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 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 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 semi-synthetic 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.

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.

Quantitative determination of cytokines was performed as previously described (46). Male BALB/c mice (n=6) were sacrificed, and serum was collected and stored at –20°C until further quantification of cytokine levels. Using the Flow Cytomix Mouse Th1/Th2 10-plex test system, cytokine levels were measured by adding beads coated with monoclonal antibodies to IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-21 and IL-22, as well as IFNγ and TNFα, following the manufacturer’s instructions (eBioscience, San Diego, USA) using a Cytomix FC-500 flow cytometer (Beckman Coulter, Brea, USA).

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 μg, BSA ~200 μg; aluminum hydroxide, as an adjuvant, standardized for aluminum, was added in an amount of 250 μg. 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 μg of polysaccharides from serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, and 23F, as well as 4.4 μg of the polysaccharide from serotype 6B. The vaccine also contains 32 μg of the carrier protein, CRM₁₉₇, and 125 μg of aluminum as aluminum phosphate. Mice were immunized twice with a single dose of 1.1 μg of CP from S. pneumoniae type 3 per inoculation (equivalent to half of the recommended human dose). Control mice were injected with saline.

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 0.08–0.11 EU/mL of endotoxin (LAL-Center, Moscow, Russia).

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 μL) were added, followed by incubation for 20 minutes at 22°C. The reactions were quenched with 1 M H₂SO₄. 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.

Splenocytes were isolated from mice that had been vaccinated with the glyconjugate either in the absence of or in the presence of aluminum hydroxide, one and seven days after primary and booster immunizations. Single-cell suspensions of splenocytes were prepared by manually mashing the spleens using the plunger from a disposable syringe. The ground spleen was then passed through a nylon mesh and the cells were suspended in PBS. Splenic single-cell suspensions were then stained with antibodies conjugated to phycoerythrin (PE) or fluorescein isothiocyanate (FITC) to detect specific proteins in the cells: CD3e-FITC (clone 145–2C11), CD4-FITC (clone GK1.5), CD8a-FITC (clone 53–6.7), CD19-FITC (eBio1D3), CD5-PE (clone 53–7.3), NK.1.1 (clone PK136), CD3/CD16/CD32 (NKT), CD25-PE (PC61.5), CD4/CD25/Foxp3 (Treg), γδT (clone γδ TCR-PE, eBioGL3), and MHCII-PE (I-EK) (clone 14–4-45). Treg cells were stained with CD4-FITC (clone GK1.5), together with CD25-PE (PC61.5), and after fixation with the fixation/permeabilization buffer, with Foxp3- APC (clone FJK-16s). Splenocytes were incubated with 50 µL of appropriate monoclonal antibodies (eBioscience, US) at 4°C for 30 minutes. Erythrocytes were then lysed using red blood cell lysis buffer (BioLegend, US). After washing with phosphate-buffered saline (PBS), the samples were fixed using a fixation solution (BioLegend, US) and analyzed by flow cytometry (Cytomix FC-500, Beckman Coulter, USA, with the CXP software). The cell population gate was determined based on forward and side scatter and cell size. 10,000 cells were recorded per gate.

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 10⁵ colony-forming 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.

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 μL). 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 non-adjuvanted with aluminum hydroxide, were used as controls.

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).

Although the di-BSA conjugate adjuvanted with aluminum hydroxide was found to be less immunogenic than adjuvanted tri- and 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/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).

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).

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-1α, IL-1β, IL-6, IL-13, IL-17A, IL-21, IFNγ, and TNFα 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-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, IL-22, IFNγ, and TNFα 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 IFNγ (from 23.1 to 50.8 pg) after double immunization (data not shown). CP-CRM₁₉₇ (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, IFNγ, and TNFα (47, 48). Free aluminum hydroxide did not elicit cytokine production when administered at the same time points (data not shown).

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 non-adjuvanted conjugate did not induce any change in the number of CTLs during the entire observation period. An interesting result was revealed in relation to γδ T cells. One day after prime immunization of mice with the adjuvanted and non-adjuvanted di-BSA conjugate, the number of γδ 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⁺ γδ 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 21 (7 d after boost) relative to the non-adjuvanted conjugate. 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 CRM₁₉₇-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⁺) γδ 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⁺ γδT 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.

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 non-adsorbed on aluminum hydroxide, Prevnar 13, BSA, and free aluminum hydroxide ( Figure 6 ).