Synthetic BSA-conjugated disaccharide related to the Streptococcus pneumoniae serotype 3 capsular polysaccharide increases IL-17A Levels, γδ T cells, and B1 cells in mice

The disaccharide (β-D-glucopyranosyluronic acid)-(1→4)-β-D-glucopyranoside represents a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 3. A conjugate of the disaccharide with BSA (di-BSA conjugate) adjuvanted with aluminum hydroxide induced — in contrast to the non-adjuvanted conjugate — IgG1 antibody production and protected mice against S. pneumoniae serotype 3 infection after intraperitoneal prime-boost immunization. Adjuvanted and non-adjuvanted conjugates induced production of Th1 (IFNγ, TNFα); Th2 (IL-5, IL-13); Th17 (IL-17A), Th1/Th17 (IL-22), and Th2/Th17 cytokines (IL-21) after immunization. The concentration of cytokines in mice sera was higher in response to the adjuvanted conjugate, with the highest level of IL-17A production after the prime and boost immunizations. In contrast, the non-adjuvanted conjugate elicited only weak production of IL-17A, which gradually decreased after the second immunization. After boost immunization of mice with the adjuvanted di-BSA conjugate, there was a significant increase in the number of CD45+/CD19+ B cells, TCR+ γδ T cell, CD5+ В1 cells, and activated cells with MHC II+ expression in the spleens of the mice. IL-17A, TCR+ γδ T cells, and CD5+ В1 cells play a crucial role in preventing pneumococcal infection, but can also contribute to autoimmune diseases. Immunization with the adjuvanted and non-adjuvanted di-BSA conjugate did not elicit autoantibodies against double-stranded DNA targeting cell nuclei in mice. Thus, the molecular and cellular markers associated with antibody production and protective activity in response to immunization with the di-BSA conjugate adjuvanted with aluminum hydroxide are IL-17A, TCR+ γδ T cells, and CD5+ В1 cells against the background of increasing MHC II+ expression.

The disaccharide (b-D-glucopyranosyluronic acid)-(1→4)-b-D-glucopyranoside represents a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 3. A conjugate of the disaccharide with BSA (di-BSA conjugate) adjuvanted with aluminum hydroxide inducedin contrast to the non-adjuvanted conjugate -IgG1 antibody production and protected mice against S. pneumoniae serotype 3 infection after intraperitoneal prime-boost immunization.Adjuvanted and non-adjuvanted conjugates induced production of Th1 (IFNg, TNFa); Th2 (IL-5, IL-13); Th17 (IL-17A), Th1/Th17 (IL-22), and Th2/ Th17 cytokines (IL-21) after immunization.The concentration of cytokines in mice sera was higher in response to the adjuvanted conjugate, with the highest level of IL-17A production after the prime and boost immunizations.In contrast, the non-adjuvanted conjugate elicited only weak production of IL-17A, which gradually decreased after the second immunization.After boost immunization of mice with the adjuvanted di-BSA conjugate, there was a significant increase in the number of CD45+/CD19+ B cells, TCR+ gd T cell, CD5+ В1 cells, and activated cells with MHC II+ expression in the spleens of the mice.IL-17A, TCR + gd T cells, and CD5+ В1 cells play a crucial role in preventing pneumococcal infection, but can also contribute to autoimmune diseases.Immunization with the adjuvanted and non-adjuvanted di-BSA conjugate did not elicit autoantibodies against double-stranded DNA targeting cell nuclei in mice.

Introduction
Streptococcus pneumoniae (pneumococcus) cause pneumonia, bacteremia, septic arthritis, meningitis, sinusitis, otitis media and some other diseases in humans (1,2).The incidence of communityacquired pneumonia is one per one thousand adults.The mortality rate for pneumococcal pneumonia among hospitalized patients is 5-7% (3)(4)(5)(6)(7).Symptoms of pneumococcal infection depend on the localization of the infection.These may include fever, cough, chest pain, a stiff neck, chills, ear pain and others.
Pneumococcal polysaccharide and conjugate vaccines, which contain capsular polysaccharides (CPs) from clinically significant S. pneumoniae serotypes, are available (8). S. pneumoniae serotype 3 is predominant among other serotypes in various countries (9)(10)(11)(12).Epidemiological data suggests a high incidence of disease caused by S. pneumoniae serotype 3 (13-15).However, the widespread use of pneumococcal vaccines should help to reduce the incidence of this disease (16)(17)(18)(19).Improving the quality of S. pneumoniae type 3 in the composition of pneumococcal vaccines is essential.
Bacterial CPs contain a diverse mixture of oligosaccharides with varying chain lengths and frame shifts (20).Although their chemical preparation is practically possible (see, for example (21),), synthetic oligosaccharide derivatives represent more convenient antigenic components for the design of conjugate carbohydrate vaccines (22)(23)(24)(25).Currently, a number of semisynthetic vaccines are under development, including those against Staphylococcus, Clostridium, Klebsiella, Shigella, and Enterococcus (25-33).The semi-synthetic glycoconjugate vaccine, Quimi-Hib, for the prevention of H. influenzae type b infection is licensed for use in Cuba (34).Optimization of the composition of pneumococcal vaccines using synthetic oligosaccharides conjugated with a protein carrier is a priority in contemporary vaccinology (25,(35)(36)(37)(38).
Moreover, synthetic oligosaccharides with precisely defined chemical structures enable the study of the effect of bacterial antigens (39,40), yielding a better understanding of the innate and cellular immunity, the antibody (Ab) response, and protective activity of CPs.
Immunization with glycoconjugate vaccines partially mimics the development of natural infection without actually causing the disease.In a mouse model, gd T cells and natural killer T cells (NKT) have been shown to play a crucial role in anti-pneumococcal immunity by producing Th1 and/or Th17-related cytokines (41).The ability of semisynthetic glycoconjugates to stimulate cytokine production in vivo and their influence on the activation of cellular immunity remain unknown.Here, we report on the effect of a conjugate of the synthetic disaccharide, which represents a repeating unit of S. pneumoniae serotype 3 (42), on production of Th1/Th2/Th17 cytokines in mice, changes in expression of surface molecules on splenocytes, antibody response, and protection against S. pneumoniae infection.We also investigated the production of autoantibodies against double-stranded (ds) DNA.

The synthetic disaccharide and its conjugate
The synthetic disaccharide (35, 43) was coupled to BSA (Sigma-Aldrich, St. Louis, MO, USA), as previously described (35,44).The structure of the conjugate is illustrated in Figure 1.BSA is often used  The structure of the BSA conjugate with the disaccharide that corresponds to a repeating unit of the CP from S. pneumoniae serotype 3.
as a protein carrier in engineered immunogenic glycoconjugates and other biological systems (45).Previous studies using MALDI-TOF mass spectrometry have shown that the di-BSA conjugate contains, on average, 19 oligosaccharide ligands per protein molecule, which corresponds to a 9% carbohydrate content by weight (43,44).The lyophilized di-BSA conjugate remains stable at +4°C, with no decrease in activity, for at least three years (i.e., observation period).

Bacterial capsular polysaccharide
Bacterial CP was isolated from the S. pneumoniae type 3 laboratory strain, #10196, which was isolated on June 30, 2011, from the blood culture of a child suffering from bacteremia in the microbiology department of the "Scientific Center for Children's Health" in Moscow, Russia.The strain had been grown in a semisynthetic growth medium.The isolation process for CP has been previously described elsewhere (46).The presence of CP in the preparation was confirmed by NMR spectrometry.

Animals
BALB/c male mice, aged 6-8 weeks (n=162), were purchased from the Scientific and Production Centre for Biomedical Technologies in Moscow, Russia, and kept in the vivarium at the Mechnikov Institute for Vaccines and Sera.Housing, breeding, blood collection, and euthanasia conditions followed European Union guidelines for laboratory animal care and use.Experimental designs were reviewed and approved (Protocol No. 2, dated February 12th, 2019) by the Ethics Committee at the Institute.

Immunization
Mice were intraperitoneally immunized with the di-BSA conjugate, either adjuvanted or not, with aluminum hydroxide (Sigma-Aldrich).The amount of carbohydrate in 0.5 mL of the experimental semisynthetic vaccine was 20 mg, BSA ~200 mg; aluminum hydroxide, as an adjuvant, standardized for aluminum, was added in an amount of 250 mg.The single immunizing dose per mice was 0.5 mL of the di-BSA conjugate.Animals were given the vaccine twice, on days 0 and 14 of the study.
Similar immunization schedules were used for the pneumococcal conjugate vaccine Prevnar 13 (Pfizer, New York, NY, USA), which contains aluminum phosphate as an adjuvant.A 0.5 mL dose contains 2.2 mg of polysaccharides from serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, and 23F, as well as 4.4 mg of the polysaccharide from serotype 6B.The vaccine also contains 32 mg of the carrier protein, CRM 197 , and 125 mg of aluminum as aluminum phosphate.Mice were immunized twice with a single dose of 1.1 mg of CP from S. pneumoniae type 3 per inoculation (equivalent to half of the recommended human dose).Control mice were injected with saline.

Content of bacterial endotoxins in glycoconjugates
Detection of bacterial endotoxin impurities in the di-BSA conjugate was performed using the Limulus amebocyte lysate ENDOCHROME ™ (Charles River Endosafe Div. of Charles River Laboratories, Inc., Charleston, US) test obtained from the Collective Usage Center of the Mechnikov Research Institute for Vaccine and Sera (Moscow, Russia), in accordance with the manufacturer's instructions.The di-BSA conjugate contained 0.08-0.11EU/mL of endotoxin (LAL-Center, Moscow, Russia).

Measurement of antibody response to the disaccharide conjugate
Antibody titers for CP in post-immunization sera were measured using ELISA.Briefly, plates coated with S. pneumoniae type 3 CP were incubated with antisera from 6 immunized mice (42).Wells were washed and secondary antibody was added, followed by incubation and washing.The results were then analyzed.Enzyme substrate aliquots (100 mL) were added, followed by incubation for 20 minutes at 22°C.The reactions were quenched with 1 M H 2 SO 4 .Optical densities (ODs) were determined using an iMark microplate absorbance reader (Bio-Rad, Osaka, Japan) at a wavelength of 450 nm.Antibody titers are expressed as the dilution of serum in which the antibody was detected.

Di-BSA-induced active protection in immunized mice
BALB/c mice were intraperitoneally immunized with the di-BSA conjugate adsorbed or non-adsorbed on aluminum hydroxide on days 0 and 14 (twenty animals per conjugate).The same animals were intraperitoneally challenged after 2 weeks with 10 5 colonyforming units of S. pneumoniae type 3/0.5 mL.Non-immunized control mice (twenty animals per conjugate) were also exposed to the bacteria.Mortality rates were determined at seven days post-infection.

Antibodies against doublestranded DNA
The analysis of antibodies against ds DNA in the immune sera of mice was conducted using ELISA.Salmon sperm DNA (Behringer GmbH, Germany), dissolved in a carbohydrate buffer solution at a concentration of 20 g/mL, was adsorbed onto the bottom of the wells.The plates were incubated for 2 hours at 37°C and then for additional 18 hours at 6°C.The serums were analyzed using dilutions of 1:10 to 1:1280.As secondary antibodies, secondary rabbit anti-mouse peroxidase conjugated IgG (Rockland Immunochemicals Inc., Pottstown, PA) was utilized (100 mL).After adding tetramethylbenzidine for 15 minutes, the reaction was terminated with 1 M sulfuric acid.Results were obtained utilizing a multi-channel automatic photometer (TiterTek Multiscan MC from Flow Laboratories, England), with excitation at 490nm.Serums from non-immunized mice, as well as mice immunized with either Prevnar-13 or BSA adjuvanted or nonadjuvanted with aluminum hydroxide, were used as controls.

Statistical analysis
Between-group comparisons were performed using Mann-Whitney rank sum tests for independent samples.Fisher exact tests were conducted to evaluate survival of mice after pneumococcal challenge.P values ≤0.05 were considered to indicate statistical significance.Statistical analyses were performed using the Statistical data analysis software system version 10 (StatSoft Inc., Tulsa, OK, USA).

Antibodies induced by the di-BSA conjugate
Although the di-BSA conjugate adjuvanted with aluminum hydroxide was found to be less immunogenic than adjuvanted triand tetra-BSA conjugates, it was still able to induce the production of opsonizing antibodies and was sufficient for the development of serotype 3-protective immunity in mice (42).
In this study, we explored the ability of the di-BSA conjugate to induce antibodies capable of binding to the CP of S. pneumoniae serotype 3 in ELISA after primary and booster immunization with and without the adjuvant (Figure 2).The di-BSA conjugate without adjuvant did not induce Ab production after the prime and boost immunizations and no difference was observed relative to the control.The glycoconjugate adjuvanted with aluminum hydroxide induced no Ab production after prime immunization; however, after booster injection, the level of Abs increased in seven days (21 d) and was significantly elevated up to 28 d (14 d after boost).Prevnar 13 (1.1 µg/ IgG1 antibody production induced by the adjuvanted and nonadjuvanted di-BSA conjugate.BALB/c mice (n = 6 per conjugate) were intraperitoneally injected with the di-BSA conjugate (20 µg/ dose of carbohydrate) adjuvanted and non-adjuvanted with aluminum hydroxide, on days 0 and 14.The IgG1 Ab titer in the blood of mice was determined on days 0 (before prime immunization), days 7, 14, 21, and 28 (7 and 14 days after booster immunization, respectively), by ELISA, using CP of S. pneumoniae serotype 3 as the well-coating antigen.Mice (n=6) injected with saline at the same time served as a control group.AL -aluminum hydroxide.The data are presented as mean ± standard deviation (M ± SD).The Mann-Whitney rank sum test was used to determine significance, *P < 0.05.
dose of carbohydrate of CP of S. pneumoniae serotype 3) induced IgG Ab production on day 14 after boost immunization (the time of the study) at a titer of 1:800 (data not shown).

Active protection upon challenge of mice immunized with the di-BSA conjugate
Mice immunized with the di-BSA conjugate and di-BSA conjugate adjuvanted with aluminum hydroxide were challenged with S. pneumoniae serotype 3 on day 28 (14 d after booster immunization).All control mice injected with saline and 18 out of 20 mice immunized with the non-adjuvanted di-BSA conjugate died on the second day after the challenge (Figure 3).
The non-adjuvanted di-BSA conjugate that failed to induce Ab production also did not elicit any protection against challenge with S. pneumoniae serotype 3.However, the same conjugate administered to mice with aluminum hydroxide induced protection against S. pneumoniae serotype 3. Thus, aluminum hydroxide is indispensable for inducing protective immunity to the disaccharide conjugate.Prevnar 13 (1.1 µg/dose of carbohydrate of CP of S. pneumoniae serotype 3) protected all mice (n = 6) from the challenge (42).

Cytokine production in mice
To evaluate cytokine production, mice were intraperitoneally injected with the di-BSA conjugate adjuvanted or non-adjuvanted with aluminum hydroxide at a single dose of 20 µg (carbohydrate content).Serum cytokine levels were determined before injection of the glycoconjugate (d 0) and on days 1, 7, 15, and 21 (1 and 7 days after boost immunization, respectively) (Figure 4).
After prime immunization, the non-adjuvanted di-BSA conjugate induced an increase in the levels of IL-1a, IL-1b, IL-6, IL-13, IL-17A, IL-21, IFNg, and TNFa compared with that in the control (0 d).After booster immunization with the conjugate, IL-5, IL-10, and IL-22 production was induced in addition to these cytokines.The concentration of IL-4 did not increase in any of the study periods.
After prime immunization, the di-BSA conjugate adjuvanted with aluminum hydroxide stimulated higher production of IL-1a, IL-1b, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, IL-22, IFNg, and TNFa compared with the conjugate without the adjuvant.After booster immunization, all cytokines were found to be produced at high levels.When the conjugate was administered with the adjuvant, a very high level of IL-17A production was noted at all time points.In contrast, when mice were immunized with the conjugate without the adjuvant, the IL-17A level gradually decreased even after booster immunization.Regardless of the presence of the adjuvant, the levels of IL-2 and IL-12p70 did not increase during all follow-up periods.Free CP of S. pneumoniae serotype 3 (5 µg/mouse) elevated only the level of IFNg (from 23.1 to 50.8 pg) after double immunization (data not shown).CP-CRM 197 (Prevnar 13) is able to induce the production of IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IFNg, and TNFa (47,48).Free aluminum hydroxide did not elicit cytokine production when administered at the same time points (data not shown).

Expression of cell-surface molecules on splenic mononuclear cells
After first immunization with the di-BSA conjugate adjuvanted and non-adjuvanted with aluminum hydroxide, the number of CD45 + /CD3 + T cells and CD45 + /CD4 + T helper cells increased compared with that in the control.After booster immunization, regardless of the presence of adjuvant, there was no difference relative to the control (Figure 5).
After primary and booster immunization with the adjuvanted di-BSA conjugate, the number of CD45 + /CD8 + cytotoxic T cells (CTLs) increased compared with that in the control.The nonadjuvanted conjugate did not induce any change in the number of CTLs during the entire observation period.An interesting result was revealed in relation to gd T cells.One day after prime immunization of mice with the adjuvanted and non-adjuvanted di-BSA conjugate, the number of gd T cells increased compared with that in the control and decreased to the initial levels on day 7.However, after booster immunization with the adjuvanted conjugate, the number of TCR + gd T cells increased on day 15 (1 d after boost), reaching high values on day 21 (7 d after boost).In contrast, in the absence of aluminum hydroxide, their values did not differ from the control level.After booster immunization with the di-BSA conjugate, the number of CD45 + /CD19 + B cells increased only following booster immunization in the presence of aluminum hydroxide.After injection of the non-adjuvanted conjugate, the level of CD45 + /CD19 + B cells did not differ from that in the control.The number of CD5 + B1 increased on day 1 after the first immunization with adjuvanted and non-adjuvanted conjugate compared with that in the control and then decreased on day 7. Booster immunization with the adjuvanted di-BSA conjugate led to an increase of number of CD5 + B1 cells on day 15 (1 d after boost) compared with that in the control, and on day The administration of the adjuvanted and non-adjuvanted conjugate increased the number of CD16 + /CD32 + natural killer cells (NK) and CD3 + /CD16 + /CD32 + natural killer T cells (NKT) after primary and booster immunization.The adjuvanted and non-adjuvanted di-BSA conjugate led to increase in the number of cells expressing CD25+ and the IL-2 receptor and CD4 + /CD25 + / Foxp3 + T regulatory cells (Treg).The number of cells expressing MHC II + increased only after booster immunization-to a greater extent on day 21 (7 d after boost)-and was higher than that in the case of conjugate administration without aluminum hydroxide.Prevnar 13, containing a CRM 197 -CP of S. pneumoniae serotype 3 conjugate, induced similar changes on day 28 (14 d after booster immunization) in the number of cells expressing cell-surface molecules.Specifically, there was an increased number of (TCR + ) gd T cells, CD45 + /CD8 + CTLs, CD5 + B1 cells, CD45 + /CD19 + B cells, CD4 + /CD25 + /Foxp3 + Tregs, cells expressing CD25 + , and cells expressing MHC II+.The number of NK-and NKT-cells did not differ from that in the control.
An elevation in Ab production and protection against S. pneumoniae serotype 3 was detected only after double immunization with the adjuvanted di-BSA conjugate.This finding suggests that the cells whose number showed a large increase after booster immunization (TCR + gdT cells and CD5 + B1 cells), against the background of an increase in the number of activated cells expressing MHC II + , play a crucial role in the protective activity of the conjugate.

Antibodies against double-stranded DNA
No difference was observed in the level of Abs against ds DNA relative to the control at the dilution of 1:80 in sera of mice immunized with the di-BSA conjugate adsorbed and nonadsorbed on aluminum hydroxide, Prevnar 13, BSA, and free aluminum hydroxide (Figure 6).

Discussion
In contrast to the conjugate without adjuvant, the di-BSA conjugate adjuvanted with aluminum hydroxide, induced production of IgG1 antibodies and protected mice against S. pneumoniae serotype 3 after prime-boost immunization.The role of adjuvants in enhancing the adaptive immune response to antigens, including semisynthetic glycoconjugates corresponds to the data of other authors (49)(50)(51).Cytokine production in mice induced by the adjuvanted and non-adjuvanted di-BSA conjugate.BALB/c mice were immunized with the di-BSA conjugate (20 mg of carbohydrate per mouse) adjuvanted or non-adjuvanted with aluminum hydroxide (n = 24 for each conjugate).Control mice (n = 6) were injected with saline 24 hours before the start of immunization (0 d). Serum was collected from mice (n=6 for each time point) after immunization.Cytokine levels were analyzed using flow cytometry.No increase in IL-2 or IL-12 p70 levels was observed in any of the time points (data not shown).The data is presented as the mean ± SD.Mann-Whitney rank sum tests were used to determine significant differences between control and other experimental groups; *P <0.05.
The concentrations of IL-1a, IL-1b, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, IL-22, IFNg, and TNFa in mice sera in response to the di-BSA conjugate adjuvanted with aluminum hydroxide were higher compared with those in response to the non-adjuvanted glycoconjugate.Free aluminum hydroxide is known to stimulate the production of IL-1b and IL-18, and, when administered with antigens, the spectrum of cytokines expands (52-58).IFNg, IL-17A, and IL-22 (a member of the Th17 cytokine family) plays a role in the early stages of controlling S. pneumoniae infections (59-67).IL-17 has an important function in protecting against bacterial carriage and lung infection 65, 68-71).The di-BSA conjugate adjuvanted with aluminum hydroxide induced a very high level of IL-17A after the prime and boost immunizations, while the conjugate without adjuvant caused a weak production of IL-17A that gradually decreased after the booster injection.A high level of Th2 cytokines (IL-4 and IL-5) was revealed in mice immunized with the adjuvanted di-BSA conjugate.Th2 cytokines promote switching from IgM to IgG, which is associated with high production of IgG1 antibodies (72).The conjugate without adjuvant did not elicit IL-4 production, only weakly stimulated the production of IL-5 even after boost immunization, and did not induce the antibody response.Prevnar 13 is known to induce the production of Th1/ IgG antibodies to double-stranded DNA in immunized mice, analyzed by ELISA.ds DNA was used as the well-coating antigen.Sera to each conjugate, BSA, aluminum hydroxide, and control (non-immunized mice) (n = 6 for each antigen) was added to each well in dilutions from 1:10 to 1:1280.AL -aluminum hydroxide; control -mice injected with saline.After prime-boost immunization, autoantibodies to ds DNA, which target the cell nuclei, were not detected.The number of splenocytes expressing membrane molecules in mice immunized with the di-BSA conjugate with and without adjuvant.BALB/c mice were immunized with the di-BSA conjugate (20 mg/dose of carbohydrate per mouse) adjuvanted or non-adjuvanted with aluminum hydroxide and with Prevnar 13 (1.1 mg/dose of carbohydrate of CP S. pneumoniae serotype 3 per mouse) adjuvanted with aluminum phosphate.Splenocytes were isolated from mice (n = 6 for each conjugate and each time point) on the indicated days after immunization.Control mice (n = 6) were injected with saline 24 hours before the start of immunization (0 d). Spleen cell suspensions were stained using antibodies against mouse CD3e-FITC (clone 145-2C11), CD4-FITC (clone GK1.5), CD8a-FITC (clone 53-6.Th2 and Th17 cytokines (47,48).In our previous studies, we have shown that Prevnar 13 induced anti-CP S. pneumoniae type 3 IgG1antibodies and protected immunized mice from the challenge with S. pneumoniae type 3 (42).
The di-BSA conjugate and CPs, including that of S. pneumoniae serotype 3, are not Toll-like receptor (TLR) ligands (46).Purified CP from S. pneumoniae can bind to macrophages through the carbohydrate-recognition domains on the mannose receptor, leading to the production of proinflammatory cytokines such as IL-1, IL-6, and TNFa, as well as chemokines (73).Another receptor, the C-type lectin, also known as carbohydrate-binding protein, SIGN-R1, is expressed by macrophages, particularly in the marginal zone of the mouse spleen.This receptor is able to bind carbohydrates from several different serotypes of S. pneumoniae (73).Other carbohydrate-recognition receptors of macrophages remain to be identified (74).It is likely that macrophages play a significant role in the initial stage of the immune response to the di-BSA conjugate (36,59,(74)(75)(76)(77).
Regardless of the presence of the adjuvant, the number of CD4 + T helper cells involved in the adaptive immune response to the antigen increased only after the first immunization with the di-BSA conjugate.The number of CD4 + T cells after booster immunization with the BSA-conjugated synthetic hexasaccharide related to S. pneumoniae serotype 14 CP adsorbed on aluminum hydroxide did not differ from that in the control either (46).However, the number of CD4 + T helper cells increased on day 14 after booster immunization in mice immunized with CP of S. pneumoniae serotype 3 conjugated to CRM 197 and adsorbed on aluminum phosphate (Prevnar 13).This result may be attributable to the multicomponent composition of the vaccine and the presence of a small amount of bacterial impurities remaining even after purification of CPs.The number of CD8 + cytotoxic cells (CTL) in response to the disaccharide conjugate and Prevnar 13 increased.
Both the adjuvanted di-BSA conjugate and Prevnar 13 significantly increased the number of (TCR + ) gd T cells among the splenocytes after booster immunization.gd T cells play a crucial role in prevention of pneumococcal infection owing to their ability to recognize unprocessed non-peptide antigens (41).A large number of gd T cell ligands remain unknown to date (78, 79).In mice, most gd T cells are found in the body's barrier tissues, with a small proportion in the blood and spleen (46, 80-83).The activation of gd T cells through TCRs can be mediated by non-classical MHC molecules (e.g., T10/T22 and members of the CD1 family) and MHC-unrelated molecules (e.g., viral glycoproteins and butyrophilin 3A1) (79,(84)(85)(86)(87). Putatively, gd T cells bind the oligosaccharide portion of the glycoconjugate without processing in antigen-presenting cells (APCs) in combination with MHC-like molecules activate cytokine production.gd T cells produce a large variety of cytokines and exhibit potent cytotoxic activity against pathogens through apoptosis-inducing receptors (FAS and TRAIL), as well as cytolytic proteins such as perforin and granzyme (88,89).Furthermore, gd T cells can function as professional APCs that require surface interactions with opsonized cells (90).The di-BSA conjugate has been shown to induce the formation of opsonizing antibodies (42).Certain subsets of gd T cells express CD4.These cells have a Th1 or Th2 phenotype and produce IL-2, IL-4, IL-17A, IFNg, and TNF (70).The di-BSA conjugate induced the production of IFNg and TNFa (Th1 cytokines); IL-4, IL-5, and IL-13 (Th2 cytokines); IL-17A (Th17 cytokines); IL-21 (Th2 and Th17 subsets); and IL-22 (Th1 and Th17 subsets).gd T cells play a crucial role in immune protection against extracellular respiratory bacteria (41, 91, 92).The potential role of gd T cells in pneumococcal infection has only been investigated in animal models using S. pneumoniae serotype 3 (41).During infection, the number of gd T cells can significantly increase, accounting for up to 50% of all peripheral lymphocytes (93,94).In the mouse model, gd T cells accumulate and become activated in the lungs during S. pneumoniae infection (95, 96).Mice with a lack of gd T cells exhibit a higher bacterial load in their lungs and lower survival rates compared to control mice (66,95,97).The absence of gd T cells is associated with impaired secretion of MIP-2, TNFa, and IL-17, as well as a poor recruitment of neutrophils (66,95,97).In addition, gd T cells produce IFNg during S. pneumoniae infections of serotypes 3 and 1.Along with their early role in defense against S. pneumoniae, gd T cells participate in the resolution stage of pneumococcal pneumonia, eliminating inflammatory mononuclear phagocytes (98).Therefore, gd T cells are essential for the host's defense against S. pneumoniae (66, 95).
The di-BSA conjugate adjuvanted with aluminum hydroxide and Prevnar 13 adsorbed on aluminum phosphate induced a significant increase CD5 + B1 cells after booster immunization.CD5 + B1 cells are mainly located in the peritoneal and pleural cavities, but very small amounts were also found in the spleen (99, 100).CD5 + В1 cells are activated primarily by T-independent antigens (101, 102) and play an important role in protecting against pneumococcal infections (103) This role may be attributed to their production of natural antibodies as well as possible participation in the T-dependent immune response (102,(104)(105)(106)(107)(108)(109).The B cell receptor (BCR) is involved in the phagocytosis of bacteria by B1 cells (110).CD5 + B1, isolated from the spleens of mice, primarily induce IL-17 production by T cells (111).B1 cells present antigen to antigen-specific T cells and induce more efficient proliferation than conventional CD19 + B cells (107, 108).After immunization with the di-BSA conjugate, the number of CD19 + B cells in the blood increased, regardless the presence of the adjuvant.The number of CD19 + B cells increased during all observation periods.Ovalbumin-presenting B1 cells were found to express a higher level of MHC class II compared to naïve B1 cells.
Immunization with either the adjuvanted or the nonadjuvanted di-BSA conjugate increases the number of natural killer (NK) cells and natural killer T (NKT) cells.NK cells, through the production of IFNg, participate in the early immune response to pulmonary S. pneumoniae infection.NKT cells have a key role in protecting against pneumococcal infection.When mice lacking NKT cells were infected with S. pneumoniae serotype 3, they exhibited a higher mortality rate and bacterial load in their lungs compared to wild-type mice.It has been suggested that IFNg derived from NKT cells has a critical function in protecting mice against pneumococcal pneumonia.Using S. pneumoniae serotype 1, it has also been found that NKT cells are an important innate immune effector in clearing pneumococci from the body.NKT cells can indirectly or directly assist B cells in mounting antibody responses and have a crucial role in the production of antibodies against pneumococcus and in the switch of classes in response to the administration of pneumococcal vaccines (112-114).
IL-17A, gd T, and CD5 + В1 cells can also contribute to autoimmune diseases (115).In response to infection or immunization, autoreactive clones of B1 cells can be produced in the body's own tissues (109,(116)(117)(118).The expansion of autoreactive clones of B cells is controlled by IL-10, leaving the BCR in a state of anergy.After booster immunization, there was an increase in the number of CD4 + /CD25 + /FoxP3 + T regulatory cells (Tregs) on the background of interleukin-10 (IL-10) production, which regulates the development of the immune response.After prime-boost immunization with the di-BSA conjugate or Prevnar 13, no formation of autoantibodies against ds DNA targeting cell nuclei was detected.

Conclusion
The key effectors of the immune response in mice following immunization with aluminum hydroxide adjuvanted di-BSA conjugate, associated with antibody response and protection from infection by S. pneumoniae serotype 3, were IL-17A, gd T, and CD5 + B1 cells, with an increase in the number of MHC II-expressing cells after booster immunization.The roles of non-conventional gd T cells, B1 cells, and production of IL-17A upon pneumococcal immunization with the semisynthetic glycoconjugate may provide an in-depth understanding of post-vaccination defense mechanisms, enabling the development of novel efficient therapies and improvement of existing vaccine formulations.

FIGURE 3
FIGURE 3Protective activity of the adjuvanted and non-adjuvanted di-BSA conjugate.BALB/c mice (n = 20 per conjugate and control group) intraperitoneally injected with the di-BSA conjugate (20 µg/dose of carbohydrate) adjuvanted and non-adjuvanted with aluminum hydroxide on days 0 and 14 were challenged with 10 5 colonyforming units of S. pneumoniae serotype 3 on day 28.Mice injected with saline were used as a control.AL -aluminum hydroxide.The results of two experiments are summarized.The difference between mice immunized with the adjuvanted di-BSA conjugate and nonadjuvanted/non-immunized mice (control) is shown.Fisher exact test; ***P < 0.001.