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3 Carbohydrate-Based Antiviral Vaccines

(a)

(c)

(e)

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importance of the terminal Manα1→2Man moiety for 2G12 recognition and in agreement with previous crystallographic studies[26]. Based on these observations, several groups have focused on the development of different synthetic structures that can mimic the 2G12 epitope and have assessed their binding affinity in vitro (antigenicity) as well as their ability to elicit bnAbs invivo (immunogenicity).

3.2.1.2 Synthesis and Immunological Evaluation of 2G12 Epitope Mimics

In 2004, Wang and Li synthesized several oligomannoside clusters by conjugating highmannose glycans with cholic acid[32] and galactoside moieties[27] through a maleimide–thiol coupling reaction (Scheme3.1a). Multivalent Man

GlcNAc2 struc-

tures (Figure 3.1a,b) presented relatively high binding affinity to 2G12, but they were not comparable to that of native gp120. In a subsequent study, the tetravalent galactosidescaffolded moiety was conjugated through a maleimidebased linker to keyhole limpet hemocyanin (KLH, a carrier protein that promotes Thelper responses and multivalent antigen presentation) (Figure3.1b) in order to evaluate its capacity to induce bnAbs in rabbits[33]. Unfortunately, most of the antibodies were raised against the maleimide linker, and only modest titers of carbohydratespecific antibodies were elicited, which showed weak crossreactivity against gp120 and no HIVneutralizing activity.

Meanwhile, the Danishefsky laboratory performed the total synthesis of hybrid

type and Man

GlcNAc2 glycan structures and incorporated them into a gp120 pep-

tide fragment via Lansbury aspartylation (Scheme 3.1b), obtaining fully synthetic N322glycosylated gp120 (A316R355) fragments [34, 35]. Although surface plasmon resonance (SPR) experiments showed weak binding of the compounds with 2G12 bnAb, subsequent dimerization of the structures through a disulfide bond (C331) resulted in enhanced antigenicity[36].

Collectively, the studies by the groups of Wang and Danishefsky suggested a crucial effect of glycan multivalency in binding the 2G12 bnAb. Therefore, later efforts have been focused on the design and synthesis of carbohydrate clusters by

(b)

(d)

Scheme 3.1 Conjugation reactions and linker chemistry applied in the synthesis of 2G12 epitope mimics. (a) Thiol–maleimide coupling/thioether linkage, (b) Lansbury aspartylation/ amide linkage, (c) Copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC)/triazole linkage, (d)Amide linkage (via NHS ester), (e)Thiourea linkage.

3.2 Human Immunodeficiency Virus

Table3.1 Significant examples ofsynthetic 2G12 epitope mimics.

Coupling reaction/

Year(s) Author(s)

linkage Template/scaffold

Reference(s)

2004–2006 Wang Maleimide–thiol/

2004 Danishefsky Lansbury

2007 Wang CuAAC/triazole Cyclopeptide (c) [37]

2007–2008 Danishefsky Lansbury

2008 Wong CuAAC/triazole Glycodendrimer (e) [40]

2010 Costantino NHSbased/amide Glycodendrimer (f) [41]

2008 Burton Thiourea BSA protein (g) [42]

2019 Kosma Thiourea BSA protein (h) [43]

2010 Finn, Burton CuAAC/triazole Qβ viruslike particle

2010 Wilson,

Davis

2011–2021 Krauss CuAAC/triazole DNA, peptide (k),

a) Corresponding author(s). b) Structures indicated in Figure3.1.

thioether

aspartylation/amide

CuAAC/triazole Qβ viruslike particle

Cholic acid (a) and galactoside (b)

Gp120 peptide [34–36]

Cyclopeptide (d) [38, 39]

(i)

( j)

and RNA

[27, 32, 33]

[44]

[45]

[46–52]

incorporating glycan moieties into different scaffolds using several conjugation strategies (Scheme3.1). Table3.1 and Figure3.1 illustrate significant examples of synthetic 2G12 epitope mimics developed over the last 15 years.

The synthetic Man

(D1 arm of Man9GlcNAc2) tetravalent constructs (Figure3.1c)

synthesized by the Wang group using CuAAC (Scheme3.1c) afforded relatively high 2G12 affinities, albeit decreased binding was observed for the fluorinated derivative[37]. In parallel, Danishefsky and coworkers prepared a range of Man

GlcNAc2

Nglycan clusters on a modular cyclic peptide (Figure3.1d) via Lansbury aspartylation (Scheme3.1b), with the divalent and trivalent structures showing significantly higher affinities compared to monovalent ones, which confirmed the importance of multivalent presentation. Subsequently, they attached the bivalent compound to the outer membrane protein complex (OMPC, an immunostimulatory carrier protein) via thiol–maleimide coupling (Scheme 3.1a) (~2000 glycopeptide monomers per conjugate) (Figure3.1d)[38], in order to perform immunogenicity studies in guinea pigs and rhesus macaques. Although high levels of carbohydratespecific antibodies were elicited in both species, they were not able to recognize a recombinant HIV gp120 precursor, thus failing to elicit a 2G12like bnAb response[39]. These results show that despite their antigenicity, the synthetic highmannose structures do not mimic the 2G12 antibody epitope realistically, and lack the ability to induce bnAbs, presumably due to a suboptimal oligosaccharide conformation/presentation necessary for efficient immune recognition.

3 Carbohydrate-Based Antiviral Vaccines

(a)

(d)

(g)

(j) (k)

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Other multivalent structures, such as Wong’s Man9 glycodendron (Figure3.1e) obtained via CuAAC (Scheme3.1c)[40] or Costantino’s Man

 and Man9containing

glycodendrimers (Figure3.1f) synthesized using amide coupling (Scheme3.1d)[41], both exhibited significant binding affinities with 2G12. The latter were linked to the CRM

carrier protein via amide linkage (Scheme3.1d) for subsequent immuniza-

197

tion studies invivo[41]. Analogously, Astronomo etal. synthesized a (Man (bovine serum albumin) conjugate via a thiourea linkage (Scheme3.1e) using BSA as a carrier protein for multimeric presentation (Figure 3.1g) and evaluated its immunogenicity in rabbits [42]. In both cases, carbohydratespecific antibodies were generated, but the antisera were not crossreactive to HIV1 gp120 [41, 42]. Notably, Clark etal. showed that a bacterial lipooligosaccharide (LOS) derived from Rhizobium radiobacter Rv3 that included a Man4 D1like arm was bound with

4)14

–BSA

(b) (c)

(e) (f)

(h)

Figure3.1 Synthetic 2G12 bnAb epitope mimics.

(i)

3.2 Human Immunodeficiency Virus

reasonable affinity by 2G12. Mouse immunization with heatkilled Rv3 bacteria elicited glycanspecific antibodies that recognized monomeric gp120 but could not neutralize HIV1 virions[53]. Based on these findings and on the crystallographic structure of the 2G12/LOS complex[54], Kosma and coworkers conjugated several LOSderived, penta or heptamannose structures to BSA and assessed their antigenicity, with a thiourealinked (Scheme3.1e), βanomeric Man

glycan (Figure3.1h)

showing the highest 2G12affinity[43].

In other studies, Finn and collaborators investigated the use of viruslike particles (namely, bacteriophage Qβ), as scaffolds to facilitate a multivalent, ordered presentation of highmannose glycans via triazole linkers (Scheme3.1c) with the purpose of mimicking the oligomannose clustering on gp120. The highest 2G12 affinities were obtained with Qβ–Man

constructs (Figure 3.1i). The first two conjugate types (Qβ–Man4 and Qβ–

Man

) induced mannosespecific antibodies in rabbits that recognized the respec-

Man

, Qβ–Man9, and especially with a mixture of Qβ–Man8/

tive glycans, but they did not crossreact with native gp120 or show HIV1neutralizing activity[44].

In a rationally designed strategy to enhance the immunogenicity of synthetic 2G12 epitope sugar mimics, Davis and coworkers synthesized a number of unnatural mannosederived monosaccharides and their respective D1arm tetrasaccharides for antigenicity and immunogenicity studies [45]. Interestingly, the nonself Man

glycan incorporating a terminal C6methylated mannose (see

Scheme 3.2 for synthesis) showed the highest binding to 2G12, and its corresponding triazolelinked (Scheme3.1c) Qβ conjugate (Figure3.1j) was evaluated in rabbits. Although considerably increased titers of mannosespecific antibodies were induced, they were not crossreactive with native gp120 and failed to neutralize HIV1.

In the last decade, Krauss and coworkers have applied a directed evolutionbased approach to the development of multivalent carbohydrate clusters as effective 2G12 epitope mimics. Following their early work with DNAscaffolded Man

and Man9

Scheme 3.2 Synthesis of unnatural C6-methylated mannose and assembly of a nonself

tetrasaccharide D1-arm mimic.

3 Carbohydrate-Based Antiviral Vaccines

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glycoclusters[46, 47, 55], they developed a method to select Man9bearing multivalent glycopeptides as glycocluster scaffolds by combining mRNA presentation, incorporation of alkynecontaining unnatural amino acids, and subsequent glycan coupling via CuAAC (Scheme3.1c)[48]. This strategy led to invitro selected glycopeptides incorporating 3–5 oligosaccharides that showed binding affinities comparable with natural 2G12–gp120 interactions (in the picomolar to low nanomolar range), which represents the most antigenic 2G12 glycopeptide epitope mimic reported to date. The best Man functionalized CRM

(Scheme 3.1a) (Figure 3.1k) [49] for immunological

197

bearing peptides were conjugated to maleimide

evaluation in rabbits[50]. While glycopeptidereactive antibodies targeting the carbohydrate part were generated, low binding to the nativelike soluble trimeric HIV Env protein (SOSIP) was observed (only in two cases), with negligible HIV1 neutralizing activity. Moreover, the induced antibodies were found to bind mainly to the core mannoses rather than the Manα1→2Man termini recognized by 2G12, which might result from serum mannosidase trimming invivo before immunogen presentation to Bcell receptors[50]. In a subsequent study to assess the effect of the vaccination regimen with a view to promote Manα1→2Manspecific antibodies, an evolved Man

glycopeptide immunogen was coadministered with the QS21 adju-

vant in liposomes using standard bolus dosing, an exponential series of mini doses, or continuous infusion[51]. The two latter regimens led to higher overall IgG titers to the glycopeptide, whereas bolusimmunized mice showed the strongest HIV Env binding antibody response. Nonetheless, Manα1→2Manbinding antibodies were not induced in either case, suggesting that mannosidase activity might be saturated under the bolus immunization protocol, resulting in an increased presentation of intact Man

to B cells, albeit still insufficient to elicit antibodies to the Manα1→2Man

motif. These findings highlight the need for an improved understanding of these biological processes in order to rationally develop optimal bnAbeliciting HIV1 vaccines. Recently, the Krauss group has also developed a novel directed evolution platform for the selection of stable2′fluoromodified RNAsupported Man

glycoclusters

that bind to 2G12with low nanomolar affinities[52].

Taken together, while the synthetic glycoconjugates developed so far could induce oligomannosespecific antibodies with high 2G12binding affinities (as described above), they have not proven to be effective HIV immunogens, failing to elicit 2G12 like antibodies that are crossreactive with the native Env protein to lead to an HIV1neutralizing response. The lack of immunogenicity of these 2G12 epitope mimics can be attributed to several reasons. First and foremost, the conformation of the glycan and its presentation as part of the synthetic immunogen may be different from that of the natural gp120 protein, making it, therefore, unable to recapitulate the native spatial orientation in the Env spike. This may be due to their distinct, inherent physicochemical properties as well as the subsequent glycan processing invivo. For instance, the inner GlcNAc

core that is missing in most of the con-

structs developed may have an important effect in defining the optimal carbohydrate orientation, which together with the high flexibility of the synthetic glycans may lead to unproductive immune recognition of irrelevant oligosaccharide conformations. This notion is supported by observations from Doms and coworkers, who

3.2 Human Immunodeficiency Virus

used yeastderived highmannose glycoproteins as immunogens, presenting

GlcNAc2 glycans in a more dense, nearnative form[56, 57]. Immunization in

Man

rabbits generated carbohydratespecific antibodies that recognized gp120 and efficiently neutralized HIV1 virions expressing highmannose Nglycans but did not neutralize the wildtype virus. Second, considering the rare domainexchange 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 HIV1infected 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 HIV1 Env trimer[58–63]. These bnAbs contain a long, anionic CDRH3loop to penetrate the glycan shield and bind a quaternary motif within the first and second variable loops (V1V2). So far, synthetic glycanbased vaccine development has centered on the binding sites of PG9, PG16, and CH01 bnAbs, which include similar glycandependent 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 highmannose glycans at N160 and N156/N173 and a connected V1V2peptide βstrand[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 carbohydratebased immunogens as synthetic epitope mimics.

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 HIV1 strains with different glycosylation profiles, CAP45 (N156, N160) and ZM109 (N160, N173) (Scheme3.3). Binding analysis by SPR and ELISA revealed that a Man

GlcNAc2

glycan at N160was critical for recognition by PG9 and PG16, while the presence of an additional sialylated complextype oligosaccharide at N156 or N173 further increased the binding affinity[66]. A more efficient chemoenzymatic approach was later developed for the siteselective glycosylation of the peptides with two distinct Nglycans by using orthogonally protected GlcNAcAsn 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 hybridtype glycans bearing oligomannose and

3 Carbohydrate-Based Antiviral Vaccines

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Scheme 3.3 Chemoenzymatic synthesis of V1V2 glycopeptides.

α2,6sialylated 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 HIV1 A244 strain (I148–I184) using chemical synthesis (Scheme3.4). The corresponding glycosyl amines derived from Man GlcNAc

and Man3GlcNAc2 were incorporated at the N156 and N160 residues of

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 mannosebearing Nlinked oligosaccharides[71]. In a followup study, these glycopeptides were dimerized through a disulfide bond (C157), which resulted in even higher binding affinities to bnAbs (in the low

Scheme 3.4 Chemical synthesis of V1V2 glycopeptide.

3.2 Human Immunodeficiency Virus

nanomolar range) and their unmutated common ancestors [72]. In addition, circular dichroism experiments suggested that not only the Man

/Man3 Nglycans

but also the disulfide bondmediated dimerization may contribute to the more stable, βstranded 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 V1V2directed 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 HIV1infected elite neutralizers were found to target epitopes on the V3loop involving the N332 glycan and the V3 peptide backbone [60]. Further detailed characterization of the antibodybinding sites was obtained by crystallographic studies, which revealed an epitope formed by a V3 βstrand and 2 oligomannoses at N332 and N301 for PGT128[73] and a preference toward complextype 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 βlinked Man7 BSA conjugate (see Figure3.1h) 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 highmannose glycan epitope[73]. In rat immunizations, this glycoconjugate induced reasonable levels of carbohydratespecific IgM antibodies but low IgG titers, suggesting weak immunogenicity with Bcell activation in the absence of Tcell involvement. Interestingly, immune sera were crossreactive with native gp120 and even exhibited neutralizing activity against some HIV1 strains, presumably due to avid interactions with polymeric IgMs[75].

3.2.3.3 Synthetic V3 Glycopeptides as bnAb Epitope Mimics

In 2017, Wang and coworkers applied their glycosynthasebased chemoenzymatic strategy for the synthesis of a gp120 miniV3 glycopeptide derived from the HIV1JRFL strain (E292N339). 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

PGT124like bnAbs [76]. In a related study, the same group pinpointed the fine epitopes of some V3 bnAbs by exploiting differently glycosylated synthetic V3 glycopeptides (Figure3.2a). Thus, PGT128was found to exhibit binding affinity toward oligomannose glycopeptides with glycosylationsite 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 complextype oligosaccharide [77]. Later, they conjugated the highmannose V3 glycopeptide (E293N339) to a Thelper epitope from the tetanus toxoid (TT) carrier protein and

(a)

(b)

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(d)

Figure3.2 Synthetic V3-directed bnAb epitope mimics.

3.3 Influenza A Virus

to the Pam3CysSK4 TLR2ligand (as an adjuvant) (Figure3.2b)[78]. In addition to this threecomponent “selfadjuvanting” construct, they also synthesized a trivalent analog presenting three copies of the V3 glycopeptide (Figure3.2c)[79] as well as a monovalent variant with another V3 glycopeptide fragment derived from a different HIV1 strain (A244) (Figure3.2d)[80]. Rabbit vaccination studies with these structures showed induction of glycanspecific antibodies that crossreacted with HIV1 gp120/gp140 but did not neutralize HIV1 virions[78–80].

Separately, Alam etal. designed and synthesized, through a twostep Lansbury aspartylation/NCL strategy, a minimal highmannose V3 glycopeptide (Figure3.2e) that was bound by PGT128 and PGT125[81]. This Man

construct served to isolate

V3 glycan bnAbs from an HIV1infected individual and elicited highmannose targeted antibodies in vaccinated rhesus macaques, thus mimicking the V3glycan bnAb epitope. However, no HIV1neutralizing activity was observed. In a subsequent study, Seder and collaborators immunized nonhuman primates with a designed, dendrimerbased star nanoparticle system presenting several copies of a minimal synthetic immunogen consisting of a related V3 glycopeptide and a univer-

sal CD4

Tcell helper epitope (Pan DRbinding epitope, PADRE). Although high titers of V3sitedirected antibodies were generated, they showed weak affinity for nativelike Env trimers and were not able to neutralize HIV1 virions[82].

Despite important recent progress on the design and evaluation of minimal immunogens based on synthetic V3 Nglycopeptides, V3targeted 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 Bcell 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 Bcelllineage design strategy, and fine structural determination of the epitopes recognized by intermediate Bcell receptors could yield critical insights for the development of effective minimal immunogens for HIV1[83].

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

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