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3.2 Human Immunodeficiency Virus
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used yeastderived highmannose glycoproteins as immunogens, presenting
GlcNAc2 glycans in a more dense, nearnative form[56, 57]. Immunization in
Man
8
rabbits generated carbohydratespecific antibodies that recognized gp120 and efficiently neutralized HIV1 virions expressing highmannose Nglycans but did not
neutralize the wildtype virus. Second, considering the rare domainexchange structure of 2G12, this class of bnAbs might be intrinsically difficult to induce, and some
animal species may not have the ability to generate this unique and complex antibody type. Taking these concepts into account, a deeper knowledge of the key features of 2G12 bnAb evolution in HIV1infected individuals would be essential to
design improved synthetic glycoconjugate immunogens that can elicit 2G12 bnAbs,
leveraging a suitably devised vaccination approach.
3.2.2 Vaccine Constructs Derived from gp120 First and Second
Variable Loops (V1V2)
3.2.2.1 V1V2-Targeting bnAbs
Since 2009, researchers have been continuously discovering new potent human
bnAbs, which represents an important springboard toward the identification of new
targets for HIV vaccine design. The PG9, PG16, CH01–04, and PGT141–145 antibodies were found to target the gp120 V1V2 apex of the HIV1 Env trimer[58–63]. These
bnAbs contain a long, anionic CDRH3loop to penetrate the glycan shield and bind
a quaternary motif within the first and second variable loops (V1V2). So far, synthetic glycanbased vaccine development has centered on the binding sites of PG9,
PG16, and CH01 bnAbs, which include similar glycandependent conformational
epitopes in the V1V2 region [59, 64]. Specifically, crystal structure studies of the
complexes between PG9 and scaffolded V1V2 domains revealed that the antibody
interacts with two highmannose glycans at N160 and N156/N173 and a connected
V1V2peptide βstrand[65]. While the fine glycan specificities of the bnAb epitopes
were yet uncertain, the available structural insights provided an important framework for the development of V1V2 carbohydratebased immunogens as synthetic
epitope mimics.
81
3.2.2.2 Synthetic V1V2 N-Glycopeptide Antigens as bnAb Epitope Mimics
Wang and coworkers designed and chemoenzymatically synthesized a number of
gp120 V1V2 cyclic glycopeptides (V154–Y177) based on two HIV1 strains with different glycosylation profiles, CAP45 (N156, N160) and ZM109 (N160, N173)
(Scheme3.3). Binding analysis by SPR and ELISA revealed that a Man
glycan at N160was critical for recognition by PG9 and PG16, while the presence of
an additional sialylated complextype oligosaccharide at N156 or N173 further
increased the binding affinity[66]. A more efficient chemoenzymatic approach was
later developed for the siteselective glycosylation of the peptides with two distinct
Nglycans by using orthogonally protected GlcNAcAsn residues[67]. The important role of the sialylated N‐glycan at the second glycosylation point was also corroborated by crystallographic studies with the PG16 bnAb[68]. Subsequently, Wu
and coworkers synthesized unusual hybridtype glycans bearing oligomannose and
GlcNAc2
5
3 Carbohydrate-Based Antiviral Vaccines
82
Scheme 3.3 Chemoenzymatic synthesis of V1V2 glycopeptides.
α2,6sialylated branches and analyzed their binding to PG9 and other bnAbs using
glycan arrays. The high affinity obtained for these structures highlighted the critical
role that the spatial distance between both glycan arms plays on antibody binding
and provided uncommon glycans as potential epitope mimics for vaccine
development[69, 70].
Meanwhile, Danishefsky and coworkers prepared several differently glycosylated
gp120 V1V2 peptides based on the HIV1 A244 strain (I148–I184) using chemical
synthesis (Scheme3.4). The corresponding glycosyl amines derived from Man
GlcNAc
and Man3GlcNAc2 were incorporated at the N156 and N160 residues of
2
two individual peptides via Lansbury aspartylation, and the resulting fragments
were coupled together in unprotected form by native chemical ligation (NCL).
Binding studies confirmed the multivalent simultaneous interaction of PG9 with
both the peptide backbone and the mannosebearing Nlinked oligosaccharides[71].
In a followup study, these glycopeptides were dimerized through a disulfide bond
(C157), which resulted in even higher binding affinities to bnAbs (in the low
-
5
Scheme 3.4 Chemical synthesis of V1V2 glycopeptide.
3.2 Human Immunodeficiency Virus
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nanomolar range) and their unmutated common ancestors [72]. In addition,
circular dichroism experiments suggested that not only the Man
/Man3 Nglycans
5
but also the disulfide bondmediated dimerization may contribute to the more stable, βstranded conformation necessary for bnAb binding. These results signal the
promise of these rationally designed glycopeptide antigens for further development
as potential synthetic immunogens to elicit V1V2directed bnAbs.
3.2.3 Vaccine Constructs Derived from gp120 Third Variable
Loops (V3)
3.2.3.1 V3-Targeting bnAbs
A majority of PGT bnAbs isolated from HIV1infected elite neutralizers were found
to target epitopes on the V3loop involving the N332 glycan and the V3 peptide backbone [60]. Further detailed characterization of the antibodybinding sites was
obtained by crystallographic studies, which revealed an epitope formed by a V3
βstrand and 2 oligomannoses at N332 and N301 for PGT128[73] and a preference
toward complextype glycans for PGT121[74].
3.2.3.2 Synthetic Glycoconjugates and N-glycopeptides as V3-Directed
bnAb Epitope Mimics
Kosma and collaborators observed that the previously mentioned βlinked Man7
BSA conjugate (see Figure3.1h) bound with high affinity to bnAbs of the PGT128
class and to their common germline precursor[75], likely because of the structural
similarities of the bacterial LOS [54] and the PGT128 highmannose glycan epitope[73]. In rat immunizations, this glycoconjugate induced reasonable levels of
carbohydratespecific IgM antibodies but low IgG titers, suggesting weak immunogenicity with Bcell activation in the absence of Tcell involvement. Interestingly,
immune sera were crossreactive with native gp120 and even exhibited neutralizing
activity against some HIV1 strains, presumably due to avid interactions with polymeric IgMs[75].
83
3.2.3.3 Synthetic V3 Glycopeptides as bnAb Epitope Mimics
In 2017, Wang and coworkers applied their glycosynthasebased chemoenzymatic
strategy for the synthesis of a gp120 miniV3 glycopeptide derived from the
HIV1JRFL strain (E292N339). By using enzymatic transglycosylation and CuAAC
cycloaddition, they prepared di and trivalent constructs incorporating the high
mannose Man
GlcNAc2 glycan at N332, which were recognized by the PGT128 and
9
PGT124like bnAbs [76]. In a related study, the same group pinpointed the fine
epitopes of some V3 bnAbs by exploiting differently glycosylated synthetic V3 glycopeptides (Figure3.2a). Thus, PGT128was found to exhibit binding affinity toward
oligomannose glycopeptides with glycosylationsite flexibility (N301/N332),
whereas the PG124 homolog recognized only the peptide having a high mannose at
N332 and PGT121 required the presence at N301 of a sialylated complextype oligosaccharide [77]. Later, they conjugated the highmannose V3 glycopeptide
(E293N339) to a Thelper epitope from the tetanus toxoid (TT) carrier protein and
(a)
(c) (e)
(b)
(d)
Figure3.2 Synthetic V3-directed bnAb epitope mimics.
3.3 Influenza A Virus
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to the Pam3CysSK4 TLR2ligand (as an adjuvant) (Figure3.2b)[78]. In addition to
this threecomponent “selfadjuvanting” construct, they also synthesized a trivalent
analog presenting three copies of the V3 glycopeptide (Figure3.2c)[79] as well as a
monovalent variant with another V3 glycopeptide fragment derived from a different
HIV1 strain (A244) (Figure3.2d)[80]. Rabbit vaccination studies with these structures showed induction of glycanspecific antibodies that crossreacted with HIV1
gp120/gp140 but did not neutralize HIV1 virions[78–80].
Separately, Alam etal. designed and synthesized, through a twostep Lansbury
aspartylation/NCL strategy, a minimal highmannose V3 glycopeptide (Figure3.2e)
that was bound by PGT128 and PGT125[81]. This Man
construct served to isolate
9
V3 glycan bnAbs from an HIV1infected individual and elicited highmannose
targeted antibodies in vaccinated rhesus macaques, thus mimicking the V3glycan
bnAb epitope. However, no HIV1neutralizing activity was observed. In a subsequent study, Seder and collaborators immunized nonhuman primates with a
designed, dendrimerbased star nanoparticle system presenting several copies of a
minimal synthetic immunogen consisting of a related V3 glycopeptide and a univer-
+
sal CD4
Tcell helper epitope (Pan DRbinding epitope, PADRE). Although high
titers of V3sitedirected antibodies were generated, they showed weak affinity for
nativelike Env trimers and were not able to neutralize HIV1 virions[82].
Despite important recent progress on the design and evaluation of minimal
immunogens based on synthetic V3 Nglycopeptides, V3targeted antibodies with
broadly neutralizing activity have also not been generated. In part, this may be due
to a different, irrelevant conformation/presentation of the V3 synthetic structures in
comparison to that of the native Env glycoprotein epitopes, preventing elicitation of
fully functional antibody responses. Moreover, in addition to the unique features of
bnAbs, a potential reduction of the Bcell precursor pool because of immune tolerance could further limit the induction of these bnAbs by vaccination[16]. Preferably,
an optimal immunogenic construct should activate these rare naive B cells in a
selective manner, whereas further boost immunizations using rationally designed
immunogens should ultimately produce bnAbs by driving B cells along desirable
maturation pathways. With that objective in mind, the identification of clonally
related bnAbs, a suitable Bcelllineage design strategy, and fine structural determination of the epitopes recognized by intermediate Bcell receptors could yield critical insights for the development of effective minimal immunogens for HIV1[83].
85
3.3 Influenza A Virus
Influenza virus affects between 10% and 15% of the global population every year. In
most cases, infection in healthy individuals results in a mild illness in the upper
respiratory tract that does not require any type of surgery. However, it is estimated
that between three and five million of these infections cause severe disease that
progresses to the lower tract and viral pneumonia, resulting in up to 650 000 deaths
3 Carbohydrate-Based Antiviral Vaccines
86
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
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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].
87
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)
88
(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
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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].
89
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
90
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 10 000 times
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.
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
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