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Vaccinology Gets Help from Chemistry
Please cite this article as: Adamo, Vaccinology Gets Help from Chemistry, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.003
Cell Chemical Biology
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Vaccinology Gets Help from Chemistry Roberto Adamo1,* 1GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2016.09.003
A recent report on the immunological activity of protein conjugates of synthetic lipoteicoic fragments from Clostridium difficile underpins the use of these molecules for the development of a vaccine. In a recent issue of Cell Chemical Biology, Broecker et al. (2016) illustrate the utility of glycoarray-based selection of bacterial carbohydrates with the potential to become vaccine candidates. Clostridium difficile is a Gram-positive anaerobic bacterium commonly found in the environment that infects both humans and animals. Its intrinsic capacity to survive in a vegetative state by forming spores under harsh conditions, including high temperatures, ultraviolent light, and alcohol, makes C. difficile a seriously dangerous pathogen. In the human host, C. difficile germination, the process of switching from the dormant to live vegetative state, takes place in response to chemical factors present in intestinal epithelial cells. In this context, use of wide-spectrum antibiotics that alter the normal commensal gut flora has an impact on the diffusion of C. difficile infection and has been linked to infection flair up. The incidence and severity of C. difficile infection (CDI) appears to have increased over the last two decades, particularly in the US, Canada, and Europe, where it has been associated with higher casefatality rates, and the emergence of the hyper-virulent strain NAP1/027/BI, which is linked to some of the most deadly outbreaks, has been attributed to overuse and misuse of antibiotics, leading to the emergence of severe antibiotic resistance. Two large enterotoxins, TcdA and TcdB, both able to severely damage the intestinal mucosa, have been identified as the major cause of C. difficile disease (Leuzzi et al., 2014). A third toxin called CDT, or binary toxin, composed of the ADP-ribosyltransferase subunit CDTa and the binding subunit CDTb, is also known, but the role of CDT in human disease is still unclear. Vaccination with detoxified toxins or non-toxic peptide fragments has been pursued as a way to fight CDI (Leuzzi et al., 2014); however, a vaccine is not yet available.
The structures of surface carbohydrates from C. difficile, PSI, PSII and PSIII (Figure 1) have been elucidated recently, which has led to increased interest in using these sugars as targets for vaccine discovery (Ganeshapillai et al., 2008; Reid et al., 2012). It is well known that polysaccharides coating the surface of bacterial pathogens are weak immunogens, because they are T cell-independent antigens (Pace, 2013); however, conjugation to a carrier protein turns them into molecules able to evoke a T cell memory characterized by robust levels of anti-carbohydrate antibodies, which can be further boosted by subsequent doses of the vaccine. Efficacious vaccines against Streptococcus pneumonia, Haemophilus influenzae type b, and Neisseria meningitidis are now available on the market and they all exploit this strategy. In most cases, carbohydrates used for developing these vaccines are obtained through the process of bacterial fermentation. However, in the case of carbohydrate-based vaccines against C. difficile, the focus has been on synthetic glycans, a strategy that minimizes the likelihood of possible bacterial contaminants and offers access to well-defined carbohydrate structures. Scaling this approach for industrial production purposes would also avoid fermentation of pathogens. Among these C. difficile carbohydrates, first efforts focused on investigating PSII immunogenicity, as PSII was found to be abundant in the hypervirulent strain NAP1/027 and other clinical isolates (Danieli et al., 2011; Oberli et al., 2011). Using glycoarray analysis, antiPSII IgA antibodies (a class of antibodies present in mucosal secretion) have been identified in the stool samples of hospital patients (Oberli et al., 2011). Phosphory-
lated PSII fragments are immunogenic after conjugation to carrier protein, and specific antibodies bound to the bacterial surface (Adamo et al., 2012). Higher levels of IgA antibodies against PSI were found in CDI patients with less severe disease compared to asymptomatic controls, indicating that conjugates able to raise this antibody production could aid the development of an anti-C. difficile vaccine (Martin et al., 2013a). PSIII is a lipoteicoic (LTA)-like polymer, which is typically present in Gram-positive bacteria underneath the capsular polysaccharide. It is not clear yet whether PSI and PSII are capsules and what is their role on the bacterial surface. Anti-LTA PSIII antibodies have been detected in the blood of infected patients (Martin et al., 2013b), and immunizations with either intact or de-O-acylated LTA fraction of PSIII conjugates were shown to elicit IgG antibodies that clearly recognized PSIII on C. difficile cellular surface (Cox et al., 2013). The recent publication from Seeberger’s group (Broecker et al., 2016) reports significant advancements in the field. As opposed to what has been observed for PSI and PSII, IgA antibodies in fecal samples from CDI patients did not recognize synthetic LTA fragments on glycoarray. However, serum anti-LTA IgG were found, possibly due to the preexposure of the hospitalized individuals. This indicated that the synthetic LTA epitopes resembled natural epitopes. To test whether the selected glycan epitopes can efficiently produce antibodies, the largest synthesized LTA fragment was conjugated to CRM197, a genetically detoxified form of the diphtheria toxin, which is a carrier protein used in commercial vaccines. The glycoconjugate was administered with Freund’s adjuvant
Cell Chemical Biology 23, September 22, 2016 ª 2016 Published by Elsevier Ltd. 1047
Please cite this article as: Adamo, Vaccinology Gets Help from Chemistry, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.003
Cell Chemical Biology
Previews
screen biological samples form patients and identify potential candidates. The selected synthetic structures are then tested in vivo using animal models. It remains to be understood whether the LTA epitope identified by Seeberger and colleagues is the optimal glycan as is, or whether longer structures would be even more immunogenic. These followup questions notwithstanding, the article undoubtedly represents an important milestone in the preclinical development of a carbohydrate-based vaccine against C. difficile. R.A. is a GlaxoSmithKline employee and owner of a patent on anti-C. difficile vaccines. REFERENCES
Figure 1. Chemical Structures of C. difficile Surface Carbohydrates Recent work by Broecker et al. (2016) elucidates the potential of PSIII for vaccine development.
(FA), alum, or without adjuvant. LTA-specific antibodies were elicited after two boosts in mice, and the quality of the antibody response was dependent on the adjuvant used. IgG1 and IgG2a levels were highest when FA was co-administered, whereas alum promoted highest levels of IgG3. As alum is known to induce primarily anti-carbohydrate IgG1, the authors carefully ascertained that the glycoconjugate was fully adsorbed onto the inorganic salt and that it was stable. This indicated that the adjuvant can vary the repertoire of raised antibody. By comparing the IgG levels induced with no adjuvant and in the presence of FA, the authors concluded that antibodies were primarily directed to the largest synthetic structures, rather than to portions of it. The antibodies elicited against the largest fragment in the absence of adjuvant or with alum co-administration significantly bound to the surface of a series of
C. difficile strains. Most importantly, the alum-adjuvanted CRM197 conjugate reduced the bacterial colonization in mice that were rendered susceptible to infection with clindamycin and orally challenged with live C. difficile cells. There are two main advancements achieved by the work of Broecker et al. (2016). First, the authors put the antiC. difficile glycoconjugates to the test and provide formal evidence for functional activity of antibodies induced against surface glycan, which was missing from previous studies. Moreover, until this work, carbohydrate-based vaccine candidates have been selected by purifying polysaccharides and then conjugating them or portions of them to proteins and testing the glycoconjugates in mice to assess immunogenicity. The strategy employed by Broecker et al., which could be defined as a reverse glycoarray-based approach, starts from synthetic glycans that are applied onto a microarray to
1048 Cell Chemical Biology 23, September 22, 2016
Adamo, R., Romano, M.R., Berti, F., Leuzzi, R., Tontini, M., Danieli, E., Cappelletti, E., Cakici, O.S., Swennen, E., Pinto, V., et al. (2012). ACS Chem. Biol. 7, 1420–1428. Broecker, F., Martin, C.E., Wegner, E., Mattner, J., Baek, J.Y., Pereira, C.L., Anish, C., and Seeberger, P.H. (2016). Cell Chem. Biol. 23, 1014–1022. Cox, A.D., St Michael, F., Aubry, A., Cairns, C.M., Strong, P.C., Hayes, A.C., and Logan, S.M. (2013). Glycoconj. J. 30, 843–855. Danieli, E., Lay, L., Proietti, D., Berti, F., Costantino, P., and Adamo, R. (2011). Org. Lett. 13, 378–381. Ganeshapillai, J., Vinogradov, E., Rousseau, J., Weese, J.S., and Monteiro, M.A. (2008). Carbohydr. Res. 343, 703–710. Leuzzi, R., Adamo, R., and Scarselli, M. (2014). Hum. Vaccin. Immunother. 10, 1466–1477. Martin, C.E., Broecker, F., Oberli, M.A., Komor, J., Mattner, J., Anish, C., and Seeberger, P.H. (2013a). J. Am. Chem. Soc. 135, 9713–9722. Martin, C.E., Broecker, F., Eller, S., Oberli, M.A., Anish, C., Pereira, C.L., and Seeberger, P.H. (2013b). Chem. Commun. (Camb.) 49, 7159–7161. Oberli, M.A., Hecht, M.L., Bindscha¨dler, P., Adibekian, A., Adam, T., and Seeberger, P.H. (2011). Chem. Biol. 18, 580–588. Pace, D. (2013). Expert Opin. Biol. Ther. 13, 11–33. Reid, C.W., Vinogradov, E., Li, J., Jarrell, H.C., Logan, S.M., and Brisson, J.R. (2012). Carbohydr. Res. 354, 65–73.
Cell Chemical Biology
Previews
Vaccinology Gets Help from Chemistry Roberto Adamo1,* 1GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2016.09.003
A recent report on the immunological activity of protein conjugates of synthetic lipoteicoic fragments from Clostridium difficile underpins the use of these molecules for the development of a vaccine. In a recent issue of Cell Chemical Biology, Broecker et al. (2016) illustrate the utility of glycoarray-based selection of bacterial carbohydrates with the potential to become vaccine candidates. Clostridium difficile is a Gram-positive anaerobic bacterium commonly found in the environment that infects both humans and animals. Its intrinsic capacity to survive in a vegetative state by forming spores under harsh conditions, including high temperatures, ultraviolent light, and alcohol, makes C. difficile a seriously dangerous pathogen. In the human host, C. difficile germination, the process of switching from the dormant to live vegetative state, takes place in response to chemical factors present in intestinal epithelial cells. In this context, use of wide-spectrum antibiotics that alter the normal commensal gut flora has an impact on the diffusion of C. difficile infection and has been linked to infection flair up. The incidence and severity of C. difficile infection (CDI) appears to have increased over the last two decades, particularly in the US, Canada, and Europe, where it has been associated with higher casefatality rates, and the emergence of the hyper-virulent strain NAP1/027/BI, which is linked to some of the most deadly outbreaks, has been attributed to overuse and misuse of antibiotics, leading to the emergence of severe antibiotic resistance. Two large enterotoxins, TcdA and TcdB, both able to severely damage the intestinal mucosa, have been identified as the major cause of C. difficile disease (Leuzzi et al., 2014). A third toxin called CDT, or binary toxin, composed of the ADP-ribosyltransferase subunit CDTa and the binding subunit CDTb, is also known, but the role of CDT in human disease is still unclear. Vaccination with detoxified toxins or non-toxic peptide fragments has been pursued as a way to fight CDI (Leuzzi et al., 2014); however, a vaccine is not yet available.
The structures of surface carbohydrates from C. difficile, PSI, PSII and PSIII (Figure 1) have been elucidated recently, which has led to increased interest in using these sugars as targets for vaccine discovery (Ganeshapillai et al., 2008; Reid et al., 2012). It is well known that polysaccharides coating the surface of bacterial pathogens are weak immunogens, because they are T cell-independent antigens (Pace, 2013); however, conjugation to a carrier protein turns them into molecules able to evoke a T cell memory characterized by robust levels of anti-carbohydrate antibodies, which can be further boosted by subsequent doses of the vaccine. Efficacious vaccines against Streptococcus pneumonia, Haemophilus influenzae type b, and Neisseria meningitidis are now available on the market and they all exploit this strategy. In most cases, carbohydrates used for developing these vaccines are obtained through the process of bacterial fermentation. However, in the case of carbohydrate-based vaccines against C. difficile, the focus has been on synthetic glycans, a strategy that minimizes the likelihood of possible bacterial contaminants and offers access to well-defined carbohydrate structures. Scaling this approach for industrial production purposes would also avoid fermentation of pathogens. Among these C. difficile carbohydrates, first efforts focused on investigating PSII immunogenicity, as PSII was found to be abundant in the hypervirulent strain NAP1/027 and other clinical isolates (Danieli et al., 2011; Oberli et al., 2011). Using glycoarray analysis, antiPSII IgA antibodies (a class of antibodies present in mucosal secretion) have been identified in the stool samples of hospital patients (Oberli et al., 2011). Phosphory-
lated PSII fragments are immunogenic after conjugation to carrier protein, and specific antibodies bound to the bacterial surface (Adamo et al., 2012). Higher levels of IgA antibodies against PSI were found in CDI patients with less severe disease compared to asymptomatic controls, indicating that conjugates able to raise this antibody production could aid the development of an anti-C. difficile vaccine (Martin et al., 2013a). PSIII is a lipoteicoic (LTA)-like polymer, which is typically present in Gram-positive bacteria underneath the capsular polysaccharide. It is not clear yet whether PSI and PSII are capsules and what is their role on the bacterial surface. Anti-LTA PSIII antibodies have been detected in the blood of infected patients (Martin et al., 2013b), and immunizations with either intact or de-O-acylated LTA fraction of PSIII conjugates were shown to elicit IgG antibodies that clearly recognized PSIII on C. difficile cellular surface (Cox et al., 2013). The recent publication from Seeberger’s group (Broecker et al., 2016) reports significant advancements in the field. As opposed to what has been observed for PSI and PSII, IgA antibodies in fecal samples from CDI patients did not recognize synthetic LTA fragments on glycoarray. However, serum anti-LTA IgG were found, possibly due to the preexposure of the hospitalized individuals. This indicated that the synthetic LTA epitopes resembled natural epitopes. To test whether the selected glycan epitopes can efficiently produce antibodies, the largest synthesized LTA fragment was conjugated to CRM197, a genetically detoxified form of the diphtheria toxin, which is a carrier protein used in commercial vaccines. The glycoconjugate was administered with Freund’s adjuvant
Cell Chemical Biology 23, September 22, 2016 ª 2016 Published by Elsevier Ltd. 1047
Please cite this article as: Adamo, Vaccinology Gets Help from Chemistry, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.003
Cell Chemical Biology
Previews
screen biological samples form patients and identify potential candidates. The selected synthetic structures are then tested in vivo using animal models. It remains to be understood whether the LTA epitope identified by Seeberger and colleagues is the optimal glycan as is, or whether longer structures would be even more immunogenic. These followup questions notwithstanding, the article undoubtedly represents an important milestone in the preclinical development of a carbohydrate-based vaccine against C. difficile. R.A. is a GlaxoSmithKline employee and owner of a patent on anti-C. difficile vaccines. REFERENCES
Figure 1. Chemical Structures of C. difficile Surface Carbohydrates Recent work by Broecker et al. (2016) elucidates the potential of PSIII for vaccine development.
(FA), alum, or without adjuvant. LTA-specific antibodies were elicited after two boosts in mice, and the quality of the antibody response was dependent on the adjuvant used. IgG1 and IgG2a levels were highest when FA was co-administered, whereas alum promoted highest levels of IgG3. As alum is known to induce primarily anti-carbohydrate IgG1, the authors carefully ascertained that the glycoconjugate was fully adsorbed onto the inorganic salt and that it was stable. This indicated that the adjuvant can vary the repertoire of raised antibody. By comparing the IgG levels induced with no adjuvant and in the presence of FA, the authors concluded that antibodies were primarily directed to the largest synthetic structures, rather than to portions of it. The antibodies elicited against the largest fragment in the absence of adjuvant or with alum co-administration significantly bound to the surface of a series of
C. difficile strains. Most importantly, the alum-adjuvanted CRM197 conjugate reduced the bacterial colonization in mice that were rendered susceptible to infection with clindamycin and orally challenged with live C. difficile cells. There are two main advancements achieved by the work of Broecker et al. (2016). First, the authors put the antiC. difficile glycoconjugates to the test and provide formal evidence for functional activity of antibodies induced against surface glycan, which was missing from previous studies. Moreover, until this work, carbohydrate-based vaccine candidates have been selected by purifying polysaccharides and then conjugating them or portions of them to proteins and testing the glycoconjugates in mice to assess immunogenicity. The strategy employed by Broecker et al., which could be defined as a reverse glycoarray-based approach, starts from synthetic glycans that are applied onto a microarray to
1048 Cell Chemical Biology 23, September 22, 2016
Adamo, R., Romano, M.R., Berti, F., Leuzzi, R., Tontini, M., Danieli, E., Cappelletti, E., Cakici, O.S., Swennen, E., Pinto, V., et al. (2012). ACS Chem. Biol. 7, 1420–1428. Broecker, F., Martin, C.E., Wegner, E., Mattner, J., Baek, J.Y., Pereira, C.L., Anish, C., and Seeberger, P.H. (2016). Cell Chem. Biol. 23, 1014–1022. Cox, A.D., St Michael, F., Aubry, A., Cairns, C.M., Strong, P.C., Hayes, A.C., and Logan, S.M. (2013). Glycoconj. J. 30, 843–855. Danieli, E., Lay, L., Proietti, D., Berti, F., Costantino, P., and Adamo, R. (2011). Org. Lett. 13, 378–381. Ganeshapillai, J., Vinogradov, E., Rousseau, J., Weese, J.S., and Monteiro, M.A. (2008). Carbohydr. Res. 343, 703–710. Leuzzi, R., Adamo, R., and Scarselli, M. (2014). Hum. Vaccin. Immunother. 10, 1466–1477. Martin, C.E., Broecker, F., Oberli, M.A., Komor, J., Mattner, J., Anish, C., and Seeberger, P.H. (2013a). J. Am. Chem. Soc. 135, 9713–9722. Martin, C.E., Broecker, F., Eller, S., Oberli, M.A., Anish, C., Pereira, C.L., and Seeberger, P.H. (2013b). Chem. Commun. (Camb.) 49, 7159–7161. Oberli, M.A., Hecht, M.L., Bindscha¨dler, P., Adibekian, A., Adam, T., and Seeberger, P.H. (2011). Chem. Biol. 18, 580–588. Pace, D. (2013). Expert Opin. Biol. Ther. 13, 11–33. Reid, C.W., Vinogradov, E., Li, J., Jarrell, H.C., Logan, S.M., and Brisson, J.R. (2012). Carbohydr. Res. 354, 65–73.