Thymus dependent (TD) antigens, such as proteins, are taken up by antigen presenting cells (APCs). The endocytosed antigen is processed through a series of catalytic steps which liberate small peptide fragments. These peptide fragments form complexes with MHCII molecules and thereby are able to be displayed on the MHCII molecules of the APC. T cells specific to the displayed MHCII-peptide complex are co-stimulated by the APC (Avci and Kasper, 2010). The activated T cells go on to “help” the antigen-specific B cells, promoting their proliferation, affinity maturation, antibody isotype switching and long lasting immunological memory (Pulendran and Ahmed, 2011).

Carbohydrate antigens are classed as thymus independent (TI) antigens and are poorly immunogenic compared to TD antigens (Weintraub, 2003). Carbohydrate antigens are recognized by APCs through pathogen recognition receptors (Blander and Sander, 2012), endocytosed, and processed into oligosaccharide epitopes. These oligosaccharides are not presented on MHCII molecules, but instead are presented directly on the APC cell surface to activate carbohydrate antigen-specific B cells. Due to the lack of T cell activation, B cells are activated in the absence of affinity maturation and isotype switching (Mond et al., 1995) which leads to the predominant production of low affinity IgM antibodies, and only low levels of IgG (Mond and Kokai-Kun, 2008; Astronomo and Burton, 2010; Berti and Adamo, 2013). The IgM antibodies bind in the micromolar range, compared to TD antigen-derived IgG antibodies that bind in the nanomolar range (Broecker et al., 2016; Geissner et al., 2016). B and T cell memory is often not achieved and the immune response is short lived (Adams et al., 2008; Mond and Kokai-Kun, 2008; Hütter and Lepenies, 2015). Moreover, children less than 2 years of age fail to mount an antibody immune response to TI antigens (Weintraub, 2003; Landers et al., 2005).

In the 1920s and 1930s, covalent conjugation of carbohydrate antigens to protein scaffolds was first explored to investigate the interaction between the TD antigen properties of proteins with TI carbohydrate antigens (Avery, 1931). This early work formed the basis of the first glycoconjugate carbohydrate vaccines produced in the 1980s (Schneerson et al., 1980; Beuvery et al., 1982; Wessels et al., 1993), where bacterial capsular polysaccharides (CPS) were covalently linked to so-called carrier proteins. Immunization with these CPS-protein glycoconjugates enabled T cell-mediated B cell activation against the target carbohydrate antigen, and induced long term immune memory even in infants (Stein, 1992).

The mechanism whereby TI carbohydrate antigens can activate the immune system like TD protein antigens via glycoconjugation remains an open area of research. A long-standing mechanistic explanation argues that the carbohydrate-protein conjugate is recognized by carbohydrate-specific B cells. The protein component of the conjugate is subsequently endocytosed, processed and presented on MHCII molecules of the carbohydrate specific B cell. Through the MHCII-peptide complex a peptide-specific T cell can activate the carbohydrate-specific B cell, resulting in TD immune responses against the desired carbohydrate antigen (Figure 1; Lucas et al., 2005). A more recently proposed second mechanism argues that after uptake by carbohydrate-specific B cells the glycoconjugate is processed into glycopeptide fragments. Thus, the peptide portion of the fragment can form an MHCII-peptide complex, enabling the simultaneous presentation of the hydrophilic carbohydrate portion to carbohydrate-specific T cells. The T cell interaction activates the presenting B cell accordingly (Avci et al., 2011).

Five TD carrier proteins are currently used in licensed carbohydrate vaccines (Pichichero, 2013). Of these licensed carrier proteins, a non-toxic mutant of diphtheria toxin (CRM₁₉₇) is often used in vaccine development research for parasitic diseases. Another carrier protein often used in parasite vaccine development is keyhole limpet haemocyanin (KLH). Unlike many other carrier proteins KLH is itself highly glycosylated and is a good promotor of TD and TI responses (Nyame et al., 2004), however it is not licensed for human use.

The linker conjugating the carrier protein to the carbohydrate antigen is another key consideration of carbohydrate vaccines. Synthetic carbohydrates with defined carrier protein binding sites are advantageous for generating well-defined glycoconjugates with predictable conjugation sites. To minimize the immunogenicity of the linker (Buskas et al., 2004; Gotze et al., 2015) short linkers with no functional groups are best used to ensure that immune responses against the desired antigen epitope are not detrimentally affected. The processing of the linker after uptake by APCs is also an important consideration, to allow for the release of vaccine oligosaccharide and peptide moieties for antigenic display.

Severe malaria pathology is largely considered to arise from toxic effects of P. falciparum GPI (PfGPI), which exists either as protein free glycolipids, or as the major carbohydrate modifications for proteins essential for erythrocyte invasion (Gowda et al., 1997; Schofield and Grau, 2005). PfGPI-specific antibodies are found in adults of malaria-endemic areas, and may be inhibiting the ability of PfGPI to induce the hyper-inflammatory response associated with severe malaria. This is still up for debate, since studies that find an association between PfGPI-specific antibody titer and protection from severe malaria (Brasseur et al., 1990; Naik et al., 2000; Gowda, 2002; Keenihan et al., 2003; Perraut et al., 2005) are balanced by studies that find no such association (de Souza et al., 2002; Boutlis et al., 2005; Cissoko et al., 2006; Gomes et al., 2013; Mbengue et al., 2016). However, PfGPI-specific antibodies are reported to show relevant action in modulating immune responses by protecting immune cells against severe P. falciparum-induced inflammatory responses in-vitro (Schofield et al., 1993; de Souza et al., 2010).

To evaluate the effect of using PfGPI in an anti-toxin vaccine, a PfGPI hexasaccharide was synthesized, conjugated to KLH carrier protein and emulsified in CFA (Figure 2). C57BL/6 mice were immunized with the glycoconjugate, and then challenged with P. berghei ANKA in a mouse model of malaria. Immunization resulted in significant protection against severe malaria, with clearly reduced death rates at 75% survival. The immunized mice were also protected from acidosis, pulmonary oedema, cerebral syndrome and fatality characteristic of the disease model. Apparently, the induction of protective, PfGPI-specific antibodies ameliorated a hyper-inflammatory response against the PfGPI toxin, specifically shown to neutralize the parasite-induced production of TNF-α by macrophages in vitro. Furthermore, vaccination did not change parasitaemia levels, demonstrating that the effect of the carbohydrate vaccine candidate was through the neutralization of GPI's toxic effects, rather than interfering with parasite replication (Schofield et al., 2002).

Following this work, synthetic PfGPI were utilized as biomarkers to improve our understanding of the PfGPI-specific antibody response (Kamena et al., 2008; Tamborrini et al., 2010). The synthesis of PfGPI molecules continues to be explored (Gurale et al., 2016).

In healthy adults, up to 1–5% of circulating IgG and IgM are specific to the carbohydrate known as α-Gal (Figure 3; Macher and Galili, 2008). In contrast to other mammals, humans do not express α-Gal (Galili and Swanson, 1991) allowing for immune reactivity to this carbohydrate (Galili et al., 1984). Exposure to microbiota expressing this glycan drives production of α-Gal-specific antibodies (Macher and Galili, 2008) which are believed to contribute to immune resistance against α-Gal-expressing pathogenic microbes (Bishop and Gagneux, 2007; Cywes-Bentley et al., 2013). P. falciparum and other animal model Plasmodium spp. express the α-Gal carbohydrate (Galili et al., 1998) possibly bound to GPI-anchored surface proteins (Yilmaz et al., 2014). Early studies already uncovered an association between α-Gal-specific IgM and protection from P. falciparum infection in humans. Thus, the effect of immunization on the production of α-Gal-specific antibodies was investigated (Yilmaz et al., 2014).

Genetically modified mice unable to express α-Gal (Yang et al., 1998) were immunized for the production of α-Gal IgG and IgM. The immunizations were either inoculation with α-Gal expressing E. coli bacteria, α-Gal rich rabbit RBCs, or synthetic α-Gal conjugated to BSA. Adjuvants used were CFA and toll-like receptor 9 agonists that enhanced the immunogenicity. The mice were then challenged with P. berghei ANKA infection by Anopheles mosquito bites. It was found that α-Gal immunization reduced the risk of parasite transmission, thereby providing sterile immunity. This sterile immunity appears to be due to the cytotoxic action of α-Gal specific IgM and subclasses of IgG against the inoculating parasites. Moreover, the antibodies were shown to inhibit hepatocyte transmigration of the parasite (Yilmaz et al., 2014).

Sterile immunity against malaria infection through the induction of α-Gal-specific antibodies is not similarly present for people living in malaria endemic regions, possibly due to low levels of naturally acquired, protective α-gal-specific antibodies. Promoting a T cell-dependent immune response against the α-Gal carbohydrate was shown to enhance the protective effect of these antibodies in the mouse model (Yilmaz et al., 2014). Moreover, naturally acquired α-gal-specific antibodies may enhance the immunogenicity of antigens enriched with α-gal epitopes by augmenting the T cell response following immunization. This suggests that coupling the α-Gal carbohydrate to existing malaria vaccine candidates could enhance their immunogenicity (Benatuil et al., 2005; Yilmaz et al., 2014). The effect of α-Gal carbohydrate vaccination against other protozoan parasites expressing α-Gal, such as Trypanosoma spp. and Leishmania spp., may also be considered (Yilmaz et al., 2014).

Glycolipid GPI anchors of T. gondii (TgGPI) have been considered as possible vaccine antigens due to their effect in modulating inflammatory TNF-α responses against the parasite (Debierre-Grockiego, 2010). In 2015, TgGPI were explored as possible vaccine candidates for the first time.

GPI anchors are highly abundant on T. gondii (~10⁶ copies per cell) (Tsai et al., 2012) and exist as a protein-attached or protein-free glycoform (Figure 4). Both glycoforms induce inflammatory reactions like macrophage TNF-α production (Debierre-Grockiego et al., 2003) through TLR-2 and TLR-4 signaling (Debierre-Grockiego et al., 2007), which exacerbates toxoplasmosis in mice (Hunter et al., 1996). A vaccine that induces TgGPI-specific antibodies could ameliorate TgGPI-mediated inflammatory effects and lower disease burden (Debierre-Grockiego, 2010).

The two major TgGPI glycoforms were synthethized and covalently conjugated to CRM₁₉₇. BALB/c mice were then immunized with either one of the glycoconjugates before challenge with virulent T. gondii RH strain. Immunization did not provide protection for mice in the lethal challenge model. Furthermore, the induced antibodies failed to exhibit any significant effect on the inflammatory response for either group (Gotze et al., 2015). Analysis of the immune response indicates that antibody induction was directed away from the desired carbohydrate side branch of the TgGPI, and more toward the linker used to attach the carbohydrate to the carrier protein. In a potential next step, the formulation has to be adjusted (Gotze et al., 2015).

Ongoing research utilizing synthetic TgGPI molecules has helped to identify biomarkers of acute and latent infection. During acute infection, high levels of TgGPI-specific IgM and IgG are present, while latent infection shows a reduced IgM response (Gotze et al., 2014).

Leishmania parasites express lipophosphoglycans (LPG) on their cell surface. This molecule is composed of a GPI anchor, a repeating phosphorylated disaccharide fragment, and variable cap oligosaccharides (Seeberger, 2007). The LPGs are important for survival and virulence of the parasite (Spath et al., 2000) and vaccination preparations with purified LPG is protective against cutaneous leishmaniasis (McConville et al., 1987; Russell and Alexander, 1988; Moll et al., 1989). Regarding the variable capping oligosaccharide, a unique capping tetrasaccharide (Figure 5) was identified as vital for parasite invasion of macrophages (Descoteaux and Turco, 2002) and was the key component of a synthetic carbohydrate vaccine for leishmaniasis (Liu et al., 2006).

The capping oligosaccharide moiety of LPG was synthesized in initial immunological studies exploiting this antigen for vaccination. The carbohydrate was loaded onto virosomes that served as an integrated carrier and adjuvant, before being used to immunize BALB/c mice. Oligosaccharide-specific IgM and IgG1 responses were produced, indicating that the immune response to the carbohydrate antigen was T cell-dependent. Furthermore, the antibodies against the synthetic carbohydrate were cross-reactive with natural carbohydrate antigens of Leishmania parasites indicating its possible utility as a vaccine antigen (Liu et al., 2006). A further study immunized BALB/c mice with synthetic LPG capping oligosaccharides conjugated to CRM₁₉₇ and emulsified in CFA. This vaccine candidate produced IgG antibodies specific for the parasite. The oligosaccharides were used to evaluate immune responses of infected humans and dogs as the basis for a diagnostic test (Anish et al., 2013). Given the carbohydrate vaccines currently in production, animal model challenge studies to test the protective effect of these synthetic molecules is the next logical step.

LPG vaccination to protect against cutaneous leishmaniasis employed animal challenge studies that evaluated the LPG component of whole L. amazonensis antigen (LaAg). It was known that intramuscular, systemic immunization of LaAg results in deleterious disease outcomes after challenge, however LPG depletion rendered LaAg protective against Leishmania infection with respect to lesion growth and parasite load compared to non-depleted LaAg. This indicated that LPG was the component responsible for deleterious effects of LaAg inoculation. During further investigations, mice were intranasally vaccinated with LPG alone to determine whether intranasal vaccination of the disease-promoting component could promote protection against cutaneous leishmaniasis (Pinheiro et al., 2007). Intranasal vaccination with LPG was protective, and provided further evidence for the potential of utilizing LPG in a carbohydrate antigen vaccine that would protect against cutaneous leishmaniasis.

Recently, the role of L. mexicana protein-free GPI molecules (GIPL) in disease has been elucidated, shedding more light on immunologic pathways affecting glycolipid-specific antibody responses (Figure 6). After it was determined that L. mexicana infection induces GIPL-specific IgG1 responses, a monoclonal antibody against GIPL was shown to bind to the surface of parasites, and promote IL-10 production in macrophages co-cultured with parasite. Production of IL-10 is deleterious as it blocks an effective immune response that is needed to kill parasites and resolve skin lesions in cutaneous leishmaniasis. In humans, GIPL-specific antibodies are produced in response to infection with cutaneous leishmaniasis. Opsonization of parasites with these antibodies was shown to promote deleterious IL-10 production in macrophages. The role of GIPL-specific antibodies in both mouse and humans opens the possibility of generating a carbohydrate vaccine that can induce competing, non-pathogenic antibody isotypes that can protect against L. mexicana infection (Buxbaum, 2013).