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3 Carbohydrate-Based Antiviral Vaccines
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each year, according to the World Health Organization [1]. Influenza viruses are
formed into three different types: A, B, and C, albeit only A and B types seem to be
pathogenic in humans. In fact, Influenza A viruses (IAVs) are responsible for the
four pandemics that occurred in the last 100
years (1918, 1957, 1968, and 2009).
IAV is a highly mutable virus mostly associated with relatively mild diseases.
However, IAV can be lethal to individuals with cardiac or pulmonary affections. On
occasion, influenza viruses are transmitted from wild waterfowl to domestic poultry
and are able to cause a human influenza pandemic [84]. IAV belongs to the
Orthomyxoviridae family of enveloped, singlestranded RNA viruses with a genome
that encodes several viral proteins, most notably hemagglutinin (HA) and neuraminidase (NA), and is classified based on 18HA subtypes and 11NA subtypes[85]. Both
glycoproteins form the virus surface and are carbohydraterecognizing proteins that
function by recognizing sialic acid molecules on the host cell. HA binds sialylated
receptors to enable attachment, whereas NA hydrolyzes sialic acid residues to help
viral release and infection[86]. There exists a functional crosstalk between both proteins in viral attachment/release that depends on the HA glycans[87]. These carbohydrates play a role in immune evasion and constitute a dynamic glycan shield. While
influenza virus vaccines exist, their efficacy is suboptimal due to mismatches between
vaccines and circulating viral strains, requiring the development of a broadly protective vaccine to improve overall protection. Approaches are being attempted to attain
such a universal vaccine aim to induce Tcell responses, or bnAbs, by targeting different internal or surface viral antigens, respectively [88]. The latter include the viral
surface glycoproteins HA and NA, which are the main targets for the humoral
immune response and the basis of currently available influenza vaccines, generating
neutralizing antibodies.
3.3.1 Vaccine Constructs Based on Hemagglutinin (HA)
HA is the most abundant protein on the IAV surface. It plays a role in viral entry, is
involved in receptor binding and membrane fusion, and is the major target of protective antibody responses upon infection or vaccination. HA is structured as a
homotrimer on the surface of the virion, with each monomer comprising two subunits (HA1 and HA2) that originate from a distinct polypeptide precursor (HA0)[89].
The HA2 subunit presents a transmembrane region with a rather conserved cytoplasmatic stem that attaches the HA protein to the virion envelope. The receptor
binding site is located in the more variable head domain of the HA1 subunit, which
binds sialic acid residues on the hostcell surface, enabling viral entry[90]. Once the
virion is internalized, a conformational change in HA exposes the Nterminus
fusion peptide of the HA2 subunit stem, facilitating membrane fusion and release of
the viral RNA into the cytoplasm of the host cell [91]. As the virion head domain
contains the major immunodominant antigenic determinants, neutralizing antibodies against influenza are generally addressed to this region, interfering with HA
binding to sialic acid and inhibiting its hemagglutination activity [92]. Thus, the
hemagglutination inhibition test is used as a surrogate measurement to titrate the
antibody response (antiIAVneutralizing antibodies) against influenza[93].
3.3 Influenza A Virus
3.3.1.1 Hyperglycosylated HA Vaccines
In the design of vaccines directed to the more conserved HA stem domain, one strategy to increase stemspecific responses is to hyperglycosylate the variable HA1 head
region in order to hide their immunodominant epitopes, thus directing the response
to the stem[94]. Some studies showed that hyperglycosylated HA1induced stronger
antistem antibodies against the homologous H1 stem than wildtype HA[95]. The
hyperglycosylated H1 also stimulated more crossreactive antibodies to two heterologous H1 viruses and a heterosubtypic H5 virus. This strategy was also applied to
the H5 stem, but it did not result in relevant antibody responses to other group1
subtypes (H1, H3, and H9)[96]. Despite the increased antistem antibodies compared to wildtype HA, vaccination did not protect mice from critical influenza morbidity. Another example of this hyperglycosylation strategy has been used with the
highly pathogenic avian influenza (HPAI) H5N1 viruses. The transmission capability of this virus from birds to humans has raised global concerns about a potential
human pandemic. In these studies, hyperglycosylated HA vaccines were designed
using Nlinked glycan masking on highly variable sequences in the HA1 head
domain[97]. Immunization with these hyperglycosylated HA DNA vaccines, followed by a flagellincontaining viruslike particle booster, was conducted in mice to
evaluate neutralizing antibody responses against various clades of HPAI H5N1
viruses. However, no significant differences in antiHA total Ig titers were found
with these hyperglycosylated HA compared to the wildtype control.
A general approach to improve influenza vaccine’s potency is the addition of adjuvants that increase antigen immunogenicity [98]. Thus, aluminum phosphate
(alum) has been coadministered in HAbased DNA vaccines to enhance antibody
production[99], and together with the oilinwater MF59 and AS03, these are the
three main adjuvants incorporated in licensed flu vaccines[100]. Other immunopotentiating substances, such as stimulatory glycolipids functioning as invariant natural killer T (iNKT) cell activators, have also been reported to exhibit adjuvant
activities in protein and DNA vaccines[101].
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3.3.1.2 α-Gal-Based Vaccine Constructs
An important stage for stimulating an adaptative immune response is the presentation of antigen fragments on the surface of antigenpresenting cells (APCs).
αGalactosylceramide (αGalCer) was the first synthetic iNKT activator discovered,
which was derived from a natural product extracted from marine sponges
[102, 103]. Some research studies have included the αGalCer glycolipid as a vaccine
adjuvant against influenza[104, 105], while various delivery systems have also been
developed (e.g. poly(lacticcoglycolic acid) [PLGA] particles) to enhance its immunostimulatory properties and boost the immune response[106]. Moreover, recent
developments in glycoconjugate vaccines are based on chemical combination of
adjuvants (e.g. αGalCer) and relevant carbohydrate antigens, which enables
codelivering of both vaccine components to the same immune cell for boosting the
immune response[107].
This strategy of covalent conjugation was used by Anderson etal. for the development of a synthetic, influenzatargeting vaccine [108]. As opposed to antibodies
3 Carbohydrate-Based Antiviral Vaccines
(a)
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(b)
Figure3.3 Antigen–α-GalCer prodrug conjugate vaccine against influenza
challenge[108]. (a) CuAAC-coupled α-GalCer prodrug linker–SLP conjugate. (b) SPAACcoupled α-GalCer prodrug linker–SLP conjugate. R represents the synthetic long peptide
(SLP) containing an immunogenic sequence that involves the T-cell CD8
and CD4
sequence (FFRK).
+
OVA
(ISQAVHAAHAEINEAGR) epitopes together with a protease cleavage
323
+
OVA
(SIINFEKL)
257
binding cell surface HA, IAVspecific T cells recognize primarily conserved epitopes
from internal viral proteins[109]. Thus, Painter and coworkers used click chemistry
(CuAAC, Figure3.3a; strainpromoted alkyne–azide cycloaddition SPAAC, Figure3.3b)
to link an αGalCer prodrug derivative to a synthetic long peptide (SLP) from a virus
+
associated protein incorporating a wellknown CD8
Tcell epitope from ovalbumin
(OVA, see “R” substituent in Figure3.3). In vivo studies in mice vaccinated with the
SPAACcoupled αGalCer prodrug–SLP conjugate (Figure3.3b) and challenged with a
recombinant OVAmodified influenza virus showed induction of peptidespecific,
memory Tcell responses that were protective against IAV infection[108].
In another approach, Galili and coworkers developed a carbohydratebased
method that leveraged the mechanism of antibodydependent antigen uptake with
a view to enhancing the immunogenicity of influenza vaccines [110]. Given the
abundance of natural antiGal antibodies in humans, the authors incorporated a
synthetic αGal epitope into the Nglycans of HA by applying a chemoenzymatic
strategy that used α1,3galactosyltransferase (α1,3GT)[111]. As such, modification
of the virus Nglycans using recombinant α1,3GT generated an influenza virus
strain incorporating the αGal epitope. This engineered HA glycoprotein was bound
by natural antiGal antibodies, leading to the formation of immune complexes,
which results in targeting and uptake of the modified vaccine virus by APCs for
stimulation of virusspecific T cells in the lymph nodes. In their study, mice vaccinated with this αGalcoated viral construct induced substantially increased antibody and cellular responses with higher protection than those immunized with the
unmodified virus strain[110].
3.3.2 Vaccine Constructs Based on Neuraminidase (NA)
NA is the second most abundant glycoprotein on the viral surface and is implicated
in viral delivery and propagation from infected cells. NA enables the release of lineage viruses from the host cell by cleaving terminal sialic acid from glycans both on
the host cell and on the emerging virion[86]. Due to its role in the virusreplication
3.3 Influenza A Virus
cycle, NA has traditionally been a key target for antivirals or therapeutics based on
inhibition of NA activity, including the commonly used oseltamivir (Tamiflu) and
zanamivir (Relenza)[112]. Nonetheless, accumulated scientific evidence is leading
to growing interest in considering NA as a target for vaccine design in the context of
humoral immunity [113]. Thus, infectioninduced human antibodies against NA
were crossreactive and able to inhibit the protein sialidase activity, blocking viral
egress and providing protection from lethal influenza virus in mice[114, 115]. The
major advantage of NA is its slower antigen evolution and the subsequent ability to
induce longer lasting immunity and crossprotection than that offered by HA vaccines [116]. While the promise of natural NAbased immunity warrants further
investigation into the inclusion of this glycoprotein in nextgeneration influenza
vaccines targeting NA, there are still many knowledge gaps, including those regarding its immunogenicity, protection breadth, and mechanism, as well as the nature of
the antigenic sites [117]. Notably, recent structural advances have yielded key
insights into targeted epitopes and the basis of protection of antiNA antibodies,
providing templates for the rational design of NAbased vaccines and therapeutic
agents[118, 119].
3.3.3 Acetalated Dextran asAdjuvant Carrier
Acetalated dextran (AcDex) is a pHresponsive polysaccharide that can be readily
synthesized from dextran through acetal formation with 2methoxypropene.
AcDex is not soluble in water but is able to form microparticles loaded with different encapsulated cargoes by using emulsion techniques, releasing its content under
acidic conditions [120]. It has been used as a vaccine carrier system to increase
immune activation[121], whereby its microparticles can deliver the immunostimulatory agents (e.g. antigens and/or adjuvants) as a combined vaccine formulation, as
shown against influenza and other infections[107].
This combination strategy was later used to apply the cyclic guanosine
monophosphate–adenosine monophosphate (cGAMP)encapsulated AcDex together with soluble HA from the H1N1 subtype for antiinfluenza vaccination [122].
This vaccine system induced a potent Th1skewed neutralizing antibody response in
mice, providing more than sixmonth protection from a lethal H1N1 challenge. In
another example, Ainslie and coworkers coformulated cGAMP and the ectodomain
of the surface protein matrix 2 (M2e), both encapsulated within separate AcDex particles, as an antiinfluenza vaccine, which induced protective antibody and cellular
immune responses[123].
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3.3.4 Multivalent Constructs asAnti-Influenza Inhibitors
The multivalent interactions between HA and sialic acid residues on cellsurface
receptors are the first step leading to viral internalization and consequent infection.
Thus, for competitively blocking virus attachment to cells, synthetic multivalent
glycoconjugates have been developed as inhibitors of influenza infection by using
3 Carbohydrate-Based Antiviral Vaccines
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different scaffolds (e.g. dendrimers, proteins, and gold nanoparticles) decorated
with numerous copies of sialic acid[124, 125].
Whitesides and coworkers introduced the first multivalent entry blockers in the
1990s. They developed α
-sialoside–polyacrylamide copolymers that inhibited IAV
adhesion to erythrocytes (measured as hemagglutination) more than 10
more efficiently than its α
-methyl sialoside monomer[126], in line with their greater
binding affinity observed to the viral surface due to cooperative multivalent interactions[127]. Optimization studies led to high
-affinity (in the nanomolar range) sialic
aciddecorated polyacrylamidebased polymers[128] that, despite their considerable inhibitory potential, showed high cytotoxicity associated with their polyacrylamide backbone [129]. Additional designed multivalent conjugates consisted of
polyamidoamine (PAMAM) dendrimers displaying sialyllactose, which showed
invitro micromolar inhibition and protected mice from H1N1lethal challenge[130].
Haag and collaborators investigated other dendritic multivalent nanostructures
based on chemical functionalization of gold nanoparticles[131, 132] and biocompatible polyglycerol nanogels (nPG) with sialic acidterminated dendrons [133].
These carbohydrate nanosystems had high affinity for HA and showed 30% and 80%
influenza infection inhibition, respectively. Another multivalent construct involved
covalent conjugation of many copies of the antiinfluenza drug zanamivir to a poly
glutamine scaffold, which led to zanamivir glycopolymers with enhanced potency
(subnanomolar) that inhibited influenza viral fusion and release [134]. Overall,
these examples highlight the importance of HA as a key target for further development of multivalent glycoconjugates as potential antiinfluenza vaccines and drug
candidates for the inhibition of influenza infection.
Among the options available to fight IAV, the most effective means to prevent
influenza is through a universal vaccine that is broadly protective and does not need
seasonal modification. With this aim, the development of such vaccine constructs
should focus on conserved viral glycoproteins that are shared between virus strains
and subtypes and can induce both antibody and cellular responses, providing effective crossreactive immunity and prophylactic protection against IAV infection.
Despite extensive research efforts in this direction by the scientific community,
there is still a long pathway ahead to achieve more efficient and safer influenza vaccines, especially under the increasing threat of emerging global pandemics.
000 times
3.4 Hepatitis C Virus
Currently, hepatitis C virus (HCV) affects over 70 million people worldwide and
has become the most important chronic liver disease, with the risk of developing
into cirrhosis and hepatocellular carcinoma (HCC). HCV belongs to the Flavi-
viridae family and consists of an enveloped positivesense singlestranded RNA
virus that has six major genotypes and multiple subtypes[135, 136]. The HCV
genome encodes one polyprotein precursor processed into three structural proteins (core protein and envelope glycoproteins E1 and E2) and seven nonstructural
(NS) proteins[137]. The surface glycoproteins E1 and E2 contain around 5 and 11
3.5 Ebola Virus
potential Nglycosylation sites, respectively[138]. Most of them are rather conserved [139], suggesting that in addition to their role in protein structure and
activity, they could serve as vaccine candidates. A heterodimer complex is formed
by E1 and E2 on the viral particle, enabling HCV entry with involvement of E2
glycans[140]. Notably, the E2 glycoprotein includes the receptorbinding domain
(RBD) that interacts with cell surface entry receptors [141, 142] and is also the
major target for neutralizing antibodies[143]. Carbohydrates decorating HCV,
especially the conserved E2 oligosaccharides, form a nonevolving glycan shield
that protects underlying protein epitopes[144]. The E2 glycoprotein binds strongly
to the lectin DCSIGN (dendritic cellspecific intercellular adhesion molecule
grabbing nonintegrin), which may mediate viral infection in hepatocytes[145].
This is in line with the predominance of highmannose glycans in this protein[146], which signals the potential of carbohydratebinding agents (CBAs) as
antiHCV drug candidates that can disrupt glycan–protein interactions required
for HCV entry and infection.
Thus, several mannosespecific CBAs targeting the oligomannosecontaining
E1E2 heterodimer have been developed that bound to envelope glycans and inhibited capture of HCV by DCSIGN and viral entry [147]. Moreover, the lectin
c yanovirinN was also found to inhibit HCV infection, likely due to interaction with
HCV envelope glycoproteins[148], highlighting the promise of CBAs as powerful
lead drugs for blocking HCV at the cell entry phase.
In addition to enabling the screening of CBAs with antiHCV activity, the abundance of conserved highmannose glycans in the E1/E2 glycoproteins suggests the
prospect of targeting these carbohydrate epitopes for the challenge of developing a
sofar elusive HCV vaccine that can fight against the highly heterogeneous HCV
genotypes. Nonetheless, some strategies based on recombinant HCV E2 protein
have been investigated that can induce bnAbs against most HCV genotypes. For
instance, an hyperglycosylation approach leading to an engineered subunit viral
vaccine with distinct glycan patterns was employed to mask suboptimal epitopes
associated with non-neutralizing antibodies, resulting in a more immunogenic
HCV E2 protein that generated a more epitopefocused and protective neutralizing
antibody response in mice[149].
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3.5 Ebola Virus
Ebola virus (EBOV) is among the most lethal human pathogens, causing one of the
deadliest infectious diseases in the world (Ebola hemorrhagic fever, or EBOV disease). It was first described in 1976in Zaire (currently the Democratic Republic of
the Congo), followed by various subsequent outbreaks, predominantly in Central
Africa[150]. EBOV belongs to the Zaire ebolavirus species within the Ebolavirus
genus of the family Filoviridae[151]. The last Ebola epidemic caused by EBOV in
West Africa (2013–2016) reported over 28 000 cases of infected people and 11 000
deaths[152].
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3.5.1 Glycoprotein-Based Vaccines
The envelope glycoprotein (GP) on the viral surface plays a key role in viral entry,
being responsible for the interaction with hostcell receptors[153], and is the key
target of neutralizing antibodies and protective immunity. It is highly immunogenic
and has, therefore, been used as an immunogen in the development of Ebola vaccines. At present, two major vaccines exist against the Zaire ebolavirus species: the
®
FDAapproved rVSVZEBOV (called Ervebo
)[154] and the twocomponent Ad26.
ZEBOV/MVABNFilo vaccine, authorized by the European Commission [155].
Mature GP is formed by GP1 and GP2, which are presented as trimers of disulfide
linked GP1–GP2 heterodimers. The GP1 subunit comprises two highly variable
regions, the glycan cap and the mucinlike domain (MLD), that contain many N and
Olinked glycans[156]. Some of these Nglycans may serve as attachment points to
host cells, for instance, via mannosebinding Ctype lectins, which provides additional therapeutic opportunities (see below)[157]. Both heavily glycosylated domains
can prevent neutralizing antibodies fr om binding the GP by protecting critical
underlying epitopes[158]. Some research studies have investigated the influence of
GP glycosylation, showing that mutation of two Nglycosylation sites on GP1 (388,
415 sites) may increase immunogenicity, presumably by exposing protective antibody epitopes, whereas deletion of the mucin region led to reduced protective efficacy in mice [159]. Because GP antibodies are mainly generated against the least
conserved MLD, another study used a construct devoid of this domain for mouse
vaccination, which resulted in the induction of crossspecies immunity due to unmasking of more conserved regions by the immune system[160].
3.5.2 Monoclonal Antibodies and Carbohydrate Antiviral Agents
asTherapeutics
Currently, there are two therapeutic options approved in 2020 by the FDA to treat
Ebola, which are based on monoclonal antibodies that target the GP, blocking entry
of the virus into the host cell[161, 162]. In addition to these antibodybased strategies, some smallmolecule antivirals have been developed and tested in clinical trials, such as the broadspectrum nucleoside analogs BCX4430 (Galidesivir)
[163, 164] and Remdesivir[165], which act by inhibiting the viral RNA polymerase.
A general strategy exploited for the development of antiviral compounds involves
the inhibition of viral transmission through blockade of DCSIGN. This lectin interacts with highly mannosylated glycoproteins in a multivalent manner, including the
EBOV GP[166], and is one of the most important pathogenrecognition receptors,
being an important target for Ebola infection[167]. Consequently, a variety of multivalent carbohydrate structures have been developed with controlled glycan
valency, size, and shape[168] that mimic the glycan presentation on the GP surface.
Notably, some of these synthetic systems could block DCSIGN and were able to
inhibit infection by EBOV[169]. In a representative example, Davis and coworkers
used a proteic platform (multimeric Qβbased viruslike particles) for conjugation of
mannose glycodendrons through CuAAC click chemistry, assembling multivalent
3.5 Ebola Virus
glycodendrimeric nanoparticles that displayed >1600 monosaccharides in a
homogeneous manner[170]. These glycoconjugates showed potent antiviral activity, preventing DC infection by pseudotype Ebola by competitively inhibiting the
binding of DCSIGN at low nanomolar/picomolar concentrations. Subsequently,
Martin and collaborators efficiently synthesized a watersoluble multifullerene
based dendritic structure decorated with 120mannose units using click chemistry
(Figure3.4), providing nanosized globularshaped multivalent macromolecules that
blocked DCSIGN and inhibited EBOV infection in the subnanomolar range[171].
The high inhibitory potencies of these antiviral constructs open the door to investigating whether these multivalent systems could induce potent antibody responses
invivo that might potentially neutralize some of the relevant glycan–protein interactions operative at the virus–cell interface. Moreover, the modular nature and full
chemical control enabled by this synthetic approach raise the possibility of creating
dual/bifunctional structures that can incorporate immunogenic elements recognized by the immune system, leading to prospective novel molecular vaccines for
inducing immune responses against EBOV.
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Figure3.4 Glycofullerene “superballs” substituted with up to 120mannose units[171].
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3.6 SARS-CoV-2 Virus
COVID19has become a major pandemic and a global public threat since its first
outbreak in China in December 2019. It is caused by a novel coronavirus called
SARSCoV2 (WHO 2020) [172]. Coronaviruses are enveloped, singlestranded
positivesense RNA viruses that have a spike glycoprotein (Sprotein) that plays a
critical role in pathogenesis and in host immune response induction[173]. In addition to the spike glycoprotein, three other different proteins make up the SARS
CoV2 structure: an envelope protein (E), membrane protein (M), and nucleocapsid
protein (N)[174]. The SARSCoV2 spike has two functional subunits (i.e. S1 and
S2) and contains 22 putative Nlinked glycosylation sites and 4 potential Olinked
glycosylation points[175, 176]. The function of the S1 subunit is associated with
receptor binding, and the S2 subunit is responsible for membrane fusion[177]. As
with many viral envelope glycoproteins, the carbohydrates around the spike form a
SARSCoV2 glycan shield that helps evade the immune system. In addition to
shielding, two Nglycans (N165 and N234) have been found to play a structural role
in modulating the conformation of the RBD that binds to the angiotensin
converting enzyme (ACE2) hostcell receptor, priming the virus for infection[178].
Despite the structural similarity between SARSCoV2 and the first SARSCoV
virus, 5–6key amino acid residues of the RBD implicated in ACE2 recognition are
changed[179, 180], indicating distinct antigenic sites in the two viruses. The existence of different key epitopes points to the need to develop new therapeutic agents
and vaccines that are specific for COVID19[181, 182]. In this context, the SARS
CoV2 S protein serves as the main immunogen and a key target of neutralizing
antibodies[183], representing an area of focus for vaccine development [184]. In
particular, the spike glycan shield emerges, and its vulnerabilities [185] can be
exploited for smallmolecule drug development as well as for the design of potential
carbohydratebased vaccines to combat the COVID19 pandemic[186, 187].
3.6.1 Prospective Vaccine Constructs Based on α-Gal Epitope
As mentioned in the end of Section3.3.1.2 on influenza virus vaccines, endogenous
human antiGal antibodies might also be exploited for increasing SARSCoV2 vaccine immunogenicity by glycoengineering αGal epitopes on inactivated SARS
CoV2 or on Sprotein subunit vaccines[188]. Presentation of these αGal epitopes
would enable formation of antiGal/SARSCoV2α
or antiGal/Sproteinα
Gal
immune complexes, resulting in higher APC targeting and uptake via specific receptor interactions and, as a result, enhanced immunogenicity and vaccine efficacy.
This glycoengineering strategy would convert the native, proteinmasking glycan
shield into an αGalmodified carbohydrate coating that effectively directs the engineered vaccine to APCs via antiGal antibodies. Despite its promise, this αGal vaccine approach needs to be further demonstrated in a COVID19 setting to fully
assess its potential to boost antiSARSCoV2 immune responses, both in terms of
neutralizing antibody titers as well as Tcell immunity.
Gal
3.6 SARS-CoV-2 Virus
3.6.2 RBD-Based Constructs for Vaccine Development
The RBD glycoprotein within the Sprotein trimer mediates viral infection by binding to ACE2 and represents a key target for the design of vaccines able to elicit neutralizing antibodies against SARSCoV2[189]. In a notable example, a recombinant
monomeric RBD vaccine adjuvanted with alum was shown to elicit a potent and
protective antibody response in laboratory animals after invivo SARSCoV2 challenge[190]. With a view to increasing immunogenicity and favoring presentation of
the key RBD motif surface in search of enhanced neutralizing antibody induction,
VérezBencomo and coworkers chemically linked the recombinant RBD to the
highly immunogenic protein carrier TT in a siteselective manner[191]. The resulting multivalent RBD–TT conjugates induced a robust IgGneutralizing antibody
and cellular response in mice when coadministered with alum, which prompted
advancement of this conjugate vaccine candidate to clinical trials[192].
In an effort to guide the development of semisynthetic carbohydratebased RBD
vaccines, Wang and coworkers recently investigated the role of precise glycan structures on this domain (at T323, N331, and N343) by preparing homogeneous RBD
glycoproteins using chemical synthesis and recombinant protein engineering[193].
Leveraging their homogenously glycosylated RBDs, they found no differences
between various synthetic glycoforms in terms of ACE2 binding and revealed the
influence of discrete RBD glycosylation on antiSARSCoV2 RBD monoclonal antibody binding. In addition to deciphering carbohydrate structure–activity relationships, these studies have provided chemically defined glycosylated constructs as
potential epitope mimics for further immunological evaluation invivo, opening the
door to the prospective rational development of synthetic glycanbased immunogens for future antiSARSCoV2 vaccines and therapeutics.
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3.6.3 Saponins as Carbohydrate-Based Adjuvant Candidates for
COVID-19 Vaccines
The identification of novel vaccine adjuvants that can potentiate humoral, cellular,
and memory immune responses to prevent COVID19 infection is emerging as a
frontline strategy in vaccine development against COVID as well as other infectious
diseases[194, 195]. Triterpenoid and steroidal saponin natural products show antiviral activity against different viral groups. Aqueous extracts from the Chilean soapbark tree Quillaja saponaria Molina contain many physiologically active triterpenoid
saponins, some of which show high adjuvant activity, potentiating the immune
response against the coadministered antigen[196]. Purified saponin fractions (specifically QS21), either alone or integrated as part of immunostimulating complexes
(ISCOMs) and adjuvant systems, have been demonstrated to be powerful adjuvants
inducing both antibody and cellular immunity in several vaccine clinical trials
[197, 198]. AS01 is a potent adjuvant combination recently approved in herpes and
malaria vaccines that includes two immunostimulants, 3Odesacyl4′
monophosphoryl lipid A (MPLA) and the naturally derived purified saponin fraction
(QS21), in a liposomal formulation[199]. Both immunopotentiating substances in
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