-exposure [29].Nowadays, the molecular engineering methods serve a huge sector of the antigenic candidates' productions.Although those approaches were efficient in eliciting a safe and effective humeral immune response, they were also exceedingly expensive and time-consuming [30].BGCs were proven to induce efficient humeral immune response in variable in vivo models [5,28,31].That line perfectly with significant level of the released IgG in immunized challenged groups with BGCs.
In the current study we implemented one of the most novel technologies to preliminary produce an antigenic candidate, which is bacterial ghost cells (BGCs).Those cells are empty bacterial cells obtained through the formation of transmembrane tunnels within bacterial cell walls [3,32].BGCs produced in a specific manner that eventually elicit the desired immune response without losing the outermost membrane that hold the pyogenes.The X-axis represents the tested groups (negative control "control/PBS, " positive control "non-immunized challenged with ATCC 19615, " Sp-BGC/TW80 "immunized with S. pyogenes bacterial ghost cells treated with Tween 80 then challenged with ATCC 19615", and Sp-BGC/TX100 "immunized with S. pyogenes bacterial ghost cells treated with Triton X-100 then challenged with ATCC 19615".The above results are represented as opsonization percentage compared to control/PBS wells, and error is described as standard error of the calculated mean for each group (SEM).One-way ANOVA was used to statistically analyze the results and followed by performing Tukey's multiple comparison test as the probability value of p < 0.05 was considered statistically significant (***, p < 0.0001), all obtained using GraphPad Prism (8.0.2) Fig. 5 Serum antibody response (IgG) of immunized and non-immunized animal groups.A bar chart representing specific antibody responses (IgG) measured by indirect ELISA for all tested groups.The X-axis represents the tested groups (negative control "non-immunized non-challenged, " positive control "non-immunized challenged with ATCC 19615, " Sp-BGC/TW80 "immunized with S. pyogenes bacterial ghost cells treated with Tween 80 then challenged with ATCC 19615", and Sp-BGC/TX100 "immunized with S. pyogenes bacterial ghost cells treated with Triton X-100 then challenged with ATCC 19615").The Y-axis represents the recorded OD450/IgG levels for each group at dilution 12.5µl.(**) denotes a significant difference between tested groups p < 0.05.Data represent mean ± SE using (one-way ANOVA) by GraphPad Prism (8.0.2) software immunogenic epitopes.Conventionally produced BGCs were very difficult, time-consuming, and costly to produced [5].Shigella flexneri ghost cells as a Gram-negative candidate were successfully produced by our team chemically, by treating the cells with 7% TW80 for 24h or with 5% TX100 for12h.That method secured the outermost membrane of the cells even after creating the transmembrane tunnels and preserved the immunogenic epitopes that required to elicit a significant immune response.Additionally, implementing that method approved to induce a significant and safe immune response in vivo [12].In the current study, we have tested method of production on a Gram-positive candidate and assessed its antigenicity in a in vivo model.
One notable characteristic of BGCs that makes them ideal for prospective vaccine manufacture is that they Fig. 6 Histopathology images of pulmonary and tracheal tissues of negative control group "non-immunized, non-challenged." Histopathology images produced by hematoxylin and eosin staining method.(a, and c) represent pulmonary tissues of the negative control group "non-immunized non-challenged, " which show apparent intact, well-organized morphological features of lung tissue parenchyma with intact alveoli showing thin, intact interalveolar walls without abnormal infiltrates (dark blue arrows) and normal vasculatures, as well as intact bronchioles with intact lining epithelium (star) without abnormal infiltrates.(d, e, and f) represent tracheal tissue sections of the same group, which show normal histological features of the tracheal wall with intact respiratory ciliated epithelium (black arrow) and intact propria submucosa alveolar walls without abnormal infiltrates (black arrow) and normal vasculatures, as well as intact bronchioles with intact lining epithelium (star) without abnormal infiltrates, a and d sections are 200 µm, while b, c, e, and f are 50 µm are the inactivated (dead) form of bacterial cells, where growth or colonization cannot be happening [1].We have confirmed the absence of growth in vitro, where none of the cultivated BGCs showed any signs of growth when compared to untreated cells under the same cultivating conditions.On the other hand, those cells were checked for their preliminary safety in vivo, where no colonies were obtained from the entire cultivated throat swab samples collected from the immunized mouse groups with BGCs after each immunization dose.
Moreover, this protocol lacks any physical inactivation during BGC's production [11].This could have a significant impact on maintaining the majority of the important  and c) represent pulmonary tissues of the positive control group "non-immunized challenged with ATCC 19615, " showing severe hemorrhagic pneumonia with diffuse records of extravasation of blood into the alveolar lumen (red star) as well as intra-bronchiolar lumen with hyperplasia of the bronchial epithelium (black star).A moderate increase of peribronchiolar mononuclear inflammatory cells is shown (red arrow) in addition to interalveolar walls thickening by cellular infiltrates as well as congested blood vessels (dashed arrow), which show severe hemorrhagic pneumonia with diffuse records of extravasation of blood into the alveolar lumen (red star) as well as intra-bronchiolar lumen with hyperplasia of the bronchial epithelium (black star).A moderate increase of peribronchiolar mononuclear inflammatory cells is shown (red arrow) with interalveolar walls thickening by cellular infiltrates as well as congested blood vessels (dashed arrow).(d, e, and f) represent tracheal tissue sections of the same group, showing marked diffuse necrotized respiratory epithelium (red arrow) with severe mucosal and submucosal inflammatory cell infiltrates (red star).Bloody luminal discharges mixed with necrotic tissue depress are observed in some tissue sections; sections a and d are 200 µm, while b, c, e, and f are 50 µm immunogenic features of the bacterial cell wall.In addition, the cells were extensively depleted of their genetic resources, thus reducing the likelihood of horizontal gene transfer [33].That was confirmed in this study by the significant high level of released DNA from the treated cells compared to the untreated ones under the same cultivating conditions.Our results came in line with studies published by Rabea et al. [11].
Antigenicity evaluation of the prepared BGCs was a crucial step in this study.That was performed using an NIH/Ola-Hsd mouse model through immunization and intra-nasal challenge experiments using the same bacterial strain "ATCC 19615" used in BGC's production.It is well known that to evaluate the antigenicity of any potential antigenic candidate, challenging the subjects must take place after the immunization, and for that to be accomplished, the mouse strain must be sensitive to this pathogen.Previous studies reported that some strains of male mice were more sensitive to S. pyogenes infections than female ones, but in this study, we tended to use more immunocompetent and younger (6-weekold) subjects as females in order to diminish the resistance to the infection that might happen [34,35].It is well known that S. pyogenes strains are strictly human pathogens, and it is extremely rare for them to spread from animals to humans.However, human strains are more likely to infect animals (reverse zoonosis) [36].Above all, the ATCC 19615 strain had not been used before to induce intra-nasal infection in any of the mouse strains; consequently, a screening model was applied to detect the most sensitive mouse strain to intra-nasal challenge with ATCC 19615.Our results led us to conclude that the female NIH/Ola-Hsd mouse model, aged six weeks, was the most sensitive to ATCC 19615 intra-nasal infection, as it developed viable CFUs after 24 h only and reached the infection peak on day six post-infection.On the contrary, the BALB/C mouse model was resistant to intranasal ATCC 19615 infection, and that was consistent with the previous study [37].This in vivo infection model could be used in the future as a fast and secure way to induce intra-nasal challenge with S. pyogenes in mice, as the previously conducted model took eight days to visualize viable CFU colonies from swab cultures [38].
Counting the viable CFUs from mouse throat swabs and homogenized tracheal and pulmonary tissues was the first line of antigenicity evaluation of Sp-BGC/TW80 and Sp-BGC/TX100 after intra-nasal challenge with ATCC 19615 [38,39].The absence of viable CFUs of S. pyogenes in those samples was consistent with the positive and significant immune response that was found in the sera of these groups.Overall, those assessments confirmed the antigenicity of both BGCs preparations.
The primary purpose of any potential antigenic candidate against S. pyogenes is dependent on its ability to develop opsonic antibodies [40].Additionally, systemic IgG with opsonic action toward S. pyogenes has previously been linked to protection from S. pyogenes at systemic sites [41].Thus, we investigated whether sera from immunized challenged mice with the BGCs could opsonize the used bacterial candidate (ATCC 19615) or not.Both of BGCs preparations produced a significant level of opsonic antibodies.They successfully engulfed the targeted infection cells (ATCC 19615) in comparison with the non-immunized challenged group.The findings of our study align with previous research examining the efficacy and safety of recently produced S. pyogenes antigenic candidates [42,43].It is worth mentioning that there is a positive linear correlation between IgG antibody titers, opsonization assay results, and in vitro phagocytosis of S. pyogenes [44].As the opsonic activities of the immunized groups of BGCs in this study were evaluated, we noticed that the immunized sera from the Sp-BGC/TW80 and Sp-BGC/TX100 groups had significantly higher bactericidal (opsonic) activities and serum IgG levels than the non-immunized challenged group.This result ties well with previous studies investigating the opsonic activities of S. pyogenes vaccine candidates [27,42,45].
Intra-nasal administration of ATCC 19615 for the nonimmunized challenged group successfully confirmed the intra-nasal colonization of S. pyogenes and progressed to develop a severe pulmonary infection, as confirmed in the histopathological analysis of lung tissues [46].A further novel finding in this study is that the challenged Sp-BGC/TW80 showed significant pulmonary protective efficacy with more organized histological features of lung parenchyma.On the other hand, the pulmonary and tracheal tissues of Sp-BGC/TX100 showed minimal hemorrhagic records and mild injuries.These findings conclude that Sp-BGC/TX100 produces a positive immune response against S. pyogenes ATCC 19615, but with some devastating effects on the examined tissues.We speculate that this might be due to the possibility of TX100 particles being loaded inside the cells during the treatment process that, upon release, cause some tissue damage and inflammation [47][48][49].
The development of a safe and efficient antigenic candidates against S. pyogenes is a challenging endeavor [50].Due to that, this study marks the initial phase of a comprehensive investigation into the complete safety of these potential candidates.In relation to our future goals, we aim to explore the probable occurrence of autoimmune events in the heart or kidneys and the antibody cross-reactivity with cardiac myosin proteins.This investigation will allow us to determine the overall safety of Sp-BGC/TW80 as a potential antigenic candidate against S. pyogenes.Furthermore, we will assess the potential of this antigenic candidates to stimulate an immunological response and evaluate its efficacy in defending against different strains of S. pyogenes.

Conclusion
This study developed two successful S. pyogenes BGCs using TW80 or TX100, which induced a significant immune response against S. pyogenes ATCC 19615 in NIH/Ola-Hsd mice.On the other hand, the histopathological analysis showed that Sp-BGC/TW80 was safer and had a less devastating effect on mouse tissues than Sp-BGC/TX100.These findings suggest a demand for further investigation on the possibility of autoimmunity and antibody cross-reactivity with cardiac myosin proteins.It is also important to thoroughly evaluate the safety of BGCs through pre-clinical and clinical trials.

Fig. 1
Fig. 1 SEM image of Sp-BGCs/TW80.Scanning electron microscope (SEM) image of Sp-BGCs/TW80 pellet at magnification power × 16,000.White arrows point to a transmembrane tunnel formed in the cell walls of cells treated with 7% (v/v) TW80 for 24 h

Fig. 2 Fig. 3
Fig. 2 SEM image of Sp-BGCs/TX100.Scanning electron microscope (SEM) image of Sp-BGCs/TX100 pellet at magnification power × 16,000.White arrows point to a transmembrane tunnel formed in the cell walls of the cells treated with 5% (v/v) TX100 for 12 h

Fig. 4
Fig.4 Opsonization assay results of sera of immunized and non-immunized animal groups.A bar chart representing average opsonization (duplicate from two independents) of sera collected from immunized and non-immunized mice at the last day of in vivo immunization and challenge experiments against ATCC 19615 of S. pyogenes.The X-axis represents the tested groups (negative control "control/PBS, " positive control "non-immunized challenged with ATCC 19615, " Sp-BGC/TW80 "immunized with S. pyogenes bacterial ghost cells treated with Tween 80 then challenged with ATCC 19615", and Sp-BGC/TX100 "immunized with S. pyogenes bacterial ghost cells treated with Triton X-100 then challenged with ATCC 19615".The above results are represented as opsonization percentage compared to control/PBS wells, and error is described as standard error of the calculated mean for each group (SEM).One-way ANOVA was used to statistically analyze the results and followed by performing Tukey's multiple comparison test as the probability value of p < 0.05 was considered statistically significant (***, p < 0.0001), all obtained using GraphPad Prism (8.0.2)

Fig. 7
Fig.7 Histopathology images of pulmonary and tracheal tissues of positive control group "non-immunized challenged with ATCC 19615".Histopathology images produced by hematoxylin and eosin staining method.(a, b, and c) represent pulmonary tissues of the positive control group "non-immunized challenged with ATCC 19615, " showing severe hemorrhagic pneumonia with diffuse records of extravasation of blood into the alveolar lumen (red star) as well as intra-bronchiolar lumen with hyperplasia of the bronchial epithelium (black star).A moderate increase of peribronchiolar mononuclear inflammatory cells is shown (red arrow) in addition to interalveolar walls thickening by cellular infiltrates as well as congested blood vessels (dashed arrow), which show severe hemorrhagic pneumonia with diffuse records of extravasation of blood into the alveolar lumen (red star) as well as intra-bronchiolar lumen with hyperplasia of the bronchial epithelium (black star).A moderate increase of peribronchiolar mononuclear inflammatory cells is shown (red arrow) with interalveolar walls thickening by cellular infiltrates as well as congested blood vessels (dashed arrow).(d, e, and f) represent tracheal tissue sections of the same group, showing marked diffuse necrotized respiratory epithelium (red arrow) with severe mucosal and submucosal inflammatory cell infiltrates (red star).Bloody luminal discharges mixed with necrotic tissue depress are observed in some tissue sections; sections a and d are 200 µm, while b, c, e, and f are 50 µm

Fig. 8
Fig. 8 Histopathology images of pulmonary and tracheal tissues of Sp-BGCs/TX100 group "immunized with BGCs produced by TX100 and challenged with ATCC 19615".Histopathology images produced by hematoxylin and eosin staining method.(a, b, and c) represent pulmonary tissues of the Sp-BGCs/TX100 group "immunized with S. pyogenes bacterial ghost cells treated with Triton X-100 then challenged with ATCC 19615, " showing minimal hemorrhagic records allover lung lobes as well as intact bronchiolar lumens with moderate hyperplasia of the bronchiolar epithelium (star).However, persistent records of a moderate thickening of inter-alveolar walls (dashed arrow) and peribronchiolar infiltrate with inflammatory cells (red arrow).(d, e, and f) represent tracheal tissue sections of the same group, showing alternated areas of the intact luminal epithelium (black arrow) and necrotic patches (red arrow) with the persistence of mild records of mononuclear inflammatory cell infiltrates (red star), a and d sections are 200 µm, while b, c, e, and f are 50 µm

Fig. 9
Fig. 9 Histopathology images of pulmonary and tracheal tissues of Sp-BGCs/TW80 group "immunized with BGCs produced by TW80 and challenged with ATCC 19615".Histopathology images produced by hematoxylin and eosin staining method.(a, b, and c) represent pulmonary tissues of the Sp-BGC/TW80 group "immunized with S. pyogenes bacterial ghost cells treated with Tween 80 then challenged with ATCC 19615, " showing significant pulmonary protective efficacy with more organized histological features of lung parenchyma including alveolar lumens, thin interalveolar walls (dashed arrow), bronchiolar lumens (star), and intact vasculatures.These sections show similar intact tissues as the non-immunized non-challenged group (negative control).(d, e, and f) represent tracheal tissue sections of the same group, showing persistent records of intraluminal bloody discharges (yellow star).However, scanty records of submucosal inflammatory cell infiltrate are shown (black star) with almost apparent intact luminal respiratory epithelium (black arrow), a and d sections are 200 µm, while b, c, e, and f are 50 µm