Antibody responses to the HIV-1 envelope high mannose patch

CHAPTER TWO

Antibody responses to the HIV-1 envelope high mannose patch Christine N. Danielsa,b, Kevin O. Saundersa,c,d,e,* a

Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC, United States Department of Medicine, Duke University School of Medicine, Durham, NC, United States Department of Surgery, Duke University School of Medicine, Durham, NC, United States d Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, United States e Department of Immunology, Duke University School of Medicine, Durham, NC, United States *Corresponding author: e-mail address: [email protected] b c

Contents 1. Introduction 2. Categories of high mannose patch HIV-1 neutralizing antibodies 3. V3-glycan HIV-1 neutralizing antibodies against the high mannose patch 3.1 Immunogenetics of V3-glycan broadly neutralizing antibodies 3.2 V3-glycan antibody recognition of envelope 3.3 Autoreactivity and polyreactivity of protruding loop V3-glycan antibodies 3.4 The ontogeny of V3-glycan antibodies 3.5 Neutralization capacity of V3-glycan antibodies 3.6 Preclinical achievements with V3-glycan antibodies 3.7 Human clinical trials of V3-glycan antibody passive immunity 4. Domain-exchanged glycan-dependent HIV-1 neutralizing antibody 2G12 4.1 Discovery of a domain-exchanged HIV-1 antibody 4.2 Env recognition by 2G12 4.3 Structure of domain-exchanged glycan antibodies 4.4 The importance of domain-exchange conformation 4.5 Polyreactivity and autoreactivity of 2G12 4.6 2G12 neutralization breadth and potency 4.7 2G12 neutralization activity and other functions 4.8 Preclinical trials using 2G12 4.9 Clinical trials of 2G12 passive immunity 5. Mannose-restricted glycan-dependent HIV-1 neutralizing antibodies 5.1 Discovery of a mannose-cavity glycan-dependent HIV antibody 5.2 Ontogeny of a mannose-cavity glycan-dependent HIV antibody 5.3 DH501 structure 5.4 Neutralization activity is restricted to Man9GlcNAc2-enriched viruses 6. Vaccine development targeting high mannose patch antibodies 6.1 Vaccine design to elicit V3-glycan HIV-1 neutralizing antibodies

Advances in Immunology, Volume 143 ISSN 0065-2776 https://doi.org/10.1016/bs.ai.2019.08.002

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2019 Elsevier Inc. All rights reserved.

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6.2 Vaccine design to elicit domain-exchanged glycan-dependent HIV-1 neutralizing antibodies 6.3 Vaccine design to elicit mannose-cavity glycan-dependent HIV-1 neutralizing antibodies 7. Conclusions References

56 58 58 59

Abstract Neutralizing antibodies against human immunodeficiency virus subtype 1 (HIV-1) bind to its envelope glycoprotein (Env). Half of the molecular mass of Env is carbohydrate making it one of the most heavily glycosylated proteins known in nature. HIV-1 Env glycans are derived from the host and present a formidable challenge for host anti-glycan antibody induction. Anti-glycan antibody induction is challenging because anti-HIV-1 glycan antibodies should recognize Env antigen while not acquiring autoreactivity. Thus, the glycan network on HIV-1 Env is referred to as the glycan shield. Despite the challenges presented by immune recognition of host-derived glycans, neutralizing antibodies capable of binding the glycans on HIV-1 Env can be generated by the host immune system in the setting of HIV-1 infection. In particular, a cluster of high mannose glycans, including an N-linked glycan at position 332, form the high mannose patch and are targeted by a variety of broadly neutralizing antibodies. These high mannose patchdirected HIV-1 antibodies can be categorized into distinct categories based on their antibody paratope structure, neutralization activity, and glycan and peptide reactivity. Below we will compare and contrast each of these classes of HIV-1 glycan-dependent antibodies and describe vaccine design efforts to elicit each of these antibody types.

1. Introduction Human immunodeficiency virus subtype 1 (HIV-1) expresses envelope glycoprotein (Env) on its surface, which mediates virus entry into permissive cells (Klatzmann et al., 1984; Maddon et al., 1986; Wyatt & Sodroski, 1998). The HIV-1 Env consists of a heterodimer of gp120 and gp41 subunits (Checkley, Luttge, & Freed, 2011). Each gp120:gp41 heterodimer associates with other Env heterodimers to form a trimeric complex of gp120:gp41 heterodimers (Checkley et al., 2011). The HIV-1 Env trimer is a metastable protein meaning it readily undergoes conformational changes (Kong et al., 2016; Lu et al., 2019; Munro et al., 2014; Sullivan et al., 1998; Ward & Wilson, 2017). The ability of the Env to fold into its proper conformation is influenced by glycans that are post-translationally attached to its surface (Li et al., 2008; Stewart-Jones et al., 2016). There are approximately 25 N-linked glycosylation sites on Env (Cao et al., 2018;

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13

Go, Hua, & Desaire, 2014; Pritchard, Spencer, et al., 2015; Pritchard, Vasiljevic, et al., 2015) that comprise 50% of its molecular mass (Leonard et al., 1990). Crystal structures of recombinant Env trimers have shown these N-linked glycans form an interactive network that promote the stability of the HIV-1 Env trimer (Stewart-Jones et al., 2016). The presence of O-linked glycans on virus-associated Env is debated (Bernstein, Tucker, Hunter, Schutzbach, & Compans, 1994; Go et al., 2014; Stansell et al., 2015). While O-linked glycans appear on recombinant gp120, Env purified from virions have lacked such glycans (Bernstein et al., 1994; Go et al., 2014; Stansell et al., 2015). Thus, characterization of N-linked glycans on Env has been the major focus of ongoing research. Seventy percent to 90% of glycans that decorate the Env surface are various forms of high mannose (Bonomelli et al., 2011; Doores, Bonomelli, et al., 2010). In normal glycan biosynthesis, glycans that are attached to the Env polypeptide undergo serial processing by enzymes that cleave off carbohydrate moieties and enzymes that add new ones (Dalziel, Crispin, Scanlan, Zitzmann, & Dwek, 2014). Glycan processing results in a range of glycoforms from minimally processed glycans such as high mannose, intermediately processed hybrid glycans, and more processed forms referred to as complex glycans (Dalziel et al., 2014). The distribution of these different types of glycans can be influenced by the Env form (Bonomelli et al., 2011; Doores, Bonomelli, et al., 2010). Recombinant gp120 monomers tend to harbor more complex glycans, whereas virion-associated gp160 tends to be primarily high mannose (Bonomelli et al., 2011; Doores, Bonomelli, et al., 2010; Go et al., 2015). The most notable high mannose glycans are those proximal to the third variable loop. In the tertiary structure of Env protein these glycans come together to form the high mannose patch (Kong et al., 2013). The high mannose patch is thought to be the result of dense glycans whose close association creates steric hindrance for mannosidases resulting in under-processing of the glycans (Scanlan, Offer, Zitzmann, & Dwek, 2007). The removal of one of the densely packed glycans results in changes in processing of nearby glycans supporting the notion that their close proximity affects their processing (Pritchard, Spencer, et al., 2015). The unusually high density and unique orientation of these glycans makes the high mannose patch a primary target for human glycan-dependent HIV-1 antibodies. HIV-1 Env glycans are critical for virus infectivity (Dash, McIntosh, Barrett, & Daniels, 1994). Scanning mutagenesis experiments of Env glycosylation sites have shown that elimination of particular glycans inhibits the

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Christine N. Daniels and Kevin O. Saunders

infectivity of HIV-1 and chimeric simian-human immunodeficiency virus (SHIV; Dash et al., 1994; Johnson, Sauvron, & Desrosiers, 2001; Kanekiyo et al., 2013; Lee, Yu, Syu, Essex, & Lee, 1992). These mutant viruses have defective Env intracellular trafficking and tend to not be cleaved from gp160 into gp120 and gp41 (Dash et al., 1994). Thus, Env glycosylation is an essential post-translational modification for HIV-1 replication. The density and flexibility of glycans on HIV-1 Env allows them to cover the majority of HIV-1 envelope peptides (Pancera et al., 2014; Stewart-Jones et al., 2016). For this reason the dense collection of glycans on Env is called the HIV-1 Env glycan shield (Wei et al., 2003). Since HIV-1 Env is the sole target for neutralizing antibodies, glycosylation plays a critical role in antibody neutralization activity (Wei et al., 2003). It is hypothesized that any antibody that is capable of binding the fusioncompetent, native Env trimer will be able to neutralize HIV-1—termed the occupancy model (Corti & Lanzavecchia, 2013; Yang, Lipchina, Cocklin, Chaiken, & Sodroski, 2006). However, the human antibody repertoire has a limited proportion of antibodies capable of binding to high mannose glycans (Schneider et al., 2015), which limits the number of antibodies capable of binding HIV-1 Env. The proportion of mannose-reactive antibodies is low presumably to avoid autoreactivity between self antibodies and self glycoproteins (Wardemann et al., 2003). The impact of glycosylation on neutralizing antibody responses was initially highlighted in the Wei et al. longitudinal study of viruses during natural infection (Wei et al., 2003). In this study neutralizing antibodies developed 52 days after initial seroconversion, however, circulating, neutralizationsensitive virus was replaced with neutralization-resistant virus (Wei et al., 2003). Comparison of the sequences of neutralization-sensitive and resistant viruses over time found that Env glycosylation sites were added or shifted during infection concurrent with the acquisition of neutralization resistance. Mutation of the newly acquired glycosylation sites changed resistant viruses back to sensitive viruses (Wei et al., 2003). This study highlighted the presence of an evolving glycan shield, which HIV-1 virions utilized to escape contemporaneous neutralizing antibody pressure (Wei et al., 2003). It also defined that glycosylation could preclude access to otherwise neutralizing epitopes (Wei et al., 2003). Corroborating these findings, several studies have removed glycans near the receptor binding site for HIV-1 Env and found that those viruses were 1000-fold more sensitive to neutralization (Koch et al., 2003; Zhou et al., 2017). Given that HIV-1 Env encodes up to 31 potential N-linked glycosylation sites, which can have

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heterogenous occupancy and can shift locations (Wei et al., 2003), the ability of envelope to occlude neutralization sensitive sites is quite substantial. Epitope mapping studies of serum neutralizing antibodies has shown the high mannose patch to be one of the most common broadly neutralizing epitopes targeted during human natural HIV-1 infection (Landais et al., 2016; Walker et al., 2010). Advances in antibody cloning from antigenspecific, single B cells have resulted in the isolation and characterization of the responsible monoclonal glycan-dependent neutralizing HIV-1 antibodies (Bonsignori et al., 2011; McCoy & Burton, 2017). The most broad and potent neutralizing antibodies target various glycans in the high mannose patch and recognize different forms of mannose (Doores, 2015; Kong et al., 2013). Thus, the primate immune system has found multiple solutions for interacting with glycans on HIV-1 Env. Below, the different categories of HIV-1 glycan-dependent antibodies will be compared and contrasted, and the antibodies that comprise each category will be described in an effort to highlight the differences present even within each category. Passive infusion studies have shown these antibodies to be important for protective immunity against HIV-1 (Moldt et al., 2012; Nishimura et al., 2017; Pegu et al., 2014); thus, current vaccine strategies for eliciting HIV-1 high mannose patch neutralizing antibodies will be discussed.

2. Categories of high mannose patch HIV-1 neutralizing antibodies The first category of high mannose patch HIV-1 neutralizing antibodies is called V3-glycan broadly neutralizing antibodies (bnAbs). V3-glycan bnAbs are principally characterized by their interaction with HIV-1 Env glycan at asparagine 332 (N332) and polypeptide through long complementarity determining regions (CDR) loops within their paratopes (Fig. 1; Barnes et al., 2018; Bonsignori, Kreider, et al., 2017; Doores et al., 2015; Garces et al., 2015, 2014; Kong et al., 2015). Both V3-glycan and V1V2-glycan antibodies can bind the N156 glycan (Bonsignori et al., 2011; Garces et al., 2015, 2014; McLellan et al., 2011; Sok, Doores, et al., 2014; Walker et al., 2009), but V1V2-glycan antibodies will not be discussed here. Instead, antibodies that focus on the N-linked mannose glycans at asparagine residues 295, 301, 332, 339, 386, and 392 will be described. The second category of high mannose patch HIV-1 neutralizing antibodies is termed the heavy chain variable region (VH) domain-exchanged

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PGT128

A

B

PGT122

HIV-1 Env HIV-1 Env

N332 glycan

HCDR2

HCDR3 N332 glycan

N301 glycan

VH

VL VH

VL

Fig. 1 V3-glycan antibodies use protruding CDR loops to contact glycan and envelope peptide. (A) Crystal structures of PGT128 (green; PDB: 3TYG) and (B) PGT122 (orange; PDB: 4TVP) bound to HIV-1 envelope (Env, gray) respectively. The heavy chain variable (VH) regions and light chain variable (VL) regions of the antibody Fabs are shown. PGT128 is bound to the HIV-1 Env outer domain with a truncated V3 loop. PGT122 is bound to BG505 SOSIP gp140 (gray), however, only one protomer of the envelope trimer is shown for clarity. N-linked glycans (yellow) at position N301 and N332 are shown as stick structures. The protruding complementarity determining region (CDR) loops utilized by the two V3-glycan antibodies are shown in magenta. The protruding loop in PGT128 is the result of a 6 amino acid insertion in the second heavy chain CDR (HCDR2). The third heavy chain CDR (HCDR3) loop in PGT122 is formed by VHDJH recombination and protrudes out to contact peptide and glycan on HIV-1 Env.

bnAbs. At the present, this category contains only one antibody called 2G12 (Kunert, Ruker, & Katinger, 1998; Trkola et al., 1996). 2G12 was one of the earliest isolated broadly neutralizing antibodies (Kunert et al., 1998; Trkola et al., 1996). This antibody contacts only mannose glycans on Env in order to bind to it; hence it exclusively binds glycans. While 2G12 lacks the unusually long CDRs of proteoglycan HIV-1 broadly neutralizing antibodies, it possesses a unique domain-exchanged antibody structure that is requisite for the neutralizing activity of this antibody (Calarese et al., 2003; Doores, Fulton, Huber, Wilson, & Burton, 2010; Wu et al., 2013). The third category of high mannose patch HIV-1 neutralizing antibodies is mannose-cavity neutralizing antibodies. This category of antibody possesses a deep cavity on its paratope into which mannose inserts. The initial monoclonal antibody of this category was isolated from a vaccinated rhesus macaque and is called DH501 (Saunders, Nicely, et al., 2017). This type of antibody has broad neutralization activity when HIV-1 Env glycosylation is enriched for

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Man9GlcNAc2 glycans. Together, these three categories describe the glycandependent antibody response to the high mannose patch.

3. V3-glycan HIV-1 neutralizing antibodies against the high mannose patch 3.1 Immunogenetics of V3-glycan broadly neutralizing antibodies Multiple lineages of protruding-CDR loop V3-glycan high mannose patch antibodies have been isolated from numerous individuals during natural infection (Bonsignori, Kreider, et al., 2017; Freund et al., 2017; Poignard et al., 1999; Simonich et al., 2016; Sok et al., 2016; Walker et al., 2011). Notable examples of V3-glycan antibody lineages include DH270, PGT121, PGT128, BG18, PCDN33, PGDM11, PGDM21, PGDM31 and BF520 (Bonsignori, Kreider, et al., 2017; Sok et al., 2016; Walker et al., 2011). Unlike some CD4 binding site broadly neutralizing antibodies, V3-glycan antibodies are not restricted in their heavy chain or light chain gene usage (Table 1). V3-glycan bnAbs like PGT121, PGT128, and VRC29, have somatic mutation frequencies higher than the 6–10% nucleotide mutation percentage observed for most antibodies in the repertoire (Kwong & Mascola, 2012; Longo et al., 2016; Tiller et al., 2007). The notable exceptions, are BF520.1, DH270.1, and PCDN33 which developed neutralization breadth with limited somatic mutation (Bonsignori, Kreider, et al., 2017; MacLeod et al., 2016; Simonich et al., 2016). The third heavy chain CDR (HCDR3) lengths tend to be relatively long (Briney, Willis, & Crowe Jr, 2012) with PGT121, PGDM31, and DH270.6 antibodies having 24, 22, and 20 amino acid HCDR3s, respectively (Bonsignori, Kreider, et al., 2017; Sok et al., 2016; Walker et al., 2011). As with other HIV-1 broadly neutralizing antibodies (Kepler et al., 2014), nucleotide insertions and deletions are common among V3-glycan antibodies (Table 1). Mutational analysis has shown that removal of these insertion and deletions reduces antibody neutralization function (Doores et al., 2015). The long HCDR3s and insertions creates long protruding CDR loops that are characteristic of this type of HIV-1 antibody (Fig. 1). The discovery of DH270 and PCDN33 antibody lineages was particularly interesting because these antibodies were the first broadly neutralizing protruding-CDR loop high mannose patch antibodies that lacked insertions and deletions (Bonsignori, Kreider, et al., 2017; MacLeod et al., 2016) Since insertions and deletions are rare genetic events (Briney, Willis, & Crowe Jr., 2012;

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Table 1 Immunogenetics of high mannose patch HIV-1 neutralizing antibodies. V3-glycan antibodies Antibody

VH

JH

a

DH270.6

1–2

4

18

L2–23 2

10

PGT121

4–59 6

24

L3–21 3

12

BG18

4–4

6

21

L3–25 3

11

PGT128

4–39 5

19

L2–8

PGT135

4–39 5

18

K3–15 1

9

BF520

1–2

4

18

K3–15 3

11

PGDM11

3–11 3

19

K2–24 1

9

HCDR1(
2)

PGDM21

4–34 6

18

K3–20 5

9

HCDR2(+4)

PGDM31

1–8

6

22

K3–20 3

9

PCDN38B 4–34 5

22

K3–20 1

8

VRC21

3–30 4

12

L6–57 3

10

VRC22

4–34 6

19

K1–17 1

9

HCDR2(
1)/ HCDR2(+5)

VRC28

4–34 4

26

K2–28 1

9

HCDR2(+6)

VRC29

4–59 3

20

K3–20 1

9

K1–5

2

9

L1-D

2

9

HCDR3 VL

JL

LCDR3

2 or 3 10

b

Indel

LFR1(
7)/LFR3(+3)

HCDR2(+6)/ LCDR1(
5) HCDR1(+5)

Domain-exchanged antibody 2G12

3–21 3

14

Mannose-cavity antibody c

DH501

a

2-B

4

15

Kabat CDR3 definition. Nucleotide insertion or deletion. Rhesus macaque gene segments.

b c

Wilson et al., 1998), it was hypothesized that DH270 and PCDN33 may represent antibody lineages with the greatest potential to be elicited in multiple individuals by vaccination or infection (Bonsignori, Kreider, et al., 2017; MacLeod et al., 2016). At the present, there have not been genetic signatures of V3-glycan antibodies defined. However, there are some similarities among the antibodies that can be noted. First, there is an abundance of VH4 gene usage

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among the different antibodies (Table 1). Second, IGKV3–20 is utilized by many members of this antibody category. Third, an insertion of 4–6 amino acids in HCDR2 is common among these antibodies. Overabundant usage of particular genes can signify germline-encoded residues that make productive contacts with Env (Garces et al., 2014; Scharf et al., 2013; West Jr, Diskin, Nussenzweig, & Bjorkman, 2012). Future experiments should seek to understand the mechanism facilitating these three genetic similarities.

3.2 V3-glycan antibody recognition of envelope V3-glycan antibodies recognize a proteoglycan epitope composed of glycans in the high mannose patch and underlying peptide (Barnes et al., 2018; Kong et al., 2013, 2015; Pancera et al., 2014; Pejchal et al., 2011). In general, the antibodies contact the glycan at asparagine 332 (N332), although other surrounding glycans such as N295, N301, N339, N386, or N392 can also contribute to binding interactions (Kong et al., 2013; Pejchal et al., 2011). PGT124 is the exception to this statement, since its crystal structure showed it only bound to N332 glycan (Garces et al., 2014). This single glycan contact provided a structural explanation for why nearly all viruses lacking the N332 glycan are resistant to PGT124 (Garces et al., 2014). However, it should be noted that the absolute dependence on the presence of N332 glycan for neutralization varies across the different V3-glycan antibodies even when they are part of the same antibody lineage. DH270 lineage antibodies bind both N301 and N332 glycans (Fera et al., 2018), but they require the N332 glycan for neutralization activity of most primary isolates (Bonsignori, Kreider, et al., 2017). Similarly, PGT135 lacks neutralization of viruses glycosylated at N334 instead of N332 (Sok, Doores, et al., 2014). In contrast, PGT121 and PGT128 antibodies can tolerate the loss of the N332 glycan (Sok, Doores, et al., 2014). PGT121 compensates for the loss of the N332 glycan by utilizing glycans in the first variable region (V1) at position 137, 156, and 160 (Sok, Doores, et al., 2014). PGT121 and PGT124 are from the same lineage, and yet they evolved to recognize the high mannose patch glycans differently hence they have differing requirements for the N332 glycan (Garces et al., 2015; Sok, Doores, et al., 2014). PGT128 tolerates the removal of the N332 glycan by utilizing other glycans within the high mannose patch, such as N295 or N301 (Doores et al., 2015; Sok, Doores, et al., 2014). N332 glycan is the principal glycan contact made by this category of antibodies and is conserved in 73% of HIV-1 isolates

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in the Los Alamos National Laboratory sequence database (Sok, Doores, et al., 2014). However, multiple antibodies within this category have devised mechanisms to bind in the absence of the N332 glycan. The different types of glycans recognized by this category of antibodies have been investigated by glycan arrays and crystallography. In the initial description of the PGT128 lineage antibodies glycan arrays were used to show the antibodies bound well to Man9GlcNAc2 and Man8GlcNAc2 with terminal mannose on the D1 and D3 arms (Man8GlcNAc2 D1D3; Walker et al., 2011). Subsequently, the crystal structure of PGT128 in complex with Man9GlcNAc2 alone showed it relied on HCDR2 and FWR2 to contact the D1 arm and LCDR3 (N94, W95, D95a) to contact the D3 arm of the glycan (Pejchal et al., 2011). Man9GlcNAc2 and Man8GlcNAc2 D1D3 are the only high mannose glycans with mannose residues terminating the D1 and D3 arm; thus the atomic-level interactions explained the glycan specificity determined by glycan array. The recognition of glycan by PGT128 is similar to PGT135 (Kong et al., 2013). Specifically, both PGT128 and PGT135 bind the entire length of the N332 glycan, but they do some from opposite sides, and PGT135 preferentially binds Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 glycans (Kong et al., 2013). The structure of PGT128 in complex with HIV-1 Env gp120 outer domain showed that it interacted with both the N332 and N301 glycans (Pejchal et al., 2011). However, the N332 glycan appears to be the main glycan contact as the light chain-mediated interactions with N332 glycan were largely required for antibody function. The HCDR2 insertion mediated contacts with the N301 glycan while reaching toward Env peptide. Hence its deletion reduced neutralization activity and eliminated Man8GlcNAc2 binding (Pejchal et al., 2011). The DH270 lineage antibodies bind more strongly and more broadly to high mannose glycans as they undergo affinity maturation (Bonsignori, Kreider, et al., 2017). Antibodies early in the DH270 developmental pathway exhibited undetectable binding to free mannose in glycan arrays (Bonsignori, Kreider, et al., 2017). As the antibodies acquired more somatic mutations they bound strongly to Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Bonsignori, Kreider, et al., 2017). The most mutated, broad, and potent members of the DH270 lineage bound also to Man5GlcNAc2 and Man6GlcNAc2 (Bonsignori, Kreider, et al., 2017). The molecular interaction with the N332 glycan is mediated by the protruding HCDR3 of 20 amino acids (Fera et al., 2018). Specifically, D115 in HCDR3 contacts the D1 arm of Man9GlcNAc2 at N332 and appears to

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be the principal contact made with the N332 glycan (Fera et al., 2018). This D115-glycan interaction is not present in minimally affinity matured DH270 lineage members due to R98 forming a salt bridge with D115 (Fera et al., 2018). Somatic mutation of R98 to threonine allows D115 to be free to interact with glycan (Fera et al., 2018). It is hypothesized that the release of D115 from R98 may be why DH270.6 binds more strongly and broadly to mannose glycans (Fera et al., 2018). In addition to D115, W101, Y105, and Y106 exhibit hydrophobic interactions with the N332 glycan (Fera et al., 2018). Y48 in the light chain CDR2 also contributes to N332 glycan contact (Fera et al., 2018). DH270.6 also contacts the D3 arm of Man9GlcNAc2 via D31 in the HCDR1 (Fera et al., 2018). Mutation of most of these glycan contacts reduces antibody binding to Env (Bonsignori, Kreider, et al., 2017). Thus, glycan recognition requires multiple amino acids and is an essential aspect of DH270 binding to Env (Bonsignori, Kreider, et al., 2017; Fera et al., 2018). The PGT121 lineage antibodies recognize different glycans on Env as they evolve (Garces et al., 2014; Gristick et al., 2016; Mouquet et al., 2012). The PGT121 lineage includes PGT124, which does not react with glycans in glycan arrays, but has been shown to bind to Man8GlcNAc2 D1D3 in crystal structures of PGT124 in complex with HIV-1 Env outer domain of gp120 (Garces et al., 2014). In the co-complex structure PGT124 bound to a single Man8GlcNAc2 attached at the N332 glycan site. The PGT124 LCDR1, LCDR2, HCDR3, and light chain framework region (FWR)3 all made glycan contacts (Garces et al., 2014). The principle amino acids that contributed to glycan contact were R100 in the heavy chain and N50, N51, I66b in the light chain, where mutation of these residues dramatically reduced antibody binding (Garces et al., 2014). Within the PGT121 lineage, PGT124 and PGT122 bind to the N332 glycan similarly. PGT122 bound to HIV-1 Env trimer bound to N332 glycan via LCDR2 and HCDR3 ( Julien, Cupo, et al., 2013). Conversely, PGT121 binds to high mannose glycans via one site on its paratope and complex glycans via a second site (Mouquet et al., 2012). Modeling PGT121 onto the crystal structure of PGT122 and Env trimer indicates the complex glycans at N137 would precisely fill the groove into which complex glycan has been shown to bind within the PGT121 paratope (Garces et al., 2014; Julien, Sok, et al., 2013; Mouquet et al., 2012). Likewise, PGT122 interacts with the N137 glycan via HCDR1 and HCDR2 (Garces et al., 2014; Julien, Cupo, et al., 2013), and this glycan interaction is required for neutralization ( Julien, Cupo, et al., 2013). The combination of PGT124 and PGT122 in

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complex with HIV-1 Env trimer and the structure of PGT121 with complex glycan has shown that the PGT121 lineage evolved complex glycan recognition through somatic mutation at positions 31, 53, and 56 (Garces et al., 2014; Mouquet et al., 2012). The antibody interaction with glycans has been resolved for BG18. BG18 was isolated from a clade B elite controller who maintained low viral loads for over 30 years (Freund et al., 2017). BG18 used an arginineisoleucine-tyrosine-glycine-valine-valine motif in its HCDR3 to contact the N332 glycan (Barnes et al., 2018). For BG18, LCDR2, HCDR1, and HCDR3 contacted the N332 glycan (Barnes et al., 2018). In addition to the N332 glycan, BG18 also contacted N156, N392 and N386 glycans on Env (Barnes et al., 2018). The BG18 paratope possesses two characteristic clefts (Barnes et al., 2018; Freund et al., 2017). One of the clefts is important for glycan binding, since the N392 glycan inserts into one of them (Barnes et al., 2018). The conserved peptide contacts of V3-glycan antibodies usually include G324, D325, I326, and R327 amino acids (Garces et al., 2014; Pejchal et al., 2011). This collection of amino acids is called the GDIR motif, are conserved in 54% of HIV-1 isolates, and are located proximal to the N332 glycan (Pejchal et al., 2011; Sok et al., 2016). V3-glycan high mannose patch antibodies vary as to which amino acids in the GDIR motif they require (Garces et al., 2014; Sok et al., 2016). These differences are evident when neutralization is compared across the different antibody lineages. For instance, the PGT121 lineage of antibodies can tolerate the loss of D325 alone, whereas the PGDM21 lineage cannot (Garces et al., 2014; Sok et al., 2016). Similarly, D325N is a neutralization resistance mutation for DH270 (Bonsignori, Kreider, et al., 2017), whereas this mutation has little effect on PGT124 neutralization (Garces et al., 2014). Antibodies within a single lineage can also differ in their GDIR motif dependence. PGT128 and PGT130 are two clonally-related antibodies that differ in their dependence on G324 and D325 within the GDIR motif. While PGT128 neutralization is largely unaffected by alanine substitution at G324 and D325, PGT130 neutralization is reduced by >1000-fold by mutation of either the G324 or D325 site (Sok et al., 2016). Conversely, clonally-related antibodies PGDM11 and 12 both require R327 for potent neutralization activity. At the molecular level there are differences in how the antibodies interact with the GDIR motif. For PGT128, the GDIR motif fits into a groove between HCDR2 and HCDR3. PGT128 has a protruding HCDR2 loop due to a 6 amino acid insertion (Table 1, Fig. 1), and a protruding 19 amino acid

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HCDR3. The PGT128 HCDR3 R100A, Y100B, T100C, and D100D make backbone contacts with the GDIR motif. For this reason, PGT128 can tolerate amino acid substitutions within the GDIR motif. In contrast, PGT124 forms side chain-mediated hydrogen bonds with the GDIR motif. More specifically, PGT124 uses a protruding HCDR3 loop of 24 amino acids to reach alongside of the N332 glycan to contact the base of the V3 loop (Garces et al., 2014). PGT124 forms a side chain-mediated salt bridge between the E100I in its heavy chain and R327 in Env (Garces et al., 2014). The Y100B of the HCDR3 of PGT124 makes critical contact with R327 in the GDIR motif. Additionally, LCDR1 and LCDR3 both contact the D325 in the GDIR motif. Both R327 and D325 must be mutated for PGT124 to lose Env reactivity (Garces et al., 2014). BG18 and the PGT121 lineage antibodies make very similar contacts with the GDIR motif. The BG18 HCDR3 salt bridges with R327 of the GDIR motif via a glutamate residue and a tyrosine in the BG18 HCDR3 binds to D325 (Barnes et al., 2018). DH270 interacts with the GDIR motif through a combination of both backbone interactions (R57 in HCDR2 and D107 in HCDR3) and side chain interactions (N52 in HCDR2; Fera et al., 2018). In total, antibody binding modes to the GDIR motif are diverse among antibody lineages and within antibody lineages. Given that HIV-1 rapidly mutates to escape neutralizing antibodies it was an open question as to why the GDIR motif, or related sequence GDIK, was conserved in many HIV-1 isolates. Insight into this question came when the sequences of HIV-1 isolates that utilize different co-receptors were compared. HIV-1 isolates that used CCR5 as a co-receptor tended to have GDIR motifs (Sok et al., 2016). In contrast, viral isolates that used CXCR4 tended to encode other amino acids at positions 324 through 327 (Sok et al., 2016). The presence of the GDIR motif in many CCR5-dependent HIV-1 isolates suggested the site may be critical for CCR5 binding. Indeed, mutation of the GDIR motif reduced CCR5 peptide binding to Env gp120 (Sok et al., 2016). Thus, it was hypothesized that the N332 and N301 glycans are present on the Env to shield the conserved GDIR motif from antibody recognition. This hypothesis is consistent with most polyclonal antibody plasma samples being able to neutralize HIV-1 more potently when the N332 glycan is removed (Sok et al., 2016). However, the GDIR motif is likely not the primary contact site for CCR5 since the GDIR motif can be mutated in a CCR5-tropic Env sequence and the resulting virus is still infectious (Sok et al., 2016). This means that the mutant virus is still able to utilize CCR5 sufficiently well to mediate virus entry.

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Despite targeting a common epitope, each of the V3-glycan antibodies have distinct binding modes (Kong et al., 2013; Pancera et al., 2013). The initial discovery of the difference in PGT121, PGT128, and PGT135 antibody binding mode was determined with complementation experiments (Pancera et al., 2013). In these experiments the heavy chain and light chains from each of the lineages were swapped with each other. HIV-1 neutralization was used to assess function of the chain exchanged antibodies. Antibodies were capable of neutralizing HIV-1 when complemented with heavy chains or light chains from their own lineage, but the antibodies lost activity when paired with immunoglobulin chains from outside their lineage (Pancera et al., 2013). In a later study, Kong et al. resolved the structure of PGT135 in complex with envelope and showed that PGT128, PGT135, and 2G12 all bind to the high mannose patch with different orientations (Kong et al., 2013). Similarly, the negative-stain electron microscopy structure of PGDM14 bound to HIV-1 Env trimer showed a dramatically different angle of approach for this antibody as compared to PGT124, PGT128, or PGT122 (Sok et al., 2016). The variation in antibody binding modes to the same neutralizing epitope on Env is viewed as a beneficial characteristic of this type of antibody. Kong et al. asserted that this observation shows the immune system can devise multiple solutions for recognizing this bnAb epitope (Kong et al., 2013). Therefore, efforts to induce high mannose patch neutralizing antibodies with vaccination may be more easily achievable since there are fewer molecular restrictions and multiple binding modes for binding the high mannose patch and GDIR motif (Kong et al., 2013; Sok et al., 2016). Different HIV-1 neutralizing antibodies engage different forms of HIV-1 envelope (Doria-Rose et al., 2014; Falkowska et al., 2014; Pejchal et al., 2011; Walker et al., 2009). A subset of antibodies preferably bind envelope trimers versus envelope monomers consisting of only the gp120 subunit (Doria-Rose et al., 2014; Longo et al., 2016; Sok, van Gils, et al., 2014; Walker et al., 2009). Most of the well-characterized V3-glycan antibodies recognize monomeric gp120 subunits from Env (Bonsignori, Kreider, et al., 2017; Kong et al., 2013; Pejchal et al., 2011). However, antibodies VRC28 and VRC29 preferentially bind trimeric HIV-1 Env as either full-length membrane-bound gp160 trimers or soluble gp140 trimers. This preference for trimeric envelope has mostly been observed for V1V2-glycan antibodies where antibody binding crosslinks the trimers (Doria-Rose et al., 2014; Lee, Ozorowski, & Ward, 2016; Sok, van Gils, et al., 2014; Walker et al., 2009). Additionally, gp120–gp41

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interface antibodies such as PGT151 (Falkowska et al., 2014) preferentially bind Env trimer due to their gp120 and gp41 contacts (Falkowska et al., 2014; Lee et al., 2016). It is presently unclear why VRC28 and VRC29 prefer to bind to Env trimers. The preference for trimer does not result in broader neutralization activity. In fact, PGT121, PGT128, and DH270.6 all bind to gp120 and have neutralization breadth superior to VRC28 or VRC29 (Longo et al., 2016; Walker et al., 2011). Moreover, PGT128 and DH270 can bind to glycosylated peptides that mimic the base of the V3 loop and N301 and N332 glycans (Alam et al., 2017; Bonsignori, Kreider, et al., 2017; Fera et al., 2018; Pantophlet et al., 2017). While the importance of trimer preference still remains to be determined, the preference for Env trimer suggests that HIV-1 Env immunogens should be trimeric in order to ensure engagement of B cells encoding VRC29-like antibodies (Longo et al., 2016).

3.3 Autoreactivity and polyreactivity of protruding loop V3-glycan antibodies HIV-1 bnAbs have been shown to possess autoreactivity (Haynes et al., 2005; Haynes & Verkoczy, 2014). Thus, autoreactivity of PGT121, 128, and 135 lineage antibodies was tested against common autoantigens in diagnostic panels of autoimmune disease (Walker et al., 2011). These particular V3-glycan antibodies do not react with any of the normal autoantigens in this diagnostic panel suggesting they lack overt autoreactivity (Walker et al., 2011). However, a larger panel of 9400 human proteins was tested for binding to PGT121, PGT125, and PGT128 to assess polyreactivity and autoreactivity (Liu et al., 2015). While, all three antibodies were negative for autoreactivity, PGT125 and PGT128 bound to several human proteins indicating some level of polyreactivity (Liu et al., 2015). B cells expressing polyreactive B cell receptors can be eliminated during positive selection, thus relaxed immunotolerance may be a contributing factor for the immune system to generate PGT128 lineage antibodies (Haynes & Verkoczy, 2014).

3.4 The ontogeny of V3-glycan antibodies Coevolution studies have been performed to elucidate the pathway that led to induction of PCDN and DH270 antibody lineages (Bonsignori, Kreider, et al., 2017; Poignard et al., 1999). For DH270, the antibody evolution was followed in the infected individual CH848 by cloning monoclonal

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antibodies from memory B cells (Bonsignori, Kreider, et al., 2017). Moreover, next generation sequencing was performed to detect low copy numbers of DH270-related sequence throughout infection (Bonsignori, Kreider, et al., 2017). Nearly 1000 sequences of autologous envelopes were isolated throughout the course of infection to understand how the envelope changed during infection, and to identify which autologous CH848 envelopes bound to the DH270 lineage antibodies (Bonsignori, Kreider, et al., 2017). Interestingly, the precursor of DH270 did not react with the putative transmitted/founder virus isolated from the infected individual CH848 (Bonsignori, Kreider, et al., 2017). Instead, two other lineages of N332 glycan-dependent antibodies called DH272 and DH475 reacted with the transmitted founder envelope and its early intermediates (Bonsignori, Kreider, et al., 2017). These antibodies neutralized autologous viruses, but lacked broad neutralization of viruses not from CH848 (Bonsignori, Kreider, et al., 2017). These antibodies lost reactivity with the autologous envelopes upon deletion of 17 amino acids in the first variable (V1) loop of CH848 envelopes early during infection (Bonsignori, Kreider, et al., 2017). This deletion led to a V1 loop of 17 amino acids which was conducive to DH270 antibody binding (Bonsignori, Kreider, et al., 2017). This deletion of the V1 loop coincided temporally with the expansion of the DH270 lineage (Bonsignori, Kreider, et al., 2017). Thus, the DH270 lineage likely was bound and stimulated to expand by an envelope variant that escaped from DH272 selective pressure (Bonsignori, Kreider, et al., 2017). The DH270 lineage acquired neutralizing activity against envelopes from other individuals with acquisition of a critical mutation at position 57 (Bonsignori, Kreider, et al., 2017). Specifically, a G57R mutation resulted in neutralization of diverse HIV-1 isolates (Bonsignori, Kreider, et al., 2017). The G57R mutation generated interactions with the GDIR motif, which is why it was critical for HIV-1 envelope binding (Fera et al., 2018). Early in the DH270 development pathway, the heavy chain acquired a R98T mutation that was critical for antibody binding to Env (Bonsignori, Kreider, et al., 2017; Fera et al., 2018). This mutation had an indirect effect on N332 glycan binding in that R98T eliminated a salt bridge between R98 and D115 (Fera et al., 2018). D115 was then able to mediate interactions with the N332 glycan (Fera et al., 2018). The autologous Envs continued to evolve in the presence of the DH270 lineage, which ultimately led to escape variants (Bonsignori, Kreider, et al., 2017). The virus escaped DH270 with the introduction of a D325N mutation in the GDIR motif, and by replacing the N332 glycosylation site with a N334 glycosylation site (Bonsignori, Kreider, et al., 2017). Both of these mechanisms are

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common HIV-1 escape paths from V3-glycan antibodies (Klein et al., 2012; Krumm et al., 2016; Moore et al., 2012; Sadjadpour et al., 2013). The evolution of the PCDN lineage of antibodies was followed similarly by isolating monoclonal antibodies and autologous envelope sequences (MacLeod et al., 2016). The early antibodies within the lineage did not neutralize viruses isolated before month 5 of infection, but were able to neutralize viruses that occurred at month 10 post infection. Thus, the lineage of antibodies appeared to start between month 5 and 10 of infection. Monoclonal antibodies isolated from month 27 developed autologous virus neutralization breadth and potency. In contrast to the PCDN antibodies that arose early during infection, PCDN antibodies isolated after month 27 were able to neutralize month 5 viruses. Similar to the DH270 antibody virus co-evolution, the autologous viruses continued to evolve and eventually escaped all of the isolated PCDN monoclonal antibodies by month 38 of infection. The principal determinants of virus escape were mapped to positions 328, 332, and 335. The presence of K328 and removal of a glycosylation site at position 335 by introduction of an N335A change generated PCDN antibody-sensitive autologous viruses. These two changes were required to occur together in order to change virus sensitivity to PCDN antibody neutralization. The largest effects of the 328 and 335 sites were on the minimally-mutated PCDN antibodies isolated early in the development of the lineage. The broadly neutralizing PCDN antibodies were able to tolerate changes at position 328 and a N335 glycan. The PCDN antibody-resistant autologous viruses at months 38 and 44 eliminated the N332 glycosylation site by changing N332 and/or S334 amino acids suggesting these changes as an escape mechanism. To confirm the removal of the N332 glycosylation site as an escape mechanism, N332A mutant autologous viruses were generated in the context of viruses from months 5, 10, and 19. All of the wildtype viruses were sensitive to PCDN38B neutralization, but the N332A viruses were all resistant. Thus, elimination of the N332 glycosylation site was a principal mechanism for escape from the broadly neutralizing PCDN antibody lineage during infection (MacLeod et al., 2016).

3.5 Neutralization capacity of V3-glycan antibodies 3.5.1 PGT121 Protruding loop V3-glycan antibodies are among the most potent broadly neutralizing antibodies that have been isolated (Fig. 2; Walker et al., 2011). PGT121 is notable for its neutralization potency and breadth. It

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Antibody HIV-1 neutralization titer 0.0001 62%

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Fig. 2 V3-glycan antibodies potently and broadly neutralize HIV-1. Antibody neutralization of HIV-1 infection in vitro. Neutralization titers are shown as the concentration (μg/mL) of antibody that inhibits 50% of virus replication (IC50). Each symbol indicates an individual HIV-1 isolate. Horizontal bars indicate the geometric mean for all viruses. Geometric mean IC50 values for only the neutralized viruses are provided in the text. 2G12 (blue) is a domain-exchanged anti-glycan HIV-1 antibody that is included for comparison. Neutralization results were compiled by the CATNAP program (https:// www.hiv.lanl.gov/components/sequence/HIV/neutralization/main.comp).

neutralizes 63% of a 208-virus panel of currently circulating global viral isolates (Bonsignori, Kreider, et al., 2017). PGT121 is highly potent based on its geometric mean IC50 of 0.02 μg/mL against the 131 sensitive viruses in the panel (Bonsignori, Kreider, et al., 2017). The potential biochemical basis of PGT121 neutralization was investigated by isothermal calorimetry ( Julien, Sok, et al., 2013). PGT121 potentially neutralizes virus by inducing an allosteric conformational change that inhibits CD4 binding to gp120 ( Julien, Sok, et al., 2013). PGT121 has high affinity for Env consistent with its high neutralization potency. Studies from Moquet et al. reported a positive linear correlation between affinity for Env and neutralization titer (Mouquet et al., 2012). Thus, PGT121 neutralization potency increases as a result of improved binding to HIV-1 Env. Several mechanisms of escape or resistance to PGT121 have been identified. PGT121 neutralization capacity largely hinges on accessibility to N332 although it possesses some ability to recognize other glycans in the absence of N332 (Sok, Doores, et al., 2014). The ability of antibodies to utilize alternative glycans is referred to as glycan promiscuity (Sok, Doores,

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et al., 2014). Bricault et al. reported a correlation between N-linked N332 glycosylation site frequency and clade sensitivity (Bricault et al., 2019). Eighty-four percent of viruses from a panel of 119 difficult-to-neutralize (tiers 2 and 3) viruses with amino acid changes at gp120 N332 or N334 acquired resistance to PGT121 neutralization (Bricault et al., 2019). Of this panel, clade C viruses were amongst the most poorly neutralized. Clade C viruses commonly lack a N332 glycosylation site (Bricault et al., 2019). Similarly, circulating recombinant form 01 (CRF01) HIV-1 isolates typically lack N332 and are resistant to PGT121 (Bricault et al., 2019). Conversely, viruses that acquire asparagine at position 332 become sensitive (Mouquet et al., 2012). PGT121 must undergo affinity maturation through somatic hypermutation in order to acquire its potent neutralization capability (Mouquet et al., 2012). Inferred antibody precursors of PGT121 were unable to neutralize HIV-1 (Mouquet et al., 2012). Interestingly, Sok et al. was able to show that some PGT121 lineage antibodies with a moderate degree of somatic mutation are capable of neutralizing with moderate breadth (Sok, Doores, et al., 2014). A variant with nucleotide mutation frequencies as low as 6% was able to neutralize 46% of a 72 virus panel albeit with weaker neutralization titers than PGT121 (Sok, Doores, et al., 2014). 3.5.2 PGT128 PGT128 exhibits remarkable neutralization potency with a geometric mean IC50 of 0.056 μg/mL (excluding viruses not neutralized). Also, PGT128 has neutralization breadth of 61% (402 of 656) (calculated in CATNAP; Julien, Sok, et al., 2013). The PGT128 mode of neutralization is suspected to involve increasing avidity locally through Fab binding (Pejchal et al., 2011). Studies examining binding to HIV-1 Env on the cell surface showed IgG bound 70-fold more potently than Fab (Pejchal et al., 2011). Additionally, PGT128 IgG may neutralize the virus by leading to its decay, since HIV-1 decayed 11.2-fold faster in the presence on PGT128 compared to its absence (Pejchal et al., 2011). PGT128 displays similar patterns of neutralization and clade sensitivity as PGT121 with slightly expanded breadth (Walker et al., 2011). This extensive breadth is due in part to promiscuous binding of glycans near N332 and tolerance of variation within the GDIR motif. Like other V3 glycantargeting antibodies, the PGT128 epitope centers on N332 (Pejchal et al., 2011). However, PGT128 is better able to tolerate the loss of glycan at N332 than PGT121 and other V3-glycan antibodies (Sok, Doores, et al.,

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2014). In the absence of N332 PGT128 is able to utilize N301 and/or N295 (Sok, Doores, et al., 2014). Therefore, a N301A amino acid change also leads to increased HIV-1 resistance to PGT128 (Krumm et al., 2016). Using the less optimal N301 and/or N295 sites PGT128 is still able to neutralize multiple viruses at titers less than IC50 of 0.02 μg/mL (Sok, Doores, et al., 2014). In a screen against a panel of viruses that naturally express N332, N334 or neither asparagine, PGT128 and PGT130 were the most effective at neutralizing viruses with N334 instead of N332. PGT121 displayed intermediate neutralization of viruses with N334. Other V3-glycan antibodies such as PGT135, were completely reliant on the presence on N332 for potent neutralization (Sok, Doores, et al., 2014). 3.5.3 PGT135 PGT135 has a neutralization breadth of 34% (neutralized 126 of 369 viruses) with a geometric mean IC50 of 0.045 μg/mL for 126 sensitive viruses (calculated by CATNAP). PGT135 has vast differences in clade sensitivity and breadth as compared to other V3-glycan antibodies such as PGT128. PGT135 neutralized 30% of clade C viruses and close to 40% of clade B viruses from a panel of 162 viruses (Walker et al., 2011). These values are considerably lower than the 60–80% range achieved by PGT121 and PGT128 (Walker et al., 2011). On another panel of 162 viruses, PGT128 was able to neutralize 50% of strains in comparison to 33% by PGT135 (Walker et al., 2011). The differences in neutralization breadth are likely due in part to the number of contacts required for neutralization for each antibody. PGT128 requires contacts with only a few key glycans, whereas PGT135 requires extensive contacts with N332, 392, and 386 (Kong et al., 2013). PGT135 uses its two long HCDR loops, which are critical for neutralization activity, to penetrate the glycan shield and contact these glycans (Kong et al., 2013). Hence, V3-glycan antibodies vary in their neutralization breadth with PGT135 being among the least broadly neutralizing antibodies of this category. 3.5.4 DH270 The most broad and potent member of the DH270 lineage neutralizes 55% of tested viruses making it not as broad as PGT128 or PGT121, but more broadly neutralizing than PGT135 (Bonsignori, Kreider, et al., 2017; Walker et al., 2011). Bonsignori et al. tracked the ontogeny of the DH270 lineage from the time of natural infection to 245 weeks post infection. These studies demonstrated that autologous viruses were completely

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resistant to DH270 UCA (IC50 > 50 μg/mL), but with affinity maturation HIV-1 neutralization breadth was acquired (Bonsignori, Kreider, et al., 2017). Somatic variants of the lineage, DH270 IA3 and DH270.4, acquired potent neutralization of viruses with V1 loop lengths ranging between 17 and 24 amino acids (IC50 0.5–2 μg/mL). The ability of DH270 antibodies to neutralize was dependent on the presence of a deletion in the V1 loop of the transmitted founder virus. DH270 HIV-1 neutralization was low for viruses with V1 loops >24 residues, with near loss of neutralization of most strains with loop lengths exceeding 30 residues. As the DH270 lineage continued to affinity mature, DH270.6 was able to neutralize autologous and heterologous viruses regardless of loop length. To determine if this trend was lineage specific or a common feature of V3-glycan antibodies, they reexamined these viruses using other V3 glycan antibodies: 10–1074, PGT121 and PGT121. The ability to recognize viruses with longer V1 loops consistently coincided with a reduction in neutralization potency. Collectively these studies unveiled an inverse relationship between bnAb potency and V1 length (Bonsignori, Liao, et al., 2017). As with the antibodies described above, neutralization breadth depends on the frequency of the N332 glycosylation site within each clade (Bonsignori, Liao, et al., 2017). Of the N332 glycan-targeting antibodies, the DH270 lineage is amongst the least able to tolerate loss of N332. Viruses that undergo N-linked glycosylation site changes from N332 to N334 are almost completely resistant to DH270 lineage antibodies. Bonsignori et al. showed that only 1 of 62 viruses that lacked N332 glycosylation, was sensitive to neutralization by affinity-matured lineage members DH270.5 and DH270.6. The heterologous neutralization breadth and potency of DH270 lineage antibodies are the result of an accumulation of somatic mutations at different stages during development. While the unmutated precursor of DH270 lacked neutralization breadth, heterologous neutralization breadth was detected for DH270.3 which is 11.8% mutated based on nucleotide sequences. It neutralized 42% of a diverse 24-virus panel. DH270.6, 12.9% mutated, neutralized 71% of the same panel (Bonsignori, Liao, et al., 2017). Heterologous neutralization by DH270 early intermediate antibodies was observed if the virus was produced in kifunensine-treated cells (Saunders, Nicely, et al., 2017). This suggested that initially, V3-glycan antibodies recognize virus with abundant Man9GlcNAc2 or Man9GlcNAc2, and later evolve to tolerate glycan heterogeneity (Doores et al., 2015; Saunders, Nicely, et al., 2017). The development of neutralization potency and breadth in the DH270 lineage relied on several

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critical mutations (Bonsignori, Liao, et al., 2017). The improbable G169C nucleotide mutation encoded the G57R amino acid change that conferred neutralization against autologous virus and was shown to be necessary for heterologous neutralization breadth in the DH270 lineage (Bonsignori, Liao, et al., 2017; Fera et al., 2018; Wiehe et al., 2018). Thus, somatic mutation directly affected neutralization breadth, due to critical amino acids such as R57 that were acquired during affinity maturation (Bonsignori, Liao, et al., 2017). 3.5.5 BF520 BF520.1 is not as potent as other antibodies in the V3-glycan category, with a geometric mean IC50 of 1.95 μg/mL for viruses that are sensitive to it (Simonich et al., 2016). However, its development is intriguing. The BF520 antibody lineage developed in an HIV-infected infant in <1 year of HIV-1 infection (Simonich et al., 2016). This antibody was notable as bnAbs usually take multiple years to develop in adults (Landais & Moore, 2018). Passive transfer of the antibody from the mother to infant could have accounted for such rapid development of a bnAb. However, studies were performed to confirm BF520 developed in the baby and was not passively transferred from the mother (Simonich et al., 2016). Early intermediate antibody BF250 was capable of binding to the transmitted virus (Simonich et al., 2016). Although this early binding did not result in neutralization, a subsequent antibody BF520.1 developed 2 months afterward, and acquired the ability to neutralize multiple heterologous viruses across multiple clades and tiers (Simonich et al., 2016). When tested against a global virus reference panel, BF520.1 displayed similar neutralization breadth as several first and second generation adult bnAbs (PGT151, 4E10, 2F5) (Simonich et al., 2016). BF520.1 acquired neutralization breadth with 2–6% somatic mutation of the nucleotide sequence compared to 3.8–38.6% for typical adult bnAbs. The low number of somatic mutations needed for neutralization activity is one reason the lineage was able to develop bnAb activity so quickly (Simonich et al., 2016). Neutralization activity of BF520.1 is based on the N332 glycan, where removal of the N332 glycosylation site weakens neutralization up to 95-fold (Simonich et al., 2016). BF520.1 showed that neutralization breadth was not always reliant on the acquisition of high numbers of somatic mutations over many years of infection. 3.5.6 VRC29 VRC29 is one of three V3-glycan antibody lineages isolated from the chronically-infected individual N170 (Longo et al., 2016). Similar to

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PGT128, VRC29.03 can tolerate loss of N332 to some degree by using other nearby glycans such as N301 (Longo et al., 2016). Removal of the glycan at N301 more dramatically weakens neutralization than mutation of the N332 glycosylation site (Longo et al., 2016). Thus, the N301 glycan may be more important for antibody binding to Env than the N332 glycan. Structural analyses of VRC29 are still needed to explain the role of N301 and N332 glycans. Despite binding to the same two glycans as PGT128, the most potent member of the VRC29 lineage, VRC29.03, displays only moderate potency and breath in comparison to PGT128 (Fig. 2). In regards to clade specificity, the VRC29 lineage had poor neutralization of clade C virus with only moderate neutralization of sensitive clade B viruses (IC50 ranging from 0.1 to 1 μg/mL; Longo et al., 2016). Since VRC29 preferentially binds to Env trimer, it can be concluded that the preference for binding to Env trimer does not automatically confer extraordinary neutralization potency and breadth (Longo et al., 2016). Lastly, the VRC29 lineage is unusual in that the degree of somatic mutation does not correlate with neutralization potency (Longo et al., 2016). 3.5.7 BG18 BG18 has extensive breadth (62%, 74/119 viruses) and with a geometric mean IC50 of 0.03 μg/mL is comparable in potency to some CD4bs bnAbs (Freund et al., 2017). BG18 is more mutated and has greater potency than other V3-glycan bnAbs such as PGT121 and 10–1074. BG18 neutralization activity is largely N332 restricted (Freund et al., 2017). Removal of this glycan in epitope mapping assays resulted in undetectable levels of neutralization (Freund et al., 2017). Interestingly, studies of HIV-infected mice treated with BG18 monoclonal therapy showed a statistically significant reduction in viral load, but subsequently displayed rebound viremia, due to acquisition of mutations at the N332 site (Freund et al., 2017). This finding provides further support for the N332 dependency of this lineage (Freund et al., 2017). In summary, among the V3-glycan antibodies, the most potent and broadly neutralizing antibodies are BG18, PGT128, and PGT121 (Fig. 2). Envelope V1 length is a major contributing factor to bnAb potency (Bonsignori, Kreider, et al., 2017). In general, bnAbs are able to neutralize viruses with greater potency when V1 loops are shorter (Bonsignori, Kreider, et al., 2017). Increases in length coincide with a decrease in viral sensitivity to neutralization (Bonsignori, Kreider, et al., 2017; van den Kerkhof et al., 2016). Another key factor that influences potency and breadth is the angle of approach with which the antibody targets its epitope.

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This orientation determines which, and how many contacts with the viral Env surface can be made. Antibodies that develop orientations that enable avoidance and/or accommodation of obstructive glycans are more likely to be broad neutralizers (Garces et al., 2015). Additionally, antibodies that bind in an orientation that makes contacts with multiple glycans are more equipped to retain potency against viruses that undergo N-linked glycosylation site changes (Krumm et al., 2016; Sok, Doores, et al., 2014). Conversely, antibodies that are reliant on a single glycosylation site are more likely to experience viral escape (Krumm et al., 2016; Sok, Doores, et al., 2014).

3.6 Preclinical achievements with V3-glycan antibodies V3-glycan antibodies are promising antibodies for passive antiviral or prophylactic immunity due to their neutralization potency (Fig. 2). Initial studies using a high-dose of simian-human immunodeficiency virus (SHIV) delivered to the vagina of macaques showed that the potent V3-glycan antibody PGT121 could protect all animals when serum concentrations were as low as 15 μg/mL (Moldt et al., 2012). Sixty-seven percent protection was achievable at serum PGT121 levels 10-fold lower (Moldt et al., 2012; Rosenberg et al., 2016). The protection afforded by PGT121 is often not sterilizing immunity. Instead some innate cells become infected, but are subsequently cleared (Liu, Zhai, Zheng, & Ye, 2016). One possible explanation for the non-sterilizing protection is that V3-glycan antibodies can lack complete neutralization of HIV-1 (McCoy et al., 2015). PGT121 lacks complete neutralization of SHIV-327c and it protected only 86% of macaques challenged mucosally with this virus ( Julg, Sok, et al., 2017). The mechanisms of PGT121 protection were investigated by comparing the protective capacity of wild-type PGT121 and an effector function-silent PGT121 in macaques (Parsons et al., 2019). Comparable protection against intravenous challenge with SHIV-infected cells was observed for PGT121 capable or incapable of mediating effector functions (Parsons et al., 2019). It was concluded that PGT121 neutralization activity was sufficient for protection against cell-associated virus (Parsons et al., 2019). This observation was contradictory of earlier studies with B12, where this comparison showed wildtype antibody to be more protective than Fc effector-function-silent antibody (Hessell et al., 2007). Effector functions of PGT121 may be more important for protection against high doses of cell-associated virus. PGT121 shows only 50% protection against macaque challenge when they are

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challenged with high-doses of SHIV-infected cells compared 100% protection against low doses of infected cells (Parsons et al., 2019, 2017). Hence, an antibody with the ability to kill HIV-infected cells and neutralize free virus may provide optimal protection. Subsequent macaque studies sought to challenge the macaques with a more physiologic dose of virus and to mimic the repeated exposure to HIV-1 experienced by humans at high-risk for infection. In this experiment, 10–1074 was administered once to macaques (Gautam et al., 2016). Subsequently the macaques were challenged weekly with low doses of pathogenic SHIV (Gautam et al., 2016). Control animals that did not receive a HIV-1 neutralizing antibody required only 6 challenges for all 6 animals to become infected compared to 23 weekly challenges for all animals that received 10–1074 IgG (Gautam et al., 2016). The V3-glycan antibodies have also been effective antiviral agents in humanized mouse and macaque models of HIV-1 infection (Caskey, Klein, & Nussenzweig, 2019). In humanized mouse models reconstituted with primary PBMCs from HIV-infected humans, 10–1074 has been shown to delay the rebound of detectable virus in the reconstituted mouse (Flerin et al., 2019). In the macaque SHIV model system, PGT121 suppressed viremia below detectable levels, but when antibody levels waned most of the animals became viremic again ( Julg, Liu, et al., 2017; Julg, Pegu, et al., 2017). Intriguingly, a small subset of PGT121-treated macaques maintained undetectable viral loads after PGT121 was no longer detectable in serum (Barouch et al., 2013). Virologic control has also been achieved when V3-glycan antibodies are paired with CD4 binding site antibodies (Nishimura et al., 2017). In these experiments, virologic control after antibody administration was dependent on CD8+ T cell responses (Nishimura et al., 2017). Combinations of antibodies tended to suppress SHIV viremia in macaques and HIV viremia in humanized mice longer than single antibody administrations (Klein et al., 2012; Shingai et al., 2013). The role of antibody Fc effector functions in virus suppression is still debated. In contrast to wildtype 10–1074 IgG1, a version of 10–1074 which lacked Fc effector functions showed limited ability to prevent viral rebound (Flerin et al., 2019), suggesting Fc effector functions were key for antiviral activity. Passive infusion of PGT121 into SHIV-infected macaques has suggested Fc effector functions are not needed to suppress viremia (Parsons et al., 2019). When Fc effector functions were silenced with L234A/L235A amino acid changes in the antibody Fc, PGT121 was still able to suppress viremia (Parsons et al., 2019). Suppressing viremia may be achieved by prevention of infection by free virus through direct neutralization, whereas delaying viral rebound and decreasing

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integrated provirus may be more dependent on killing of virally-infected cells. Only the latter mechanism would require Fc effector function. While suppressing viremia is one goal for antivirals, another goal is to eliminate cells harboring latent provirus. These cells are a major barrier to curing HIV-1 infection, and current HIV-1 antivirals do not eliminate latently infected cells (Abner & Jordan, 2019). V3-glycan antibodies have been tested for their ability to eliminate the latent reservoir. SHIV infection in macaques were suppressed with antiretroviral therapy (ART) to allow establishment of latently infected cells (Borducchi et al., 2018). Subsequently, ART treatment was stopped and the SHIV-infected macaques were administered a TLR7 agonist called Vesatolimod to activate latent provirus. PGT121 was then administered to kill infected cells (Borducchi et al., 2018). Vesatolimod and PGT121 treatment delayed plasma viremia re-emergence, with a subset of macaques showing no viral rebound (Borducchi et al., 2018).

3.7 Human clinical trials of V3-glycan antibody passive immunity In initial safety and pharmacokinetic studies, 10–1074 was administered to HIV-infected and HIV-uninfected individuals. The antibody half-life was reduced twofold in HIV-1 infected individuals (Caskey et al., 2017). Nonetheless, the 10–1074 administration reduced viral load in individuals with sensitive virus at the time of antibody infusion. Sensitive virus eventually was replaced with 10–1074-escape variants and viremia returned (Caskey et al., 2017). This trial result was encouraging, but as seen in preclinical studies with 10–1074 ( Julg, Liu, et al., 2017; Klein et al., 2012), HIV-1 mutates to escape 10–1074 monotherapy (Caskey et al., 2017). Combinations of antibodies reduced viremia better in animal models than monotherapy (Bolton et al., 2016; Gautam et al., 2018; Klein et al., 2012), thus antibody combinations have been administered as antivirals to HIV-infected humans (Bar-On et al., 2018; Mendoza et al., 2018). 10–1074 in combination with 3BNC117 was administered to 7 HIV-1 infected individuals to determine their ability reduce viral load (Bar-On et al., 2018; Mendoza et al., 2018). Together the antibodies suppressed the viral load by 2 log10 (Bar-On et al., 2018). The combination therapy prevented the emergence of virus resistance, enabling virus suppression for about 21 weeks (Bar-On et al., 2018). In a similar study in HIV-infected individuals on antiretroviral drugs, had their antiretroviral drug treatment interrupted and the combination of 10–1074 and 3BNC117 was administered to them to assess viral rebound and latent reservoir elimination (Mendoza et al., 2018). The combination

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of 10–1074 and 3BNC117 suppressed viremia for a significantly longer time than 3BNC117 alone provided that the virus reservoir in the participant was sensitive to 10–1074 and 3BNC117 (Mendoza et al., 2018). The superior delay in viral rebound with the addition of 10–1074 suggested it had an effect on virus suppression. The effects of 10–1074 was evident in the amino acid sequences of viruses isolated after viremia rebound (Mendoza et al., 2018). Mutations within the 10–1074 contact sites were evident indicating immunologic selective pressure by 10–1074 most likely once 3BNC117 dropped below its effective concentration (Mendoza et al., 2018). Given the success of PGT121 in nonhuman primate preclinical trials ( Julg, Liu, et al., 2017), the antibody is planned to be used in at least four upcoming clinical trials (Clinical trials: NCT03928821, NCT03721510, NCT03205917, and NCT02960581). Currently, PGT121 is being administered alone or in combination with another HIV-1 bnAb to HIV-1 negative women via subcutaneous injection to study its safety and pharmacokinetics (Mahomed et al., 2019). A subset of women will receive one injection and another group will receive two injections. The results of the trial are expected in 2019 (Mahomed et al., 2019). Recent progress in passive immunity for glycan-independent HIV-1 antibodies has been summarized elsewhere (Caskey et al., 2019). A roadblock for passive immunity has been sustainability of the antibody in circulation. This roadblock has been resolved by two different approaches. First, the Fc of antibodies can be optimized to bind better to receptors that prevent antibody catabolism (Ko et al., 2014; Saunders, 2019). Several studies using these modified Fc versions of protruding loop V3-glycan antibodies have shown that the introduction of M428L and N434S mutations in the Fc improves antibody half-life in macaques (Gautam et al., 2018; Zalevsky et al., 2010). The extended half-life resulted in detectable 10–1074 for 7–10 weeks after a single injection (Gautam et al., 2018). The half-life enhanced 10–1074 provided more durable protection than the wildtype 10–1074 requiring 37 challenges before all of the animals were infected (Gautam et al., 2018). These results have provided the rationale for Fc optimized antibodies to be tested in humans (Clinical trials: NCT02599896, NCT02797171, NCT02840474, NCT03554408). Second, the genes expressing the V3-glycan antibody can be delivered for long-term expression (Badamchi-Zadeh et al., 2018). Adeno-associated viral vectors have been the most widely used delivery platform for these antibodies (Badamchi-Zadeh et al., 2018; Balazs & West Jr, 2013; Saunders et al., 2015). As part of an antibody cocktail, the genes for 10–1074 were

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delivered to macaques with an Adeno-associated viral vector (MartinezNavio et al., 2019). 10–1074 was expressed at 50 μg/mL serum concentrations for 2 years (Martinez-Navio et al., 2019). The gene-delivered HIV-1 antibodies suppressed viral load to undetectable levels for 3 years leading to a functional cure of infection (Martinez-Navio et al., 2019). This approach has been mostly hindered by anti-drug antibodies against the HIV-1 antibodies, but when these antibodies are controlled gene delivery of antibodies has been successful (Gardner et al., 2019; Martinez-Navio et al., 2016; Saunders et al., 2015). Methods for controlling the anti-drug antibody response have been to use IgG2 isotype antibodies instead of IgG1 or short courses of immunosuppressive drugs (Gardner et al., 2019; Saunders et al., 2015). Adenovirus serotype 5 has also been used to deliver PGT121 genes in humanized mice (Badamchi-Zadeh et al., 2018). The genes expressed detectable PGT121 in the serum within 1 day, and the antibodies suppressed viral load in infected humanized mice (Badamchi-Zadeh et al., 2018). Gene delivery of broadly neutralizing V3-glycan antibodies still requires additional development, but preclinical studies provide hope that this approach could permanently suppress viremia (Martinez-Navio et al., 2019).

4. Domain-exchanged glycan-dependent HIV-1 neutralizing antibody 2G12 4.1 Discovery of a domain-exchanged HIV-1 antibody The domain-exchanged glycan-dependent antibodies against the high mannose patch differ from the V3-glycan antibodies both structurally and phenotypically. This category is distinguished by a rare domain-exchange in the antigen binding fragment of the antibody (Fab) and its sole dependence on glycan reactivity for Env engagement. Currently, 2G12 is the only known monoclonal antibody that belongs to this category (Kunert et al., 1998). Below, the structural and phenotypic features of 2G12 will be contrasted with the features of V3-glycan antibodies. 2G12 is considered as one of the first-generation broadly neutralizing antibodies (Burton & Hangartner, 2016; Trkola et al., 1995, 1996). It is derived from VH3–21 and possesses a short HCDR3 of 14 amino acids (Table 1). These features are distinct from the protruding-loop V3-glycan antibodies discussed above that have long CDR3 loops ranging from 18 to 24 amino acids in length or insertions in HCDR2 (Table 1). Also, 2G12 lacks insertions and deletions, which are present in many of the protruding-CDR loop V3-glycan antibodies (Table 1). Thus, its genetics are more typical of most antibodies compared to V3-glycan antibodies.

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4.2 Env recognition by 2G12 The epitope of 2G12 was defined by several studies over many years. HIV-1 Env mutagenesis experiments initially identified the glycans in the high mannose patch as part of the 2G12 epitope (Sanders et al., 2002; Scanlan et al., 2002; Trkola et al., 1996). Elimination of N295, N332, N386, N392, and N448 resulted in decreased Env binding by 2G12 (Sanders et al., 2002). Subsequent electron microscopy structures of 2G12 bound to HIV-1 Env showed 2G12 contacted N295, N332, N339, and N392 glycans N386 and N448 glycans are positioned near the 2G12 epitope, but direct contacts with these glycans were not observed (Murin et al., 2014). Similarly, the N137 glycan in the V1 loop was close in proximity to the binding site but was not directly contacted (Murin et al., 2014). Due to the close proximity of the V1 and V2 loops to the 2G12 epitope their lengths regulate 2G12 binding to its epitope (Chaillon et al., 2011). Indeed, shortening the V1V2 loops confers better neutralization activity by 2G12 (Chaillon et al., 2011). 2G12 is specific for high mannose glycans (Calarese et al., 2005; Garces et al., 2014; Scanlan et al., 2002). Specifically, 2G12 binds to linear tetramannose that contain a1➔2 linkages (Calarese et al., 2005, 2003; Martines, Garcia, Marradi, Padro, & Penades, 2012; Scanlan et al., 2002). Tetramannose with a1➔2 linkages are present in Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2; thus 2G12 recognizes these glycans in microarray binding assays (Garces et al., 2014; Walker et al., 2011).

4.3 Structure of domain-exchanged glycan antibodies The hallmark of this type of antibody is its domain-exchanged Fab arms first revealed by the crystal structure of the 2G12 Fab (Calarese et al., 2003). In that complex structure, the heavy chain variable region (VH) of the two Fabs, but not the first constant domain (CH1), were shown to crossover and pair with adjacent light chains (Fig. 3). The resultant structure looked like the heavy chain Fab made an X-shape with light chains interacting with VH and CH1 from two different heavy chains. The overall consequence of the domain-exchange is an antibody with a linear I-shape, instead of a traditional Y-shape; antigen binding sites on each Fab arm that are about ˚ apart (West Jr et al., 2009; Wu et al., 2013); and three antigen bind33–35 A ing sites instead of two (Calarese et al., 2003). Negative stain electron microscopy reconstructions have suggested the third antigen binding site on the 2G12 domain-exchanged Fab binds N332 and N339 glycans and the primary VH antigen binding sites bind N295 and N392 glycans

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A

VH

B Intermolecular domain swap

VH

VL

VL

CH1

CL

CL

CH1

a b’

a’ b

b’ a

b a’

2G12

2G12

Fig. 3 Domain-exchanged 2G12 molecules. (A) Crystal structure of the domain exchanged 2G12 Fabs (PDB: 4NHH). The heavy chain variable (VH) and light chain variable (VL) regions are shown for the two Fab molecules. The domain exchange occurs where VH and the first constant domain (CH1) interact with the VL and light chain constant region (CL) on two different light chains. When the light chain is from the same IgG molecule it creates an I-shaped IgG with 3 binding sites. Two of the three binding sites are formed by VH-VL domains and the third binding site is formed between the VH-VH domains. (B) The heavy chain VH and VL can also swap between two different IgG molecules. These intermolecular domain-exchanged antibodies connect two IgG molecules together creating 6 antigen binding sites. Shown here is one 2G12 molecule in red and another in cyan. The light chains are shown in lighter shades. VH and CH1 domains are marked as a, a0 , b, and b0 for each molecule to clearly visualize the domain-exchanged arms of the IgG.

(Murin et al., 2014). The domain-exchange has been confirmed by mass spectrometry and protein unfolding analyses (Watanabe et al., 2018). To date, the domain-exchanged structure remains unique only to 2G12. The sequence of 2G12 provides favorable biochemistry for the occurrence of domain exchange. First, 2G12 has an unusual proline at the elbow region connecting the VH and CH1 domains of the Fab (Calarese et al., 2003). Second, 2G12 has weaker VH interactions with VL which allows the exchange of VH with an adjacent VL (Calarese et al., 2003). Third, the sequence is conducive for creating a third antigen binding site after domain-exchange (Calarese et al., 2003). To investigate which amino acids confer each of these aspects, Huber et al. added somatic mutations to the putative germline precursor of 2G12 in an attempt to make the antibody exchange its domains (Huber et al., 2010). Eight mutations were identified in the VH of 2G12 that were sufficient for domain-exchange (Huber et al., 2010). These amino acids include P14A, R19I, Q39R, I57R, K75E, S77F, A84V, and S113P (Huber et al., 2010). S77F made little contribution to the domain exchange and R19I was absolutely required for domain exchange (Huber et al., 2010). The obligatory nature of the I19 residue was shown by reverting it back to germline sequence. The single reversion completely

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switched the antibody back to the conventional, non-domain-exchanged Fab configuration (Huber et al., 2010). Naturally, 2G12 does not appear to switch between Fab configurations (Huber et al., 2010). Thus, 2G12 acquired only 7–8 critical amino acid changes through somatic mutation that conferred substantial domain-exchange conformation (Huber et al., 2010).

4.4 The importance of domain-exchange conformation The binding activity of 2G12 is directly affected by its domain-exchanged configuration. Only the domain exchanged form of 2G12 is able to bind to HIV-1 Env glycans (Doores, Fulton, et al., 2010). Moreover, elimination of the domain-exchange conformation with a I19R amino acid change eliminated domain-exchange and 2G12 neutralization activity (Doores, Fulton, et al., 2010; Huber et al., 2010). In some instances, two IgG molecules can domain exchange two VHs with each other resulting in an intermolecular 2G12 dimer (Fig. 3B; West Jr et al., 2009; Wu et al., 2013). The crystal structures and negative stain electron microscopy structures of these interlocked 2G12 molecules showed that dimers of IgG as well as trimers of IgG could be formed by 2G12 IgG (Stanfield, De Castro, Marzaioli, Wilson, & Pantophlet, 2015; Wu et al., 2013). The dimeric form of Fab2 of 2G12 exhibits 3-orders-of-magnitude more potent neutralization than the monomeric Fab2 (West Jr et al., 2009; Wu et al., 2013). Similarly, the dimeric Fab2 exhibited increased neutralization breadth compared to the monomeric Fab2 (West Jr et al., 2009). The mechanism underlying the improved neutralization activity is the ability of the dimer to mediate bivalent binding to gp120 (Wu et al., 2013). Although, the antibody had a nonconventional overall shape, dimeric 2G12 was still able to mediate ADCC (Klein, Webster, Gnanapragasam, Galimidi, & Bjorkman, 2010). Rational protein design efforts aiming to increase the frequency of 2G12 intermolecular dimer formation found that the deletion of T223 and H224 (Eu numbering) in the hinge region increased the percentage of intermolecular dimers formed by 2G12 (West Jr et al., 2009). The intermolecular dimers have also been shown to protect against HIV-1 infection in humanized mice (Luo et al., 2010). Thus, intermolecular dimerization of antibodies enhances their neutralization activity creating novel protective antibodies.

4.5 Polyreactivity and autoreactivity of 2G12 The mannose reactivity of domain-exchanged antibodies would suggest they could interact with multiple proteins. For 2G12, the glycan binding is relatively high affinity compared to other glycan antibodies, but 2G12

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prefers to bind to glycan clusters (Scanlan, Offer, et al., 2007; Scanlan, Ritchie, et al., 2007). The glycans within the cluster need to be in a specific spatial orientation so that they fit into the glycan binding sites on the 2G12 paratope (Scanlan, Offer, et al., 2007). Presumably, this need for a specific spatial arrangement limits 2G12 polyreactivity and autoreactivity with human proteins (Scanlan, Offer, et al., 2007; Scanlan, Ritchie, et al., 2007). Liu et al. specifically tested 2G12 polyreactivity and autoreactivity (Liu et al., 2015). 2G12—unlike PGT128 and PGT125—has been shown to lack polyreactivity when tested for binding to microarrays containing >9400 different human proteins (Liu et al., 2015). Additionally, the glycoforms on human proteins limit 2G12 reactivity since many human glycoproteins have processed glycans distinct from high mannose (Scanlan, Offer, et al., 2007). Thus, on most human glycoproteins high mannose is not sufficiently clustered or spatially organized to result in 2G12 recognition (Scanlan, Offer, et al., 2007). This hypothesis is supported by the fact that 2G12 can bind to human cells if they are treated with the glycosylation inhibitor kifunensine (Scanlan, Ritchie, et al., 2007). Kifunensine inhibits glycan processing resulting in Man8GlcNAc2 or Man9GlcNAc2 glycosylation (Scanlan, Ritchie, et al., 2007), both of which are antigenic for 2G12 (Walker et al., 2011). Thus, glycoforms present on human proteins are a principal factor limiting 2G12 autoreactivity. In addition to HIV-1, mannose glycans are found on fungi and bacteria (Clark et al., 2012; Dunlop et al., 2010; Luallen et al., 2010; Stanfield et al., 2015). For bacteria, 2G12 has been shown to bind to lipooligomannose from R. radiobacter (Clark et al., 2012; Stanfield et al., 2015). The lipooligomannose contains linear tetramannose that can bind 2G12, similar to HIV-1 Env glycans (Stanfield et al., 2015). Similar to 2G12, V3-glycan antibodies like PGT128, have also been shown to bind to lipooligomannose from R. radiobacter (Clark et al., 2012; Stanfield et al., 2015). Mannose glycans are also present on the surface of different yeast species (Hamilton et al., 2003; Wildt & Gerngross, 2005). These glycans can be bound by 2G12 (Agrawal-Gamse et al., 2011; Doores, Fulton, et al., 2010; Luallen et al., 2010; Zhang et al., 2015).

4.6 2G12 neutralization breadth and potency The neutralization activity of 2G12 is less broad and potent than most V3-glycan antibodies mentioned above (Fig. 2). Overall, the neutralization breadth of 2G12 is 40% on large panels of global viruses, with 2G12

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showing clade-specific neutralization sensitivity (Binley et al., 2004). 2G12 neutralizes 75% of clade B HIV-1 isolates (Binley et al., 2004; Trkola et al., 1995), but neutralizes very few clade C isolates (Binley et al., 2004; Bures et al., 2002; Gray, Meyers, Gray, Montefiori, & Morris, 2006). The clade C resistance to 2G12 is hypothesized to be due to a lack of the N295 glycan (Gray et al., 2006). While V295N amino acid changes improve 2G12 binding (Chen, Xu, Bishop, & Jones, 2005), 2G12 still poorly neutralizes viruses with the V295N amino acid change (Gray, Moore, Pantophlet, & Morris, 2007). 2G12 also poorly neutralizes primary clade AE HIV-1 isolates that circulate in Asia (Binley et al., 2004; Chen et al., 2016; Thida et al., 2019; Wang et al., 2018). Clade AE viruses are likely resistant to 2G12 due to their lack of a N295 or N332 glycosylation site (Chen et al., 2016). Other mechanisms of resistance for this category of antibody include adding glycosylation sites within the first variable region at positions 130 and 139; second variable region (V2) at position 160; and fourth constant region at position N397 (Malherbe et al., 2013). These glycosylation changes were identified during SHIV infection and presumably could occur during HIV-1 infection as well (Malherbe et al., 2013). Viruses also escape 2G12 by lengthening their V1V2 region loops (Chaillon et al., 2011).

4.7 2G12 neutralization activity and other functions HIV-1 can be transmitted to many different target cells as cell-free or cellassociated virus (Parsons, Le Grand, & Kent, 2018). 2G12 is able to inhibit virus infection by cell-free and cell-associated virus (McCoy et al., 2014). For inhibition of cell-associated virus the cell type matters. 2G12 inhibited cell-associated virus infection of Jurkat T cell lines far better than primary T cells (McCoy et al., 2014). It is unclear why there was a difference, but perhaps differences in CD4 or coreceptor density mediated the differences between primary T cells and T cell lines. The mechanism for 2G12 neutralization of HIV-1 infection may involve blocking the binding of the co-receptor to HIV-1 Env (Platt, Gomes, & Kabat, 2012), which makes the expression levels of the co-receptor on target cells important (Platt et al., 2012). However, 2G12 is not only effective in inhibiting infection of T cell lines, as it has shown the ability to inhibit infection of primary cells in colorectal and ectocervical tissues (Cheeseman et al., 2017). More specifically, 2G12 inhibits HIV-1 infection of macrophages and dendritic cells (Ren et al., 2018). Dendritic cells can capture HIV-1 via DC-SIGN receptors on their surface and trans-infect neighboring T cells (Geijtenbeek et al.,

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2000). 2G12 has been effective at inhibiting dendritic cell trans-infection of primary CD4 T cells by blocking the initial binding of HIV-1 to dendritic cells (Ren et al., 2018; van Montfort et al., 2015). Thus, the neutralizing activity of 2G12 is effective in preventing infection in many different tissues by many different routes that are relevant for human transmission. 2G12 can bind to infected cells and thus it can mediate antiviral functions other than direct virus neutralization (Mujib et al., 2017). 2G12 exhibits effector functions such as complement deposition (Trkola et al., 1996) and antibody-dependent cellular cytotoxicity (Klein et al., 2010; Trkola et al., 1996), but 2G12 lacks the ability to aggregate virus (Alexander, Sanders, Moore, & Klasse, 2015).

4.8 Preclinical trials using 2G12 2G12 was one of the few broadly neutralizing antibodies isolated prior to 2009. Therefore, it was included in many of the initial proof-of-concept studies alone or in combination with the other broadly neutralizing antibodies known at the time. The most prevalent mixtures of bnAbs were 2G12, 2F5, and 4E10 and TriMAb, which contained 2G12, 2F5, and B12 (Gray et al., 2006). Different variations of this combination have been used to show monoclonal antibodies could protect against HIV-1 infection in animal models. To show the protective capacity of 2G12 it was mixed with F105 and 2F5, two other neutralizing antibodies, and infused into macaques (Baba et al., 2000). The macaques were later challenged intravenously with SHIV (Baba et al., 2000). The macaques were protected from infection if they received the neutralizing antibody cocktail (Baba et al., 2000). While, this study was informative the relevance of the intravenous SHIV challenge model for sexual transmission of HIV-1 where HIV-1 crosses mucosal surfaces was debated. Therefore, mucosal transmission models were developed to mimic vaginal transmission of HIV-1 or oral transmission (Baba et al., 2000; Mascola et al., 2000; Ng et al., 2010). A vaginal SHIV challenge model was established and the ability of 2G12 to protect against mucosal routes of transmission was evaluated (Mascola et al., 2000). 2G12 was infused into macaques alone or in combination with 2F5 and polyclonal antibodies from HIV-infected individuals to demonstrate antibodies can protect macaques from simian/human immunodeficiency virus infection (Mascola et al., 2000). 2G12 infusion prevented infection of 2 of 4 macaques when administered alone (Mascola et al., 2000). 2G12 combined with 2F5 protected 3 of 5 macaques. The trivalent

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mixture of 2F5, 2G12, and polyclonal HIV-1 antibodies was most effective protecting 4 of 5 macaques (Mascola et al., 2000). The concentrations of 2G12 in the vaginal fluid were as low as 16 ng/mL in macaques protected by 2G12 infusion. Thus, low amount of 2G12 was sufficient for protection (Mascola et al., 2000). In a subsequent study, Hessell et al. compared the anti-HIV-1 activities of 2G12 and B12 to try to understand how 2G12 could protect at such low concentrations (Hessell et al., 2009). They examined antibody effector functions and antibody trafficking to the mucosa and found 2G12 was not superior to another HIV-1 bnAb that required higher concentrations of antibody for protection (Hessell et al., 2009). Thus, 2G12 was remarkably efficient for unknown reasons in protecting macaques against vaginal challenge. 2G12 is also part of a protective antibody response for oral SHIV challenge that mimics the exposure of infants to HIV-1 through breast milk (Baba et al., 2000). The mixture of 2G12 and neutralizing antibodies F105 and 2F5 was infused into pregnant dams and newly delivered infant macaques (Baba et al., 2000). Infant macaques were challenged orally 1–4 h after caesarian section. The infant macaques administered neutralizing antibodies were completely protected, whereas control macaques became infected (Baba et al., 2000). In addition to passive infusion, the protective efficacy of 2G12 has also been tested in macaques formulated as a microbicide gel (Moog et al., 2014). The gel formulation included 2G12, 2F5 and 4E10. Macaques were administered microbicide gel and challenged vaginally with SHIV. The microbicide gel protected 6 of 9 macaques from infection 1 h after gel administration (Moog et al., 2014). Comparable protection was also observed when macaques were challenged 4 h after microbicide administration (Moog et al., 2014). Nonneutralizing antibodies formulated in microbicide gel were not protective, suggesting neutralization was a key factor in the protection observed by the bnAb mixture of 2G12, 2F5, and 4E10 (Moog et al., 2014).

4.9 Clinical trials of 2G12 passive immunity The safety of 2G12 has been evaluated in uninfected and HIV-infected individuals (Armbruster et al., 2004, 2002; Joos et al., 2006). These individuals were not on HAART, but had not progressed to AIDS (Armbruster et al., 2002). The infusion of antibody was well-tolerated in all individuals with no adverse clinical effects being noted (Armbruster et al., 2002). The antibody half-life in the beta phase of the concentration curve was 16.48 days, which was double the days observed for the HIV-1 bnAb 2F5 (Armbruster et al., 2002).

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Given its safety in HIV-infected individuals, 2G12 was mixed with 2F5 and infused into HIV-infected individuals to measure viral suppression (Stiegler et al., 2002). Viral load decreased and circulating CD4 T cells increased after the two antibodies were infused into HIV-infected individuals suggesting one or both of the antibodies had antiviral effects (Stiegler et al., 2002). Testing of the virus from study participants showed the virus before and after 2G12 infusion to be resistant (Stiegler et al., 2002). Thus the virus suppression was likely due to 2F5 instead of 2G12 (Stiegler et al., 2002). The emergence of neutralization resistance to 2G12 is well documented in vitro and in vivo (Bunnik et al., 2009; Manrique et al., 2007; Nakowitsch et al., 2005; Stiegler et al., 2002). Resistance to 2G12 has also been highlighted by a study where 2G12 was shown to neutralize only 2 of 35 viruses that were reactivated from the latent reservoir of a HIV-1 infected individual (Ren et al., 2018). In addition to virus suppression, 2G12 mixed with 2F5 and 4E10 was examined for its ability to maintain viral suppression in HIV-suppressed individuals who stopped antiretroviral therapy (ART). In one study 10 individuals were administered the bnAb cocktail and ART was halted (Mehandru et al., 2007). Eight of 10 individuals had virus rebound, but 2 individuals remained virologically-suppressed (Mehandru et al., 2007). In most of the individuals with virus rebound 2G12-resistant viruses had emerged (Mehandru et al., 2007). Resistant viruses to the other two antibodies in the antibody cocktail did not emerge suggesting 2G12 exerted the most immune selective pressure on the virus (Mehandru et al., 2007). These results are comparable to those found in a second passive immunity study that investigated 14 acute and chronic HIV-infected individuals (Trkola et al., 2005). In all but one chronically-infected individual the virus rebounded at the same rate as it had done in the absence of bnAb infusion. This suggested the antibody cocktail had no effect (Trkola et al., 2005). In one person, antibody treatment delayed viral load dramatically and viral load did not reach its previous set point until 24 weeks after antibody administration (Trkola et al., 2005). At this time point passively-infused antibody concentrations had waned (Trkola et al., 2005). Antibody administration during acute infection showed a more pronounced delay in rebound with one person not rebounding for the 24 weeks of follow-up (Trkola et al., 2005). Overall, when the virus rebound occurred the viruses were resistant to 2G12 suggesting it was the main suppressive antibody in the bnAb cocktail (Trkola et al., 2005). Together the studies showed the limitations of 2G12 in maintaining virus suppression due to virus escape (Nakowitsch

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et al., 2005; Trkola et al., 2005) but suggested antibody treatment early during infection may be more effective. Prevention strategies utilizing 2G12 have mostly been in the context of microbicide gels (Morris et al., 2014). The bnAb cocktail of 2G12, 2F5, and 4E10 was formulated into a microbicide gel called MABGEL for human clinical trials (Morris et al., 2014). The MABGEL containing 50 mg of each antibody was administered over a 12-day period to 10 women. The microbicide gel application resulted in vaginal concentrations of 7 mg/mL 1 h after administration. The major pitfall was rapid antibody half-life, which was quickest for 2G12 (Morris et al., 2014). Nonetheless, the microbicide gel had only a small number of moderate side effects bolstering its safety profile. With the discovery of more potent and broadly neutralizing antibodies, such as the V3-glycan antibodies mentioned above, the clinical investigation of 2G12 as an antiviral or prophylactic drug has ceased.

5. Mannose-restricted glycan-dependent HIV-1 neutralizing antibodies 5.1 Discovery of a mannose-cavity glycan-dependent HIV antibody DH501 is the prototypical example in the category of mannose-restricted glycan-dependent antibodies. The main distinguishing features of this category of antibody is its ability to only neutralize kifunensine-treated (Man9GlcNAc2-enriched) virus, and the distinct cavity in its paratope used for glycan binding (Fig. 4). DH501 was isolated from a rhesus macaque following long-term vaccination with group M consensus envelope CON-S (Saunders, Nicely, et al., 2017). CON-S envelope was glycosylated with mannose at N332 and N301 (Saunders, Nicely, et al., 2017). Macaques were immunized with CON-S as DNA, adenovirus vector, and recombinant protein over 204 weeks. DH501 was isolated as a monoclonal antibody in the third year of vaccination, but antibody repertoire sequencing suggests the DH501 lineage was elicited during the second year of vaccination (Saunders, Nicely, et al., 2017). DH501 has normal heavy and light CDR lengths, of 14 and 9 amino acids, respectively. This HCDR3 length is shorter than all broadly neutralizing V3-glycan antibodies (Table 1). DH501 is most similar to 2G12, which has a 16 amino acid HCDR3 (Table 1). Thus, the lengths of the CDRs for mannose-cavity antibodies is one distinguishing trait of this type of glycandependent antibody. Another characteristic is the lack of nucleotide

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A

DH501

B

2G12 VH

VH

Man 9 GlcNAc 2

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Man 7 GlcNAc 2

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Fig. 4 DH501 contacts glycan via a deep cavity in its VH region. (A) Crystal structure of DH501 (PDB: 5TZ4) in complex with mannose glycan (yellow, Man9GlcNAc2). The glycan inserts into a cavity composed of heavy chain CDR loops. Only the three terminal mannose residues of the D2 arm of Man9GlcNAc2 were resolved in the structure. (B) The crystal structure of 2G12 (PDB: 6MU3) in complex with mannose glycan (yellow, Man7GlcNAc2) shows it utilizes a similar cavity to bind to glycan. The mannose residues that were resolved in the structure are shown. These cavities are distinct from the protruding CDR loops utilized by V3-glycan broadly neutralizing antibodies (Fig. 1).

insertions and deletions in the variable region of DH501, which is a hallmark of V3-glycan antibodies (Longo et al., 2016; Walker et al., 2011). The lack of this rare genetic event would suggest that mannose-cavity HIV-1 antibodies could be more readily elicited by vaccination.

5.2 Ontogeny of a mannose-cavity glycan-dependent HIV antibody The development of mannose-cavity HIV antibodies is intriguing for two reasons. First, DH501 was not observed by next generation sequencing until after the first year of vaccination (Saunders, Nicely, et al., 2017). Whether an immunologic event, such as an infection by another pathogen, occurred at that time that started the lineage is unknown. Since other antibodies with strong glycan reactivity can bind bacteria and fungi it is a plausible hypothesis that an infection with either type of pathogen could have aided in the initiation of DH501 (Clark et al., 2012; Doores, Fulton, et al., 2010; Dunlop et al., 2010; Luallen et al., 2008; Luallen et al., 2010). Second, the DH501 sequences identified by next-generation sequencing were all from the IgM isotype (Saunders, Nicely, et al., 2017). It is possible that the antibodies bind to HIV Env more avidly when expressed as pentameric IgM. Pentameric IgM would cluster mannose-cavities on five immunoglobulins that would

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theoretically make more productive contacts with HIV-1 Env glycans as compared to monomeric IgG. Also the IgM isotype of DH501 clonal members suggested DH501 B cells may more readily undergo secondary germinal center reactions upon re-stimulation (Seifert et al., 2015). A preponderance of IgM clonal member could also mean the germline precursor B cell was high affinity, which tends to result in IgM memory B cells (Pape et al., 2018). The inferred precursor of DH501 did not bind to HIV-1 Env, therefore this hypothesis seems unlikely (Saunders, Nicely, et al., 2017). Thus, it is currently unclear why DH501 antibodies do not class switch.

5.3 DH501 structure DH501 antibodies have a distinguishing paratope shape that is the hallmark of this type of antibody. The three CDR loops form a pocket that serves as a glycan-binding cavity into which glycan inserts (Fig. 4A; Saunders, Nicely, et al., 2017). Only a single glycan binding cavity was observed, which suggests DH501 Fab contacts only one glycan. PGT124 is a V3-glycan that also contacts only one glycan (Garces et al., 2014), whereas antibodies PGT121, PGT128, DH270, BG18 are able to form simultaneous contacts with multiple glycans (Barnes et al., 2018; Fera et al., 2018; Julien, Sok, et al., 2013; Kong et al., 2015). Since these antibodies utilize independent CDR loops to contact glycans they are able to bind to multiple glycans relatively easily (Barnes et al., 2018; Pejchal et al., 2011). DH501 uses all three HCDR loops to form the one cavity that binds one glycan, thus there is not a second cleft like that found in the BG18 paratope that could bind a second glycan (Barnes et al., 2018; Saunders, Nicely, et al., 2017). The glycan binding cavity is similar to the 2G12 paratope, which like DH501 lacks protruding loops (Fig. 4B). Crystal structures of 2G12 shows it creates three glycan binding sites by forming an I-shaped, domain-exchanged antibody (Fig. 3; Calarese et al., 2003; Murin et al., 2014; Wu et al., 2013). Thus a single IgG molecule can contact multiple glycans through that mechanism. The simultaneous filling of glycan binding cavities on 2G12 is likely why the domain-exchanged version binds better to Env than the non-domain exchanged version despite the fact that they both can bind to high mannose (Doores, Fulton, et al., 2010). Hence, DH501 would be most similar to non-domain-exchanged 2G12 molecules where each Fab can only bind one glycan. DH501 lacks the protruding loops of antibodies like PGT128 or PGT121 that extend to contact Env peptide (Garces et al., 2015, 2014; Pejchal et al., 2011; Saunders, Nicely, et al., 2017). Instead a binding

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mode where glycan inserts into the cavity allowing the antibody to move closer to Env and contact peptide. Although, the structure of DH501 in complex with Env has not been solved to confirm this hypothesis.

5.4 Neutralization activity is restricted to Man9GlcNAc2-enriched viruses In a study assessing the role of glycosylation in DH501 neutralization, 10 pseudoviruses were generated in either untreated cells or cells treated with the glycosylation pathway inhibitor kifunensine (Saunders, Nicely, et al., 2017). Kifunensine treatment results mostly in Man9GlcNAc2 glycosylation (Scanlan, Ritchie, et al., 2007). Based on the glycosylation synthesis pathway Man8GlcNAc2 glycosylation is also still possible (Doores & Burton, 2010). DH501 in glycan arrays and Luminex assays bound directly to Man9GlcNAc2 and Man8GlcNAc2, thus kifunensine treatment was hypothesized to improve DH501 binding to Env on viruses (Saunders, Nicely, et al., 2017). DH501 neutralized all 10 viruses when their glycosylation was restricted to Man9GlcNAc2 (Fig. 5). This neutralization breadth was remarkable since the virus panel included difficult-to-neutralize viruses from various clades of HIV-1. However, neutralization was restricted Man9GlcNAc2-enriched viruses as DH501 did not neutralize the same viruses when glycosylation was heterogenous (Fig. 5; Saunders, Nicely, et al., 2017). The mechanism was likely improved binding to the envelope on virions, since kifunensine treatment improved DH501 binding to recombinant Env (Saunders, Nicely, et al., 2017). Previous studies also reported kifunensine treatment increased the potency of 2G12 and PGT128 neutralization and envelope binding (Agrawal-Gamse et al., 2011; Doores et al., 2015; Dunlop et al., 2010; Luallen et al., 2010, 2008; Saunders, Nicely, et al., 2017; Zhang et al., 2015). However, the mannose-cavity antibodies differ from domain-exchanged antibodies and V3-glycan antibodies in that the latter two categories do not require treatment with kifunensine for neutralization (Scanlan, Ritchie, et al., 2007). While DH501 was the initial monoclonal antibody from a vaccinated primate to show kifunensine-dependent neutralization breadth, other studies have found kifunensine-dependent enhancement of neutralization by serum polyclonal antibodies (Agrawal-Gamse et al., 2011; Dunlop et al., 2010; Luallen et al., 2010, 2008). Luallen et al. immunized rabbits with a yeast strain engineered to express Man8GlcNAc2 and elicited antibodies capable of neutralizing kifunensine-treated viruses (Luallen et al., 2008,

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DH501 HIV-1 neutralization Q23.17

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JR-FL MW965.26

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CH0505.w4.3 CH505s 3016.v5.c45

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Fig. 5 DH501 neutralizes kifunensine-treated HIV-1 isolates, but not untreated isolates. DH501 neutralization of HIV-1 pseudovirus infection of the TZM-bl indicator cell line. Kifunensine treatment restricts glycosylation to Man9GlcNAc2 or Man8GlcNAc2. Neutralization titers are shown as the concentration (μg/mL) of antibody that inhibits 50% of virus replication (IC50). Each symbol indicates the virus listed in the legend.

#237). In a similar study, rabbits were immunized with purified yeast proteins that possessed Man8GlcNAc2 or Man9GlcNAc2 glycosylation (Zhang et al., 2015). Antibodies capable of neutralizing viruses produced in kifunensine-treated cells were elicited again (Zhang et al., 2015). Thus, mannose-cavity antibodies may be prevalent among vaccination responses, but monoclonal antibodies are needed to confirm their structures. As with PGT128, neutralization by DH501 was dependent on N301. Mutational analysis showed deletion of N301, but not other sites in the high mannose patch negatively impacted potency (Saunders, Nicely, et al., 2017). Deletion of this site caused the IC50 to increase from 0.046 to 30 μg/mL. The same glycosylation site deletion led to weaker PGT128 neutralization as well (Saunders, Nicely, et al., 2017). These results are consistent with previous studies that report PGT128 dependence on N301 ( Julien, Sok, et al., 2013; Kong et al., 2015). In contrast, deletion of the glycosylation site at N332 eliminated neutralization by PGT128, but had no effect on DH501. DH501 neutralization was also largely dependent on G324 in the GDIR motif at the base of the V3 loop (Saunders, Nicely, et al., 2017). In summary, DH501 has similarities with V3-glycan antibodies and domain-exchanged antibodies in terms of N301 glycan-dependence, G324-dependence, and mannose reactivity, but differs due to its lack of protruding CDR loops and domain-exchanged Fabs.

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6. Vaccine development targeting high mannose patch antibodies Current preventative HIV-1 vaccine design efforts focus on the generation of protective antibody responses (Haynes & Burton, 2017). The design effort can largely be divided into two categories: (1) induction of broadly neutralizing antibodies and (2) induction of protective nonneutralizing antibodies. The glycan-dependent antibodies discussed above are mostly neutralizing antibodies, thus they generally pertain to the induction of broadly neutralizing antibodies. However, the induction of broadly neutralizing antibodies has been difficult with vaccination (McCoy & Burton, 2017). The goal of this type of vaccine design is to elicit broadly neutralizing antibodies that are active against 50% of global difficult-toneutralize (tier 2) HIV-1 isolates (Montefiori, Roederer, Morris, & Seaman, 2018). The discovery of the high mannose patch monoclonal antibodies allows vaccines to be designed using these antibodies as a template (Astronomo & Burton, 2010; Doria-Rose et al., 2014; Escolano, Dosenovic, & Nussenzweig, 2017; Haynes, Kelsoe, Harrison, & Kepler, 2012). In practice, immunogens can be screened for binding to the high mannose patch monoclonal antibodies at different stages of their development into a bnAb. Thus, the recent explosion of HIV-1 antibody discovery has directly impacted vaccine design (McCoy & Burton, 2017). Below we will discuss vaccine design efforts to elicit neutralizing antibodies from the V3-glycan, domain-exchanged, or mannose-cavity categories of high mannose patch HIV-1 antibodies.

6.1 Vaccine design to elicit V3-glycan HIV-1 neutralizing antibodies There have been two approaches for eliciting V3-glycan antibodies. First, structure-based directed evolution has been used to design immunogens that can initiate V3-glycan broadly neutralizing antibody lineages (Steichen et al., 2016). Since the structures of many of the V3-glycan antibodies have been solved either unliganded or in complex with Env much is known about the paratope and epitope of each antibody (Barnes et al., 2018; Garces et al., 2014; Kong et al., 2015). Using this information libraries of Env immunogens have been created that possess amino acid changes at the Env-antibody interface that can bind to different members of the PGT121 lineage (Steichen et al., 2016). Through successive rounds of expressing the

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Env library on the cell surface and screening by flow cytometry for PGT121 binding, a series of HIV-1 envelopes that are antigenic for PGT121 lineage antibodies were discovered (Steichen et al., 2016). While none of the identified immunogens bound to the inferred germline precursor of PGT121, one immunogen called BG505 Mut11B was able to interact with an early intermediate antibody in the PGT121 lineage that was only minimally somatically mutated (Steichen et al., 2016). Variants of BG505 Mut11B were also designed that bound strongly to more mutated members of the PGT121 lineage (Steichen et al., 2016). To test whether the BG505 Mut11B envelopes could elicit V3-glycan antibodies, a knockin mouse model where most B cells express the heavy and light chains of a minimally-somatically mutated PGT121 antibody was generated (Escolano et al., 2016). Immunization of these mice with a sequence of BG505 Mut11B and its derivatives led to maturation of the PGT121 antibodies with substantial neutralization of viruses known to be sensitive to PGT121 (Escolano et al., 2016). The results supported the concept of immunizations with a series of envelopes that are high affinity for bnAb lineage members at different stages of maturation—a strategy called B cell lineage design (Haynes et al., 2012). However, it should be noted that the mouse did not start with a germline unmutated antibody, and the immunizations did not induce the deletions and insertions present in PGT121. Also, nearly all B cells in the PGT121 knockin mouse expressed PGT121 antibodies, which is not the case in wild-type animals. To determine whether a variant of BG505 Mut11B could elicit protruding-CDR V3-glycan antibodies in wild-type animals, immunizations of mice and macaques were performed (Escolano et al., 2019). The variant of BG505 Mut11B called RC1 had N156 removed and some hydrophobic residues added to promote 10–1074 and PGT121 binding (Escolano et al., 2019). Initial experiments did not elicit V3-glycan antibodies, but instead elicited antibodies against strain-specific epitopes (Escolano et al., 2019). To eliminate the immunogenicity of these strain specific epitopes glycans were placed at these sites to cover the epitopes (Escolano et al., 2019). Moreover, virus-like nanoparticles were created to provide a multivalent interaction between B cell receptors and the immunogen in vivo (Escolano et al., 2019). Immunization with this construct resulted in antibodies sensitive to removal of N301 and N332 glycans in the V3-glycan site (Escolano et al., 2019). Electron microscopy images of the antibodies showed they bound to a site on Env near the N332 glycan site (Escolano et al., 2019). The CDR3s of the vaccine-induced antibodies possessed a few similarities with PGT121

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(Escolano et al., 2019). Thus it was concluded that this modified version of BG505 Mut11B called RC1 was able to initiate V3-glycan antibodies similar to PGT121. None of the antibodies neutralized nor did serum from the macaques from which the antibodies were cloned. The lack of neutralization may be because only a single immunization was given (Escolano et al., 2019). Second, structure-guided design was combined with neutralization screening to modify BG505 Env trimers to activate PGT121 and CD4 binding site bnAb precursors (Medina-Ramirez et al., 2017). Based on structural analyses 2 glycans in the CD4 binding site (N276D and N462D), 1 glycan in the high mannose patch (N386D), and 1 glycan in the bridging sheet (N197D) were deleted from BG505 Env trimers. An additional, 16 other amino acid changes were added because of their presence in viruses that were neutralized by germline precursors of PG9 and VRC01 (MedinaRamirez et al., 2017). The BG505 GT1 Env trimer was designed to bind to VRC01 and PG9 and did not show detectable binding to PGT121 near-germline-intermediate antibodies. However, it was able to activate the PGT121 intermediate antibody expressed in the knockin mouse model described above for BG505 Mut11B. No serum nor monoclonal antibody neutralization was reported, thus the progress made in inducing PGT121 bnAbs was not clear (Medina-Ramirez et al., 2017). Early intermediate antibodies in V3-glycan bnAb lineages preferentially neutralize kifunensinetreated virus (Saunders, Nicely, et al., 2017). Thus, high mannose-enriched versions of the GT1 and BG505 Mut11B envelopes may enhance the efficacy of these immunogens in sequential immunization strategies. Third, minimal immunogens that recapitulate the V3 loop have been created to try to elicit V3-glycan bnAbs. One of these designs was based on the modified Env subunit whose crystal structure was solved in complex with PGT128 (Pejchal et al., 2011). The construct had a truncated V3 loop that lacked the immunodominant region targeted by poorly neutralizing antibodies (Alam et al., 2017). Alam et al. designed a 30 amino acid peptide sequence that included the N-terminal and C-terminal bases and stem of the V3 loop without the tip of the loop (Alam et al., 2017). The base of V3 loop contained the N301 and N332 position to which synthetic Man9GlcNAc2 was conjugated and cysteines that created a disulfide bonded loop similar to that made by V3 in the context of Env gp120 (Alam et al., 2017). The glycopeptide was completely synthetic allowing for homogeneous molecules that could be used as an immunogen. The crystal structure of the glycosylated peptide in complex with DH270.6 showed that its conformation

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was nearly identical to the V3 region in the BG505 SOSIP gp140 Env trimer (Fera et al., 2018). Other designs of minimal glycosylated peptides have also been designed (Cai et al., 2017, 2018b). One design differed from the Alam et al. design in that it encoded a N334 glycosylation site instead of N332. The N334 glycopeptide was modified as an immunogen by adding Pam3Csk as an adjuvant and a T cell helper epitope (Cai et al., 2017, 2018b). Vaccination of rabbits with this design elicited gp120-reactive antibodies, but none of the antibodies were neutralizing. A subset of the antibodies bound better to glycosylated peptide than peptide alone, but no monoclonal antibodies were produced to define the epitope of the antibodies (Cai et al., 2018b). Serum IgG titers could be increased by linking three of the glycopeptides together as compared to monovalent glycopeptide (Cai et al., 2018b). A pitfall of this approach has been the difficulty in creating a peptide that can bind to all V3-glycan antibodies (Orwenyo et al., 2017). Most glycopeptides have bound PGT128, but vary in their ability to bind other antibodies (Alam et al., 2013; Cai et al., 2018b; Orwenyo et al., 2017). The variation is likely due to the variation in which glycans within the high mannose patch are bound by each antibody (Orwenyo et al., 2017). Nonetheless, these glycopeptides and others like it could be useful immunogens for focusing the immune response to the V3-glycan site only. Another approach for minimal immunogens for V3-glycan antibodies was to use computational protein design to graft the V3-glycan site into peptide scaffolds (Zhou et al., 2014). These proteins would then only match HIV-1 envelope at the V3-glycan site. These scaffolds used the base and stems of the V3 loop like previous glycopeptides (Alam et al., 2017; Cai et al., 2018a). Multivalent presentation of these scaffolds on ferritin nanoparticles improved their affinity for V3-glycan bnAbs (Zhou et al., 2014). Their utility as vaccine immunogens warrants further study (Alam et al., 2017; Cai et al., 2018a). Regardless of the vaccine approach, the elicitation of potent and broadly neutralizing V3-glycan antibodies will be difficult due to the rare genetic events they possess. Vaccines will have to elicit insertions and/or deletions to create PGT121 or PGT128-like antibodies (Walker et al., 2011) or somatic mutations at sites not commonly targeted by activation-induced cytidine deaminase to create DH270 (Wiehe et al., 2018). The precursor frequency of some V3-glycan antibodies will be limited in the repertoire since antibodies with HCDR3s >20 amino acids are present at a low frequency (Briney, Willis, & Crowe Jr, 2012). There is optimism for vaccines that target this response in that there are multiple binding modes for

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recognition of the N332 glycan supersite (Kong et al., 2013). Given the genetic challenges with this type of antibody response a combination of the approaches mentioned above may be necessary to elicit broadly neutralizing V3-glycan antibodies.

6.2 Vaccine design to elicit domain-exchanged glycan-dependent HIV-1 neutralizing antibodies The unique domain-exchanged structure of 2G12 has suggested it will rarely be generated by the human immune system. Nonetheless, vaccine strategies have been tested to elicit similar neutralizing antibodies. The focus of 2G12targeted vaccines has been the display of high mannose glycans on various scaffolds (Wang, 2013). Early efforts utilized QB phage to display glycans to B cells in hopes of eliciting 2G12 antibodies (Astronomo et al., 2010). However, immunization of rabbits elicited glycan-reactive antibodies that were not HIV-1 Env reactive (Astronomo et al., 2010). A similar result was obtained when tetramannose was conjugated to bovine serum albumin (Astronomo et al., 2008; Liu et al., 2016; Trattnig et al., 2019). In one rabbit study using BSA glycoconjugates, anti-glycan antibodies were elicited, but they did not bind HIV-1 Env (Astronomo et al., 2008). When BSAglycoconjugates were used to boost gp120 immunization of mice, they failed to boost gp120 binding antibody titers (Liu, Ghneim, et al., 2016). Other scaffolds have included gold nanoparticles (Di Gianvincenzo, Chiodo, Marradi, & Penades, 2012; Marradi et al., 2011; Martinez-Avila et al., 2009), tetanus toxoid peptides (Ni, Song, Wang, Stamatos, & Wang, 2006), and Keyhole Limpet Hemocyanin (Ni et al., 2006). These constructs are antigenic for 2G12 and can compete with envelope for binding (Marradi et al., 2011). However, these approaches alone have failed to elicit 2G12like antibodies (Pashov, Garimalla, Monzavi-Karbassi, & KieberEmmons, 2009). To improve the glycopeptides used as immunogens for domain-exchanged antibodies, directed evolution of glycopeptides was used. This strategy used mRNA display to create peptides that were linked to their mRNA sequence (Horiya, Bailey, Temme, Guillen Schlippe, & Krauss, 2014; Temme, Drzyzga, MacPherson, & Krauss, 2013). The library of glycopeptides were screened for 2G12 binding in 10 or more successive rounds until glycopeptides with binding affinities of 0.54 nM for 2G12 were obtained (Nguyen et al., 2019). To present B cells with a multivalent immunogen capable of crosslinking multiple B cell receptors, the glycopeptide was linked to the CRM197 scaffold (Bailey, Nguyen, Horiya, & Krauss, 2016;

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Horiya et al., 2014; Temme et al., 2013). Rabbit immunization with the glycopeptides conjugated to CRM197 generated anti-glycan antibodies (Nguyen et al., 2019). BG505 SOSIP gp140 trimer boosts of these rabbits boosted Env-reactive antibody responses (Horiya et al., 2014; Temme et al., 2013), but the vaccine-induced antibodies recognized a part of the glycan different from 2G12 (Nguyen et al., 2019). Neutralizing antibody titers against easy and hard to neutralize viruses were all below 1:25 dilution (Nguyen et al., 2019). Hence, glycopeptide prime-Env trimer boost was not sufficient for inducing 2G12-like bnAbs. This study showed again successful induction of anti-glycan antibodies, but these antibodies differed from 2G12. Directed evolution approaches have also been used to generate peptides that can interact with 2G12 (Karpenko et al., 2012). The peptides do not aim to recapitulate the V3-glycan site, but instead are screened by phage display for their ability to bind to 2G12. The peptide VGAFGSFYRLSVLQS was selected from 2G12 binding screens of phage libraries, and fused to the T-cell and B-cell immunogen peptide called TBI to form the TBI-2G12 immunogen (Karpenko et al., 2012). Immunization of BALB/c mice with this TBI-2G12 elicited antibody responses. Whether these are glycan-dependent neutralizing antibodies remains to be determined (Karpenko et al., 2012). 2G12 cross-reacts with fungal and bacterial pathogens, which led to the hypothesis that immunization with different types of yeast or R. radiobacter would elicit 2G12-like antibodies (Agrawal-Gamse et al., 2011; Doores, Fulton, et al., 2010; Luallen et al., 2010; Zhang et al., 2015). R. radiobacter produce lipooligomannose, which can bind to 2G12 in a mode identical to Man9GlcNAc2 (Stanfield et al., 2015). 2G12 binding is facilitated by a tetramannose arm present in both lipooligomannose and Man9GlcNAc2 (Stanfield et al., 2015). Thus, immunization of mice with heatkilled R. radiobacter bacteria was investigated for eliciting anti-glycan antibodies (Clark et al., 2012). Immunization with R. radiobacter elicited Man4-reactive antibodies (Clark et al., 2012). However, the antibodies were not neutralizing (Clark et al., 2012). Similar experiments have been performed with the yeast Saccharomyces cerevisiae (Zhang et al., 2015). A glycosylation mutant of S. cerevisiae expressed five proteins that bound to 2G12 (Zhang et al., 2015). Immunization of rabbits with yeast proteins PstI or Gp38 resulted in anti-glycan antibodies that neutralized kifunensine-treated HIV-1 isolates and captured HIV-1 virions, but neutralization breadth was not elicited (Zhang et al., 2015).

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6.3 Vaccine design to elicit mannose-cavity glycan-dependent HIV-1 neutralizing antibodies Mannose-cavity antibodies can be elicited in rhesus macaques over long durations of time (Saunders, Verkoczy, et al., 2017). An improvement for eliciting these antibodies would be to elicit them with a shorter vaccination regimen. Epitope focusing is one means of eliciting these antibodies more quickly. This strategy could take advantage of the glycopeptides that are antigenic for V3-glycan antibodies (Alam et al., 2017; Cai et al., 2018a). DH501 bound the Man9-V3 glycopeptide, thus this immunogen could be used as a prime or a boost to more rapidly expand DH501-expressing B cells (Alam et al., 2017; Francica et al., 2019; Saunders, Nicely, et al., 2017). Similarly, mimetic glycoconjugates have elicited N301-dependent antibodies in humanized rats (Pantophlet et al., 2017). These glycoconjugates were generated by conjugating bacteria-like glycans onto bovine serum albumin (Pantophlet et al., 2017). These types of immunogens could be promising for expanding DH501 antibodies. The neutralization of DH501 is restricted to mannose-enriched viruses currently. Therefore, strategies to overcome this limitation are also needed. Vaccine strategies could include immunization with near-native HIV-1 Env trimers to select for mannose-cavity antibodies that can bind natively glycosylated HIV-1 Env (Go et al., 2014). Alternatively, vaccine immunogens could aim to broaden the glycans that are recognized by the mannose-cavity antibodies. In such a strategy immunogens with glycosylation restricted to high mannose or hybrid glycans by kifunensine, swainsonine, or other inhibitors could be used for vaccination (Crispin et al., 2007; Tulsiani, Harris, & Touster, 1982). These immunogens could select DH501-like antibodies that can bind high mannose and hybrid glycans. Hybrid glycans contain terminal mannose residues (Crispin et al., 2007; Higel, Seidl, Sorgel, & Friess, 2016), thus it is possible that mannose-cavity antibodies could evolve to bind to such glycans. Expanding the neutralization breadth seen for Man9GlcNAc2-enriched viruses to untreated viruses would not only improve the relevance of mannose-cavity antibodies, but would mean vaccines are able to elicit broadly neutralizing antibodies.

7. Conclusions Antibodies that target the high mannose patch are among the most potent and broadly HIV-1 neutralizing antibodies known (Kwong & Mascola, 2018). In this review, we described differences among the

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antibodies that target the high mannose patch. The antibodies against the high mannose patch can be stratified into three categories based on their neutralization activity, genetic elements, and antibody structures. Each of the three categories have hallmarks including long protruding CDR loops, domain-exchanged Fabs, or deep mannose-binding cavities. Within the V3-glycan category there is considerable variation in neutralization phenotype, HIV-1 Env glycan contacts, and immunogenetics. This variation suggests there several antibody solutions that the human immune system can use to attack the high mannose patch on HIV-1 Env (Kong et al., 2013). The lack of stereotyped antibody binding provides some freedom for vaccineelicited antibodies to develop equivalent neutralization activity without using the precise mechanism as the previously-isolated antibodies from natural infection discussed here (Kong et al., 2013). However, the low likelihood genetic events such as nucleotide insertions/deletions, somatic mutations at disfavored nucleotide positions, and unusual CDR3 recombination events pose challenges for vaccine elicitation of such antibodies (Kwong & Mascola, 2012; Schramm & Douek, 2018; Wiehe et al., 2018). Thus it is not surprising that the two most broadly neutralizing and protective categories of high mannose patch antibodies are the two with the least progress toward elicitation with vaccination. Despite the challenges these antibodies present for elicitation with active immunization, they show great promise for passive immunization. Phenotypically, the V3-glycan antibodies show the most potential for antiviral and preventative treatment due to their superior neutralization activity compared to the other types of high mannose patch antibodies (Caskey et al., 2019; Kwong & Mascola, 2012, 2018). The limitations of 2G12 and DH501 neutralization make them unlikely candidates for passive immunity. There are a number of clinical trials in progress using high mannose patch antibodies PGT121 and 10–1074, which have the potential to impact HIV-1 infection treatment (Caskey et al., 2019). Thus, the investigation of these antibodies has provided a better understanding of HIV-1 Env glycobiology as well as antibody structure, and in future clinical research will provide insight into treatment and/or prevention of HIV-1 infection.

References Abner, E., & Jordan, A. (2019). HIV “shock and kill” therapy: In need of revision. Antiviral Research, 166, 19. Agrawal-Gamse, C., Luallen, R. J., Liu, B., Fu, H., Lee, F. H., Geng, Y., et al. (2011). Yeastelicited cross-reactive antibodies to HIV Env glycans efficiently neutralize virions expressing exclusively high-mannose N-linked glycans. Journal of Virology, 85, 470.

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