Semisynthetic Glycoconjugate Vaccines To Elicit T ... - ACS Publications

Article Cite This: ACS Infect. Dis. 2019, 5, 1423−1432

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Semisynthetic Glycoconjugate Vaccines To Elicit T Cell-Mediated Immune Responses and Protection against Streptococcus pneumoniae Serotype 3 Shaojie Feng,† Chenghe Xiong,† Subo Wang,† Zhongwu Guo,*,†,‡ and Guofeng Gu*,† †

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National Glycoengineering Research Center and Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, 72 Binhai Road, Qingdao, Shandong 266237, China ‡ Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida 32611, United States S Supporting Information *

ABSTRACT: Streptococcus pneumoniae serotype 3 (ST3) is one of the main pneumococcal strains that can cause severe invasive diseases, but its current vaccines are relatively inefficient. To develop more effective ST3 vaccines, tetanus toxoid (TT) conjugates of the synthetic penta-, hexa-, hepta-, and octasaccharide analogs of ST3 capsular polysaccharide (CPS) were systematically studied. These conjugates, especially those of penta- and hexasaccharides, were demonstrated to induce extremely robust T cell-dependent immune responses in mouse. Various studies also revealed that the induced antibodies could recognize ST3 CPS and mediate in vitro opsonophagocytic killing of ST3 cells. It was proved ultimately that immunization with the hexasaccharide−TT conjugate could completely protect mice from ST3-caused infection and lung damage and significantly elongate mouse survival. It was proposed that this conjugate functions through the help of CD4+ T cells and via promoting Th cell differentiation into carbohydrate antigen-specific Th2 cells to establish humoral immunity. In conclusion, ST3 CPS hexasaccharide−TT was identified as a particularly promising ST3 vaccine candidate worthy of further investigation and development. KEYWORDS: Streptococcus pneumoniae serotype 3, semisynthetic glycoconjugate, vaccine, antibacterial activity, immunoprotection, CD4+ T cell

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he Gram-positive Streptococcus pneumoniae is an encapsulated bacterium that accounts for the majority of upper and lower respiratory tract bacterial infections.1,2 Pneumococcal infections usually have high mortality and morbidity rates in infants and in elderly and immunocompromised people,3 because antibiotic treatments are less effective in these populations. Furthermore, drug resistance has been emerging nowadays as a serious issue. As a result, vaccination against bacterial infections including pneumococcal infections has become an attractive strategy. The conserved and exposed capsular polysaccharides (CPSs) on the S. pneumoniae cell surface have been identified as valuable antigens for the development of pneumococcal vaccines.4,5 For example, antipneumonia 23-valent polysaccharide vaccine PPV23 and glycoconjugate vaccines PCV7, PCV10, and PCV13 are all based on pneumococcal CPSs. © 2019 American Chemical Society

These successfully marketed vaccines have played a critical role in controlling pneumococcal infections.1 The recently licensed 13-valent conjugate vaccine PCV13 was developed to include the CPS of S. pneumoniae serotype 3 (ST3), which is the more invasive pneumococcal strain of all serotypes found in the clinic.6 In reality, ST3 causes the majority of lung abscesses7,8 as well as other invasive diseases, such as necrotizing pneumonia and acute media in adults.9 However, the protective effectiveness of PCV13 against this epidemiologically important strain is broadly rebated owing to its hyporesponsiveness and the elicitation of low IgG responses Received: March 14, 2019 Published: May 24, 2019 1423

DOI: 10.1021/acsinfecdis.9b00103 ACS Infect. Dis. 2019, 5, 1423−1432

ACS Infectious Diseases

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against ST3.10,11 Therefore, more efficacious vaccines are urgently needed for ST3. Semisynthetic glycoconjugate vaccines made of oligosaccharides have attracted great attention recently.12 In addition to the significantly improved safety profiles as compared to conjugate vaccines made of bacterial polysaccharides, vaccines derived from structurally well-defined oligosaccharide antigens can have a number of other advantages, such as better quality control, more consistent and reliable immunological properties, etc. Moreover, they would facilitate in-depth immunological and structure−activity relationship studies for further improvement of the vaccine design to provide better and more effective vaccines that may overcome current problems such as hyporesponsiveness. Structurally, ST3 CPS consists of a repeating disaccharide containing 1,3-β-linked D-glucuronic acid (GlcA) and 1,4-βlinked D-glucose (Glc), namely, [-3)-β-D-GlcA-(1 → 4)-β-DGlc-(1-] (Figure 1). To date, several semisynthetic glyco-

Figure 2. Structures of synthetic ST3 oligosaccharide−protein conjugates (A) via a glutaryl linker and (B) via a squarate linker.

TT protein were assessed by MALDI-TOF MS analysis (Figure S1). Immunological Evaluation of ST3 CPS Oligosaccharide−TT Conjugates 2a−d. Our preliminary studies have demonstrated that all these conjugates could induce robust anti-kappa and anti-IgG antibody responses.17 In this study, individual IgG antibody isotypes of the antisera of conjugates 2a−d were evaluated. Immunizations were carried out with female BALB/c mice (6−8 weeks old), and an emulsion of each conjugate with Freund’s adjuvant (Freund’s complete adjuvant, CFA, for initial immunization and incomplete adjuvant, IFA, for boosting immunizations) was subcutaneously (s.c.) injected into a group of five mice (3 μg of carbohydrate antigen per injection per mouse) on days 1, 15, 22, 29, and 36, respectively. Blood samples were collected from each mouse on day 0 before (as blank control) and day 43 after immunization. Antisera were prepared by clotting blood samples according to standard protocols and then analyzed by enzyme-linked immunosorbent assay (ELISA) to detect oligosaccharide-specific IgG1, IgG2a, IgG2b, and IgG3 antibodies using BSA conjugates 3a−d as the capture antigens. Antibody titers were calculated according to the described methods.17 ELISA results (Figure 3) showed that all of the conjugates elicited high titers of IgG1 antibodies but low titers of IgG2a, IgG2b, and IgG3 antibodies. The production of IgG1 antibodies indicated T cell-dependent immunity,18,19 and the result was consistent with literature reports concerning the immunological properties of carbohydrate−protein conjugates. More significantly, pentasaccharide conjugate 2a and hexasaccharide conjugate 2b induced much higher titers of IgG1 antibodies than heptasaccharide conjugate 2c and octasaccharide conjugate 2d (Figure 3A), which agreed well with observed total antibody levels induced by these conjugates.17 These results again indicated that 2a and 2b possessed stronger immunogenicity for elicitation of T cell-dependent immune responses and thus might be better antigens for vaccine development against ST3. Influence of Linker, Adjuvant, and Immunization Schedule on the Immunization Result. It has been reported that the linker, adjuvant, and immunization schedule could potentially affect the efficacies of semisynthetic glycoconjugate vaccines.13,20−22 As hexasaccharide conjugate 2b elicited the strongest IgG antibody responses, we selected it to probe the influence of these factors on the designed ST3 vaccines. Therefore, conjugates 2b and 4 having different

Figure 1. Structure of ST3 CPS.

conjugates made of synthetic oligosaccharide fragments of ST3 CPS have been investigated.13−17 They were shown to elicit robust ST3 CPS-specific antibody responses and provide immunoprotection against ST3.14,15 Furthermore, structureimmunogenicity relationship studies on ST3 CPS oligosaccharides revealed that their chain length had a significant impact on the immunological activities. For example, Seeberger and co-workers15 found that ST3 CPS tetrasaccharide exhibited better binding affinity than shorter analogs to ST3specific human monoclonal antibodies that showed protective activities. Our studies demonstrated that the tetanus toxoid (TT) conjugates of ST3 CPS oligosaccharides could induce robust antibody responses in mouse and that the ST3 CPS penta- and hexasaccharide conjugates induced much stronger immune responses than that of hepta- and octasaccharides.17 Our results indicated that ST3 CPS penta- and/or hexasaccharides might be the optimal oligosaccharide antigens for anti-ST3 vaccine development. In the present report, we performed detailed immunological studies of the TT conjugates 2a−d (Figure 2A) of the synthetic ST3 CPS penta-, hexa-, hepta-, and octasaccharides. We also evaluated in vitro the bactericidal activities mediated by the antisera of conjugates 2a−d as well as in vivo the activities of these conjugates as vaccines to protect mice against ST3 infection.

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RESULTS AND DISCUSSION Preparation of ST3 CPS Hexasaccharide−TT Conjugate 4. TT conjugates 2a−d and bovine serum albumin (BSA) conjugates 3a−d (Figure 2A) of the synthetic ST3 CPS penta-, hexa-, hepta-, and octasaccharides were prepared according to our previously reported procedures.17 TT conjugated 4 (Figure 2B) was prepared from conjugation of ST3 CPS hexasaccharide with TT via the squarate linker, which was used to evaluate the influence of linkers on immunogenicity of resultant glycoconjugates. The carbohydrate loading and the average number of glycans attached to 1424



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Figure 3. ELISA results of day 43 antisera of individual mice immunized through s.c. injection of conjugates 2a−d. The titers of the corresponding hapten-specific IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) antibodies are displayed. Each graphic symbol represents the titer value of an individual mouse, and the black bar shows the mean ± SEM value. NS: not statistically significant; *: significant difference (P < 0.05).

Figure 4. Influence of immunization schedule, adjuvant, and linker on the immunological activities of the synthetic glycoconjugates. (A) Revised schedule for mouse immunization via subcutaneous injection of conjugates 2b and 4 combined with different adjuvants for only two times. (B) ELISA results of total IgG antibody titers in each group detected with hexasaccharide−BSA conjugate 3b as the coating antigen. Each graphic symbol represents the titer of an individual mouse, and the black bars show the mean ± SEM value. NS: not statistically significant; *: statistically significant (P < 0.05).

glycoconjugate vaccine 2b. About the linker, as shown in Figure 4B, conjugate 2 elicited significantly higher titers of total IgG antibody (24 080.59 ± 6896.18) than conjugate 4 (3113.72 ± 427.14), disclosing the potential impact of linker on the immunogenicity of ST3 hexasaccharide conjugates. This result was consistent with a report that adipic acid-linked glycoconjugates were more immunogenic than the corresponding squarate-linked conjugates.23 In addition, our previous studies have also proved that the glutaryl group is a superior linker, which does not induce significant antibody responses or have a detrimental impact on the immunological activity of resultant glycoconjugates.24−26 According to the literature,23 the different abilities of conjugates 2b and 4 to elicit immune responses might be because of their differences in glycan distribution on the carrier protein and/or in the distance or linkage between the oligosaccharide and the protein carrier. Cross-Reactivity of Each Antiserum with All Other Oligosaccharide Haptens. The cross-reactions between day

linkers were used together with Freund’s adjuvant (FA) or Alum adjuvant (AL) to immunize mice on days 1 and 15, respectively (Figure 4A), by the aforementioned protocols. Mice were bled on day 0 and 22 before and after immunizations, and the blood samples were used to prepare antisera for ELISA. The results (Figure 4B) showed that the day 22 antisera obtained from mice immunized with 2b two times had similar total IgG antibody titers as the day 43 antisera obtained from mice immunized with 2b five times. This indicated the potentially high efficacy of 2b as an antibacterial vaccine because it could induce robust T celldependent immune responses after only two immunizations. Concerning the influence of adjuvant, although 2b in combination with FA induced slightly higher IgG antibody titers than with AL, this difference was not statistically significant (Figure 4B). Thus, AL, which is the adjuvant approved for clinical human use, was potent enough to promote a carbohydrate-specific immune response induced by 1425



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Figure 5. Cross-reactivity of (A) overall total antibodies and (B) total IgG antibodies in the pooled day 43 antisera (1/1000 dilution) of conjugates 2a−d with various oligosaccharide haptens in the form of BSA conjugates 3a−d that were used as coating antigens for ELISA. Each column represents the average OD value of three ELISA experiments, and the black bar shows the ± SEM value.

Figure 6. FCM assay of the binding between ST3 cells and pooled antisera (1/100 dilution) derived from mice immunized with conjugates 2a−d with normal (day 0) and PBS immunized mouse sera (PBS) as negative controls. The fluorescence intensity (A) and fold of fluorescence increase (B) as a result of immunizations were shown. *: significantly different (P < 0.05) from the negative controls; #: significantly different (P < 0.05) from all other groups.

effectively bind to it. In addition, the increase of fluorescence intensity for ST3 cells treated with antiserum 2b (by 9.424 ± 0.1836-fold) was significantly higher than that of cells treated with antiserum 2a (6.850 ± 0.2024-fold), both of which were in turn significantly higher than that of cells treated with antisera 2c and 2d (4.255 ± 0.1656 and 3.432 ± 0.0833-fold, respectively) (Figure 6B). The result was consistent with that of the antibody titer assays (Figure 3) and cross-reactivity analysis (Figure 5) described above. In conclusion, glycoconjugate 2b was proved to induce higher titers of antibodies that can more effectively recognize ST3 cells than the other three glycoconjugates 2a, 2c, and 2d. Thus, it was identified as the more promising anti-ST3 conjugate vaccine candidate. In Vitro Assays of the Protective Effect of Antibodies Elicited by Conjugates 2a−d. To probe if the aboveobserved antibodies or immune responses induced by conjugates 2a−d and antibody binding to ST3 cells could be directly translated into effective protection against ST3 and related infections, we examined in vitro the bactericidal activity of these antibodies by the opsonophagocytic killing assay (OPA), which is a commonly used method to study the protective activities of antibodies against S. pneumoniae.27,28 In these studies, prefixed ST3 cells were incubated with different dilutions of the day 43 antisera of 2a−d, followed by addition of rabbit complements and neutrophil-like cells that differentiated from HL-60 cells. Thereafter, these ST3 cells were subjected to colony-forming unit (CFU) assays, namely, to plate the cell cultures and then count the bacterial colonies formed. The opsonization index (OI) of each serum, representing the serum dilution number to exhibit 50% killing of target cells as compared to the negative controls, was assessed. Clearly, the antisera of conjugates 2a, 2b, 2c, and 2d mediated significant killing of the bacterial cells with OIs of

43 antisera of TT conjugates 2a−d and all of the synthetic ST3 oligosaccharides were examined for oligosaccharide-specific total (anti-kappa) antibodies and total IgG antibodies by ELISA with BSA conjugates 3a−d as the capture antigens, respectively. The results were expressed as optical density (OD) values after subtracting the blank OD values. As shown in Figure 5, each antiserum could recognize all synthetic oligosaccharide antigens almost indifferently. On the other hand, the antisera of 2a and 2b had stronger reactions with all oligosaccharides than the antisera of 2c and 2d, which was in accordance with the observed high titers of anti-kappa and IgG antibodies in the antisera of 2a and 2b. These results suggested the majority of antibodies elicited by conjugates 2a−d could recognize a common oligosaccharide motif, mostly probably a certain length of the ST3 CPS repeating unit. Consequently, we hypothesized that these antibodies and the immune responses elicited by 2a−d should be able to recognize CPS and that 2a and 2b should be able to elicit more potent antibacterial activity than 2c and 2d. Antiserum Binding to ST3 Bacterium. To verify that the antibodies or immune responses elicited by conjugates 2a−d would indeed recognize the target CPS molecules on ST3 cells, their binding with 2a−d antisera was investigated by flow cytometry (FCM). In this regard, prefixed ST3 (ATCC 6303) cells were incubated with each pooled antiserum obtained on day 0 (as negative control) and day 43. Thereafter, fluorescein isothiocyanate (FITC)-labeled goat antimouse IgG antibody was added to the cells, which was followed by FCM analysis. The FCM results (Figures 6A and S2) revealed a statistically significant increase in fluorescent intensity of ST3 cells treated with day 43 antisera as compared to that of cells treated with day 0 sera. This demonstrated that antibodies in the antisera of conjugates 2a−d could recognize the CPS on ST3 cells and 1426



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9079 ± 396, 14 226 ± 1088, 3488 ± 327.6, and 273.3 ± 6.7, respectively (Figure 7, Tables S1 and S2). According to the

were specific to ST3 cells, as they did not induce obvious phagocytosis to S. pneumoniae serotypes 1, 5, and 19A (Table S3). The results also proved that antibodies elicited by the oligosaccharides in conjugates 2a−d could effectively recognize the CPS epitopes on ST3 cells. Furthermore, the OI of 2b was significantly higher than those of other glycoconjugates, which was consistent with the results of antibody analyses by ELISA and further demonstrated that ST3 CPS hexasaccharide might the optimal epitope for vaccine development. In Vivo Assays of the Protective Effect of Conjugates 2b against ST3 Infection. Encouraged by the potent opsonophagocytic activities of antiserum 2b, next, we evaluated its in vivo efficacies to protect against ST3 using a well-established mouse model.14 Considering that FA is not suitable for clinical use, AL was used in this study. Thus, BALB/c mice were immunized with AL emulsions of 2b (containing 3 μg of hexasaccharide per dose), TT + free hexasaccharide, and PBS, respectively, twice on days 1 and 15. All three groups of mice (6 or 10/group) were then inoculated with ST3 (1 × 105 CFU) on day 29 via intraperitoneal (i.p.) injection. Twenty-four hours later, the bacterial burdens in mice were quantified to assess the protective efficacy of 2b

Figure 7. Opsonophagocytic activities of antibodies induced by conjugates 2a−d toward ST3 cells as measured by OPA. Mean values and SDs from six independent experiments using pooled sera are given. **: very significant difference (P < 0.01) to other groups.

literature,10,29 antiserum with an OI ≥ 1:8 is considered efficient. These results indicated that conjugates 2a−d elicited highly efficient opsonophagocytic antibodies. More significantly, the opsonophagocytic activities of the 2a−d antisera

Figure 8. Glycoconjugate 2b-induced protection of mice from ST3 infection. Intraperitoneal (A) and blood (B) bacterial burdens in ST3 challenged mice (n = 6) as assessed 24 h after the challenge. (C) Survival time of mice (n = 10) challenged with ST3. (D) Lung histopathology of normal mice and an ST3 challenged mouse with PBS, TT + hexasaccharide (HexaS), and 2b immunization (red arrows show inflammation sites). NS: not significantly different statistically; **: very significantly different statistically (P < 0.01). 1427



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Figure 9. Conjugate 2b-induced long-term immunological memory. Twenty weeks after mice were immunized twice with an (A) FA or (B) AL emulsion of 2b, UV-inactivated ST3 cells were used to challenge the mice, and the elicited hexasaccharide-specific IgG antibody levels in the mouse sera were evaluated by ELISA with BSA conjugate 3b as the coating antigen. ELISA results of antibody levels before (day 0) and after (days 1 and 3) the ST3 cell challenge are presented. Each dot represents the titer of an individual mouse, and black bars show the mean ± SEM values.

Figure 10. FCM analysis of in vitro activation of CD4+ T cells derived from mice immunized with conjugate 2b. FCM profiles of (A) CD69 and (B) IL-4 and IFN-γ expression by CD4+ T cells upon stimulation with PBS, BSA + free hexasaccharide (HexaS), and BSA conjugate 3b, respectively.

immunization. It was revealed that both the blood (Figure 8A) and the intraperitoneal (Figure 8B) bacterial loads in 2btreated mice were significantly lower than that in TT + free hexasaccharide- or PBS-treated mice, suggesting that the immune responses elicited by 2b could greatly reduce ST3 colonization of intraperitoneal bacteria and also prevent bacteria transmigration into the bloodstream. On the other hand, the mixture of TT and free hexasaccharide did not show any protective activity. We have also investigated the survival of each group of mice challenged with ST3. As shown in Figure 8C, after i.p. injection of ST3, all of the mice immunized with 2b survived in the entire experimental period (2 weeks after ST3 inoculation), whereas all of the mice in the PBS and TT + free hexasaccharide groups died within 2 days after bacterial cell challenge. These results have ultimately proved that hexasaccharide−TT conjugate 2b induced highly effective immunoprotective responses in mice against lethal ST3. Furthermore, we have examined the lungs of ST3-infected mice to assess the protective effects of this synthetic vaccine. Thus, after the mice were inoculated with 2b, TT + free

hexasaccharide, and PBS two times as described above, they were challenged by nasal drip of 1 × 105 CFU ST3 on day 29. Mouse lungs were acquired 30 h after the ST3 challenge and then stained with hematoxylin-eosin. As depicted in Figure 8D, the lungs from mice immunized with 2b were rather similar to normal mouse lungs and showed a significant decrease in inflammation as compared to two other control groups. These results suggested that immunizing mice with 2b could significantly reduce the lung injuries caused by ST3 infection. Long-Term Immunological Memory Induced by Conjugate Vaccine 2b. To evaluate whether 2b could elicit immunological memory against ST3, we immunized 6 groups of mice with FA and AL emulsions of 2b, TT + free hexasaccharide, and PBS, respectively, two times on days 1 and 15, as described above. Twenty weeks after boosting immunization, ultraviolet-inactivated ST3 cells (about 1 × 107 CFU) were i.p. injected to the mice. Sera were prepared from these mice before and after ST3 challenge and eventually subjected to ELISA analysis. As shown in Figure 9, all mice immunized with 2b in combination with either adjuvant responded to the ST3 challenge swiftly to produce high levels 1428



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linker, which was consistent with literature results.23 Moreover, the antisera of 2a−d exhibited extensive cross-reactivities with all synthetic oligosaccharides, suggesting that the carbohydratespecific antibodies elicited by 2a−d recognized a common core motif, which is an interesting finding worthy of further investigation. Accordingly, it was anticipated that these antibodies should be able to recognize ST3 CPS on bacterial cells, which was eventually proved by the strong binding of the antisera with the ST3 cells. More importantly, OPA results disclosed that all of the antisera obtained with 2a−d mediated strong bactericidal activities in vitro, indicating their potential protective function against ST3 infection. Both the immunological results and the binding assay and in vitro OPA results further showed that penta- and hexasaccharide conjugates 2a and 2b were significantly more potent vaccines than hepta- and octasaccharide conjugates 2c and 2d to induce very robust antibody responses that were highly effective to mediate opsonophagocytic killing of ST3 cells. These results suggested that ST3 CPS penta- and hexasaccharides might be the optimal ST3 oligosaccharide antigens for vaccine development, particularly for its application in current advanced vaccine technologies, such as the multiple antigenpresenting system (MAPS)36 and cell-free expression system (Xpress CF Platform, SutroVax).37,38 Furthermore, in most assays, hexasaccharide conjugate 2b exhibited better properties and activities than pentasaccharide conjugate 2a. Therefore, 2b was identified as the most promising ST3 vaccine candidate and was then subjected to in vivo evaluations using an established mouse model. It was ultimately proved that immunization with 2b could effectively protect mice from ST3-induced lung damage and infection and elongate animal survival. In-depth analysis of the induced immune responses demonstrated that immunizing mice with conjugate 2b elicited long-term immunological memory. Therefore, 2b should be useful for long-term protection. In vitro studies further revealed that a significant portion of primary CD4+ T cells isolated from 2b-inoculated mice could be activated by ST3 CPS hexasaccharide and be stimulated to express IL-4, indicating their differentiation into Th2 cells. Th2 cells provided T cell help to B cells to evoke strong antibody responses39 and humoral immune responses responsible for the eradication of extracellular pathogens.35 According to the literature,40−42 a hexasaccharide hapten might be the desired fragment size for B cell receptor (BCR) recognition to facilitate B cell activation. Eventually, it is proposed that glycoconjugate 2b might mainly induce T cell-dependent humoral immune responses for the protection of mice against ST3 infection.

of IgG antibodies. On the other hand, the two control groups immunized with TT + free hexasaccharide and PBS, respectively, did not respond to the ST3 challenge or produce significant levels of ST3 CPS-specific antibodies (Figure S3). The results indicated that conjugate 2b had the ability to induce long-term immunological memory against ST3. In Vitro Activation of Primed CD4+ T Cells by Targeted Oligosaccharide Antigen. To gain insights into the functional mechanisms of the above glycoconjugates, we probed the response of CD4+ T cells primed under various conditions toward the corresponding carbohydrate antigen. Thus, 4 weeks after two s.c. injections of FA emulsions of 2b, TT + free hexasaccharide, or PBS, lymphocytes were isolated from the immunized mice and treated in vitro with PBS, BSA + hexasaccharide, and BSA conjugate 3b, respectively, for 16 h. Then, FCM was used to detect CD69 molecules expressed on the treated T cells, as CD69 is usually the marker of early T cell activation.30,31 We found that a significantly higher percentage (25.1%) of CD4+ T cells derived from mice immunized with 2b was stimulated by conjugate 3b to express CD69 (Figure 10A) as compared to that by PBS (9.5%) or BSA + hexasaccharide (15.8%). On the other hand, CD4+ T cells derived from mice immunized with TT + hexasaccharide or PBS did not exhibit any change upon treatments with PBS, BSA + hexasaccharide, and conjugate 3b (Figures S4 and S5). Since the carrier protein (TT) in conjugate 2b used for immunization was different from that (BSA) in conjugate 3b used for in vitro stimulation, the activation of CD4+ T cells must be due to the carbohydrate hapten. These results suggested that 2b and the related glycoconjugate might have elicited T cell-dependent immune responses through CD4+ T cell help, which is consistent with literature observations.32,33 We had also analyzed in vitro the cytokine profiles expressed by CD4+ T cells upon stimulation with PBS, BSA + hexasaccharide, and BSA conjugate 3b. Specifically, we probed IFN-γ and IL-4 as they are signature cytokines produced by Th1 and Th2 cells, respectively.34,35 We found that a significantly higher percentage (3.6%) of CD4+ T cells derived from mice immunized with 2b was stimulated by 3b to express IL-4 (Figure 10B) than by PBS (1.1%) or BSA + hexasaccharide (0.8%). On the other hand, the population of IFN-γ expressing CD4+ T cells was not significantly different under the three different conditions. Moreover, the populations of both IL-4 and IFN-γ expressing CD4+ T cells derived from mice immunized with TT + hexasaccharide or PBS were not affected by different stimulants either (Figures S4 and S5). IL-4 secretion by CD4+ T cells upon stimulation with 3b suggested that glycoconjugate 2b promoted Th cell differentiation and polarization to Th2 cells to help evoke strong B cell-based antibody responses and humoral immunity.

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EXPERIMENTAL SECTION Immunization of Mice. Female BALB/c mice were employed in these studies. For immunological evaluations of 2a−d, each conjugate (containing 30 μg of oligosaccharide) or the corresponding free hexasaccharide (30 μg) plus TT (∼830 μg) was dissolved in 0.5 mL of 2× PBS buffer, which was then thoroughly mixed with 0.5 mL of CFA (Sigma F5881)/IFA (Sigma F5506) or Alum adjuvant (Thermo 77161) according to the manufacturer’s instructions to generate an emulsion. Each group of five or six mice was initially immunized (day 1) by s.c. injection of 0.1 mL of the CFA or Alum emulsion (containing ca. 3 μg of carbohydrate antigen per dose). Thereafter, mice were boosted four times on days 15, 22, 29, and 36 via s.c. injection of the IFA or Alum emulsion (0.1 mL)

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CONCLUSION We had performed a systematic study on ST3 oligosaccharide−TT conjugates 2a−d as potential ST3 vaccines. The immunological results of this study together with our previous preliminary discovery17 revealed that conjugates 2a−d elicited strong carbohydrate-specific T cell-dependent immunity,18,19 which is desired for prophylactic vaccines. It was also demonstrated that immunizing mice 2 or 5 times with conjugate 2b and in combination with either FA or AL gave similar immunological results. However, conjugates with different linkers showed slightly different activities, and 2b with the glutaryl linker was better than 4 with the squarate 1429



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and the plates were incubated overnight at 37 °C in anaerobic conditions, which was followed by bacterial colony count. Challenging Mice with ST3. After mice were immunized with an Alum emulsion of conjugate 2b, free hexasaccharide plus TT, or PBS twice on days 1 and 15 as described above, 100 μL of ST3 (ca. 1 × 105 CFU) was intraperitoneally injected (for intraperitoneal and blood bacterial infections) or applied via nasal drip (for lung histopathology injury) on day 29. Bacterial elimination, bacteremia, and pulmonary histopathology injuries were monitored 24 or 30 h after the challenge. Animal survival was monitored and recorded until 14 days after the challenge. Analysis of CD4+ T Cell Activation and Cytokine Production. After mice were immunized with FA emulsion of 2b, free hexasaccharide plus TT, or PBS through s.c. injection on days 1 and 15 as described above, lymphocytes were isolated from spleens of the immunized mice using Mouse 1× Lymphocyte Separation Medium (DAKEWE 7211011) on day 43. These lymphocytes were treated in vitro with 3b, free hexasaccharide plus BSA, or PBS, harvested and washed by PBS 16 h later, and then incubated with FITC antimouse CD3, PerCP/Cy5.5 antimouse CD4, and PE antimouse CD69 (BioLegend 100203, 100433, and 104507) for 20 min on ice. Finally, the treated lymphocytes were subjected to flow cytometric analysis. For cytokine detection, the lymphocytes were treated in vitro first with 3b, free hexasaccharide plus BSA, or PBS for 20 h and then together with Brefeldin A Solution (1000×) for 6 h. The lymphocytes were harvested and washed by PBS and fixed with Fixation Buffer (BioLegend 420801) and permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend 421002). They were stained with FITC antimouse CD3, PerCP/Cy5.5 antimouse CD4, and APC antimouse IL-4 or PE antimouse IFN-γ (BioLegend 100203, 100433, 504105, and 505807) for 20 min in the dark and finally subjected to flow cytometric analysis.

of the same vaccine. Blood samples were collected via the tail vein of each mouse on day 0 before and on day 43 after the immunization and were clotted to obtain antisera by the standard protocols. The sera were stored at −80 °C before use. For other studies, the same immunization protocols were applied, but only two boot immunizations were carried out. The animal protocols involved in this research were performed in strict accordance with the National Institute for Health Guide for the Care and Use of Laboratory Animals (National Research Council, 8th ed., National Academies Press (US): Washington, DC., 2011) and approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong University. ELISA. ELISA plates were treated with a solution of each BSA conjugate 3a−d (100 μL/well, 2 μg/mL) dissolved in coating buffer (0.1 M aq bicarbonate, pH 9.6) at 4 °C overnight and then at 37 °C for 1 h. This was followed by washing with PBS buffer containing 0.05% Tween-20 (PBST) three times. Then, the plates were incubated with blocking buffer (1% BSA in PBST) at rt for 1 h followed by washing with PBST three times. Each mouse serum with serial dilutions from 1:300 to 1:218 700 in PBS (100 μL/well) was added to the coated plates, which were incubated at 37 °C for 2 h. After being washed with PBST three times, the plates were incubated at rt for 1 h with a 1:1000 diluted solution of alkaline phosphatase-linked goat antimouse IgG, IgG1, IgG2a, IgG2b, or IgG3 (Abcam 98710, 98690, 98695, 98700, and 98705) antibody. The plates were washed three times and developed with a p-nitrophenyl phosphate (PNPP) solution (1.67 mg/mL in buffer, 100 μL) for 30 min at rt, which was followed by a colorimetric readout at 405 nm wavelength using a microplate reader. After deducting the background optical density (OD) values obtained with day 0 sera, the OD values were plotted against serum dilution values, and a best-fit line was obtained. The equation of the line was employed to calculate the dilution value at which an OD value of 0.1 was achieved, and the antibody titer was calculated at the inverse of the dilution value. Protocols for FCM Assays. ST3 (ATCC 6303) was cultured in THYB Medium (Todd Hewitt Broth and 0.5% Yeast extract) and harvested. Cells (1.0 × 106 CFU) were washed twice and resuspended in 100 μL of PBS buffer and then incubated with 1 μL of day 0 or day 43 pooled antiserum at 4 °C for 30 min. Thereafter, the cells were washed with PBS buffer and incubated with FITC-linked goat antimouse IgG antibody (Biolegend 406001, 1 μL in 100 μL of PBS buffer) at 4 °C for 30 min. Finally, cells were washed and suspended in 0.5 mL of PBS buffer for FCM analysis using an ACEA NovoCyte instrument. OPA. OPAs were carried out according to the UAB-MOPA protocol of Nahm and Burton.43 In brief, 10 μL of ST3 (∼800 CFU per well) was incubated with mouse sera (20 μL, serially diluted for a titer range of 1:4 to 1:8748) in duplicate wells in a 96-well round-bottom plate for 30 min with shaking at room temperature in Opsonization Buffer B (1× Hank’s buffer with Ca2+ and Mg2+, 5% defined FBS, 1% Gelatin). Baby rabbit complement was added to HL60 cells (∼1.6 × 105 cell per well) at 20% final volume, and 50 μL of HL60/complement mixture was added to each well. The mixtures were incubated at 37 °C and 5% CO2 for 45 min with shaking. The reactions were stopped by incubating the plate(s) on ice for ∼20 min. Finally, 10 μL of each mixture was plated onto THYA plates,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.9b00103. Synthetic procedure and MALDI-TOF mass spectrum for ST3 CSP hexasaccharide−TT conjugate 4, ELISA results of TT + hexasaccharide and PBS-induced longterm immunological memory, FCM profiles of antiserum binding to ST3 bacterium, in vitro activation of CD4+ T cells derived from mice immunized with PBS or TT + hexasaccharide, and OPA data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86 (532) 5863 1408. E-mail: [email protected]. *Tel: +1 (352) 392 9133. E-mail: [email protected]fl.edu. ORCID

Zhongwu Guo: 0000-0001-5302-6456 Guofeng Gu: 0000-0002-0969-7487 Notes

The authors declare no competing financial interest. 1430



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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 21877074 and 21672129) and the Science and Technology Development Project of Shandong Province (No. 2016GGH4502). We thank Dr. Yuchuan Guo for his kind support in hematoxylin-eosin staining for the lung slices experiment.

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