Glycosylation of the ENV Spike of Primate Immunodeficiency Viruses and Antibody Neutralization

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1 Current HIV Research, 2004, 2, Glycosylation of the ENV Spike of Primate Immunodeficiency Viruses and Antibody Neutralization Cheryl A. Pikora *1,2 1 Department of Infectious Diseases, Children s Hospital, Boston, MA 02115, USA 2 Department of Microbiology, New England Primate Research Center, Harvard Medical School, Southborough, MA USA Abstract: Neutralizing antibody titers have been correlated with protection following vaccination against many viral pathogens. The logical target of protective antibody responses elicited by potential HIV vaccines should be the viral Env spike on the surface of the virion. However, the potency and titers of neutralizing antibodies that arise during HIV infection are generally discouragingly low and the antibodies that do arise recognize mainly autologous virus. This is thought to be a result of a combination of immunodominance of hypervariable regions of the Env protein that can easily escape neutralization, antibody reactivity to gp160 decoy protein in cell surface debris or monomeric gp120, conformational constraints within the Env trimer that create unfavorable antibody binding conditions and extensive glycosylation of the exposed regions of Env within the trimer. This review will describe current knowledge regarding glycosylation as a mechanism of neutralization resistance and discuss experimental approaches used to overcome this resistance. Part of the strategy toward development of an optimally immunogenic Env spike will likely require modification of Env glycosylation. Keywords: HIV, SIV, glycosylation, Env, neutralizing antibody. INTRODUCTION Urgency in the need for an effective vaccine against HIV has developed in response to the alarming rate at which infection continues to climb in many resource-poor countries. In response, a number of vaccine candidates expressing various combinations of HIV-1 antigens have entered or are preparing to enter clinical trials. Despite this, there are still many poorly understood scientific obstacles to creating a vaccine that induces sterilizing immunity. It is generally agreed that both a strong neutralizing antibody and a strong cellular immune response are required for complete protection. This, in turn, necessitates a vaccine design in which viral proteins are maximally immunogenic. Experience with the development of vaccines against other viral pathogens [7, 38, 41, 112] suggests that induction of high levels of neutralizing antibody titers might effectively block viral entry and protect against infection. The HIV Env spike, likely to be the most effective target for neutralizing antibody, will require modification to enhance immunogenicity. This review addresses one manner in which Env immunogenicity has been enhanced: by the modification of particular glycans within the carbohydrate shield. GLYCOSYLATION OF EXTERNAL VIRAL PROTEINS AND IMMUNE EVASION Early examples of the capacity of lentiviruses to cause persistent infection through antigenic variation exist in experimental infections of ponies with equine infectious *Address correspondence to this author at the Department of Infectious Diseases, Children s Hospital, Boston, MA 02115, USA; E- mail: cherylpikora@childrens.harvard.edu X/04 $ anemia virus (EIAV), of goats with caprine arthritisencephalitis virus (CAEV) and in sheep persistently infected with visna virus [18, 29, 50, 65, 71, 99]. In particular, variations in glycosylation of the two Env proteins appear to contribute to the antigenic variation seen in persistent infection caused by EIAV. In a study evaluating virus obtained during four, closely-spaced, repeat episodes of viremia within a poney infected with a prototypical EIAV strain, the Env proteins (gp90 and gp45) of the predominant viral population at each period of viremia were antigenically distinct as determined by monoclonal and polyclonal antibody reactivity. Variations in glycosylation were noted to accompany changes in antigenic reacitivity within both gp45 and gp90. Furthermore, serum neutralization indices from serum taken at earlier time points were lower against virus produced at the later viremic time points, suggesting that a correlation with glycosylation changes and neutralization escape might exist [99]. The inverse relationship of glycosylation of external viral antigens to immune recognition is not unique to lentiviral infection. The pre-s1 and pre-s2 glycoproteins of hepatitis B virus, immunogenic proteins believed to be involved in receptor recognition [80], induce enhanced levels of antibody titers in inoculated mice when deglycosylated [62]. Likewise, the outer surface glycoprotein of rotavirus, VP7, becomes resistant to neutralization when grown in the presence of neutralizing antibody in vitro [26, 60, 61] due to new sites of glycosylation arising within various antigenic regions of the protein. As a third example, the hemagglutinin of an antigenic variant of the 1968 Hong Kong (H3) influenza virus, selected by growth in the presence of a monoclonal antibody, escaped recognition of this antibody by acquisition of a single amino acid 2004 Bentham Science Publishers Ltd.

2 244 Current HIV Research, 2004, Vol. 2, No. 3 Cheryl A. Pikora substitution D63N that created an N-linked glycosylation site [106]. HIV ENV GLYCOSYLATION Translation of the Env precursor protein, gp160, takes place in the endoplasmic reticulum (ER). During its journey through the ER, it also oligomerizes into trimers in preparation for the assembly of the heterotrimer contained within the mature virion spike. The nascent Env protein is cotranslationally modified by the addition of O- or N-linked carbohydrate groups via resident glycosyltransferases. These newly added carbohydrate moieties are further trimmed prior to the protein s transport to the Golgi network. The composition of the sugar molecules within the N-linked oligosaccharide either complex or high mannose - likely differs by the type of cell in which the virus replicates [66]. Once within the Golgi network, gp160 is cleaved into the surface protein (SU), gp120, and the transmembrane fusion protein (TM), gp41. These two subunits form heterotrimers, which become incorporated into the viral membrane upon budding. Each monomer of gp120 is made up of five hypervariable regions: four that are thought to form external surface loops - V1/V2, V3 and V4 and a fifth region, V5, which does not form a looped structure. In between these regions are sequences of relative conservation designated C1-C5. Binding sites for the two receptors, CD4 and the chemokine receptor (usually either CCR5 or CXCR4), are located within gp120. Each gp120 monomer is noncovalently linked to a gp41 molecule. The gp41 monomer is composed of an ectodomain that contacts gp120, a membrane-spanning region and a cytoplasmic tail. HIV-1 Env is extensively glycosylated with more than 20 N-linked sites [34] and 8 O-linked sites [8] utilized within gp120. The gp41 protein of HIV-1 contains approximately 3 to 4 sites for N-linked glycosylation [47]. A survey performed by Myers et al. [76] of approximately 10,000 protein sequences in the SWISS-PROT library with at least one potential N-linked glycosylation site showed that the number of glycosylation sites in HIV-1 RF ranked in the top 10 proteins with this level of carbohydrate modification. Other viral proteins with high levels of carbohydrate addition include the S (spike) gene product of the pig gastroenteritis virus (a coronavirus) and the BILF2 gene product of Epstein Barr virus, a membrane-bound glycoprotein [76]. Viral protein glycosylation has been shown to play a role in proper protein folding, intracellular stabilization and protection against cellular proteases as well as receptor recognition and immunogenicity [2, 23, 31, 32, 82, 94]. Therefore, some glycosylation sites are critical to intracellular processing of the Env glycoprotein. Loss of particular glycans can also affect viral infectivity, possibly through structural alterations affecting the ability of the glycoprotein to bind its receptors, monomer interactions within the trimer or interactions of the SU and TM proteins [81, 115]. However, many N-linked glycans, in combinations, have been shown to be dispensable for viral replication within HIV gp41 [24, 47] and gp120 (as will be discussed in this review). It seems more likely that HIV and SIV use this high level of Env glycosylation as one of a variety of mechanisms that have evolved as a means of protection from neutralizing antibody. NEUTRALIZING ANTIBODY AND HIV INFECTION Numerous evaluations of the tempo and level of neutralizing antibody production in HIV infection and the stage of infection during which neutralizing antibodies exert control have yielded confusing results. Past studies examining autologous virus neutralization in HIV infected patients have demonstrated low levels of weakly neutralizing circulating antibody [13, 73, 85]. Moreover, various reports have demonstrated a range of weeks to months in the ability to detect neutralizing antibodies, post infection, in HIV-1 infection of humans [1] [5], SHIV infection of monkeys [64] and HIV-1 infection of chimpanzees [78]. Detection of strong neutralizing antibody responses to autologous virus has been more recently facilitated by assays that amplify viral Env sequences from plasma RNA. In these assays, HIV-1 is pseudotyped with DNA derived from reverse transcribed plasma RNA env sequences and antibody responses are measured in plasma derived from the same time point. Using this strategy, strong circulating neutralizing antibody responses to autologous, contemporaneous virus have been detected relatively early in infection (0-39 months) [97, 116]. The role of neutralizing antibody in control of HIV and SIV infection is unclear. A role for neutralizing antibody in control of chronic viremia is suggested by a study by Reitter, et al. [96]. In this study, rhesus macaques were inoculated with neutralization-sensitive variants of SIV239 lacking particular glycosylation sites. Although the level of primary viremia was similar between these animals and control animals inoculated with wild type (WT) SIV239, subsequent viremia in the chronic phase of infection was substantially lower than those seen in the control macaques. Furthermore, control of viremia was transiently lost with B cell-depletion of these animals [44]. Montefiore, et al. [72] also drew a correlation between neutralizing antibody and viremic control in the evaluation of viral loads observed in patients undergoing the treatment cessation phase of an early structured treatment interruption study. This group found that patients with high autologous neutralizing antibody titers also had the tightest viremic control following cessation of antiretroviral treatment. In contrast, Poignard, et al. [88] demonstrated that in the setting of established HIV- 1 infection of hu-pbl-scid mice, neutralizing antibody appeared to exert limited control of viremia. Various neutralizing epitopes of gp120 have been identified through viral mutagenesis and antibody competition analyses. Early studies identified the V3 loop as the primary antigenic determinant for antibody neutralization during HIV-1 infection [35, 79, 121]. Most of these antibodies had been characterized with T cell line adapted (TCLA) strains of HIV-1 and had been shown to be broadly reactive. However, it is now recognized that V3 loop antibodies are usually only able to neutralize autologous virus and neutralize other primary isolates of HIV little, if at all [22, 107, 113]. Other type-specific, early neutralizing antibody responses have been detected against V1/V2 loop epitopes [86]. Later, during the chronic course of HIV infection, neutralizing antibodies appear that are not only able to neutralize autologous virus but also a larger range of HIV-1 laboratory strains. A subset of these block the interaction of the CD4 binding site with its receptor and are

3 Glycosylation of the ENV Spike of Primate Immunodeficiency Current HIV Research, 2004, Vol. 2, No designated as CD4 binding site (CD4BS) antibodies [89, 118, 120]. A number of CD4BS antibodies neutralize TCLA viruses [87], however, only one human-derived monoclonal CD4BS antibody (IgGb12) demonstrates broad recognition and potent neutralization of primary isolates [22]. IgGb12 was isolated from a combinatorial phage display library from bone marrow donated by an individual who had been HIV positive for more than 6 years but who had not yet developed clinical symptoms [14]. A second group of neutralizing antibodies, found less commonly than CD4BS antibodies, are those that bind to gp120 with greater affinity when gp120 is ligated to CD4. These antibodies, referred to as CD4i antibodies [89, 118, 120], recognize an epitope that overlaps the coreceptor binding site. Although they recognize a well-conserved region of gp120 and therefore would be expected to be broadly reactive, they actually neutralize TCLA viruses very poorly and primary isolates not at all. Steric constraints imposed on antibody binding to the coreceptor site by the closely apposed viral and cellular membranes following the binding of gp120 to CD4 probably accounts for this [89, 120]. IMMUNOLOGICAL FACES OF GP120 A crystal structure of the HIV-1 gp120 core in a complex with the membrane distal two domains of CD4 and a Fab fragment derived from the CD4i monoclonal antibody 17b [57] demonstrates structural relationships useful in deciphering mechanisms of neutralization. The positions of the V1/V2 and V3 loops have been deduced in relation to the CD4 and coreceptor binding sites allowing for a visual mapping of neutralizing antibody epitopes (Fig. (1)). The gp120 core is comprised of an inner domain and an outer domain separated by a bridging sheet [57, 89]. The CD4 binding site is located within a depression formed by these Fig. (1) The structure of monomeric gp120 with variable loops V2 and V3 superimposed and with CD4BS and CD4i epitopes adapted from [118]. This illustration is based upon the crystal structure and epitope data previously published [32] [21]. The illustration represents monomeric, core gp120 in the CD4 bound conformation. The V2 and V3 loops are superimposed on the structure. Red indicates areas in which CD4BS antibodies have been mapped and green indicates areas in which CD4i antibodies have been mapped. two domains and the bridging sheet and is partially masked by the V1/V2 loop. The coreceptor binding site is located in close proximity to the CD4 binding site and involves residues near or within the bridging sheet as well as V3 loop residues. The V2 portion of the V1/V2 loop is thought to mask the bridging sheet and adjacent V3 loop [118]. CD4 binding induces a conformational change within gp120 that involves the repositioning of the V1/V2 loop. This conformational change, in turn, facilitates coreceptor binding through increased accessibility of those residues of gp120 that contact the coreceptor [119]. It is therefore easy to imagine that changes generated in one of these structures, because of their intimate association with one another, could effect adjacent structures that govern overall Env conformation and vulnerability to neutralization. From neutralizing antibody and structural data, the gp120 monomer is considered to possess three surfaces with different immunogenic properties: the non-neutralizing face, the neutralizing face and the silent face. The non-neutralizing face is that portion of the molecule which is inaccessible to antibody binding in the context of the trimer, but is readily bound by antibodies in the context of the monomer [57, 74, 102]. The neutralizing face includes the regions described above which include the CD4 binding site and CD4i epitopes. The silent face consists of the well-exposed regions of gp120 [58, 118] that are heavily glycosylated and, as such, may appear to the immune system as self. This face contains conserved regions within the gp120 core as well as the variable loops V4 and V5 [89]. NATURAL VARIATION OF GLYCOSYLATION SITES OF HIV-1 AND SIV DURING INFECTION Evidence is derived from nature that glycosylation plays an important role in the ability of HIV and SIV to escape neutralizing antibody recognition (Table 1) (Fig. (2)). One of the most thoroughly studied examples of this comes from a recent evaluation of neutralization of autologous virus in HIV-1 infected individuals [116]. This study examined neutralizing antibody in plasma samples from three patients early in infection as early as 16 days after the symptoms of acute viral infection. Early viral isolates from all three individuals were surprisingly well neutralized by contemporaneous plasma samples with a mean 50% inhibitory concentration (IC 50 ) value of (plasma dilution). However, isolates from later time points were shown to have acquired as much as 100-fold resistance against neutralization by the antibodies present in earlier plasma samples. Although the neutralizing antibody response appeared to keep pace with viral escape initially, at later time points plasma samples contained little neutralizing capacity against contemporaneous isolates. The investigators also tracked persistent changes within gp120 that both appeared early and coincident with neutralization escape. Interestingly, most fixed changes involved addition of glycosylation sites and many were within the immunologically silent face of gp120, rather than within structures known to be targeted by neutralizing antibodies (such as the receptor binding region or the V1/V2 or V3 loop). None of the isolates were atypically sensitive to monoclonal antibodies of known specificity, suggesting that the viral clones obtained from early time points behaved as

4 246 Current HIV Research, 2004, Vol. 2, No. 3 Cheryl A. Pikora Table 1. Glycosylation Changes and Neutralizing Antibody Resistance Acquired During Infection Study Viral strain Location of N-linked glycosylation changes Wei, et al. [116] Primary isolates of HIV-1 Multiple sites in silent face (201, 235, 240, 268, 301, 359 and V4 loop) Reagent used to detect neutralization resistance Autologous plasma Rudensey, et al. [98] SIVMneCL8 V1 (148), V4 (416,417) Sera from SIVMne-infected macaques Harouse, et al. [39] SHIV SF162P3 V2 (158) Autologous sera Cheng-Mayer, et al. [17] SHIV SF33 V1, V3 (301) Pooled sera from HIV+ individuals Malenbaum, et al. [70] Clade A and B TCLA HIV-1 strains V3 (301) CD4BS, CD4i antibodies Back, et al. [6] TCLA strains HIV-1 V3 (301) V3 loop, CD4BS antibodies Polzer, et al. [90] V3 loop of primary isolates in TCLA strains HIV-1 V3 (301) Pooled sera from HIV+ individuals neutralization-resistant primary isolates. This also suggests that the changes that conferred resistance represented an adaptive response to the patient s own neutralizing antibody pressure. From these findings, the authors propose the hypothesis of an evolving glycan shield in which the virus accumulates persistent glycosylation changes outside epitopes of neutralization that are capable of blocking neutralizing antibody recognition of neighboring epitopes. From this model, one would expect that elimination of one of the resistance-mediating glycosylation sites would increase viral susceptibility to neutralization. This was indeed shown to be true but in a context-dependent manner: a single glycosylation change made within one viral clone (matching the sequence of an early isolate) led to a 20-fold increase in neutralization sensitivity but the same change had no effect on neutralization sensitivity in a clone from the same individual taken from a later time point. The development of neutralization resistance during natural infection has also been seen in macaques infected with SIV. In a study by Rudensey et al. [98], evaluation of virus at late time points following infection of macaques with a macrophage-tropic, neutralization-sensitive clone of SIVMne (SIVMneCL8) (during which the animals had progressed to AIDS) revealed additions of glycosylation sites within V1 and V4. Coincident with these changes, chimeric SIVMneCL8 expressing Env from late time points were shown to be neutralization-resistant. In a further study, additional N- and O-linked glycosylation sites appeared in the V1 loop within 35 weeks following infection with SIVMneCL8. These changes conferred neutralization resistance, apparently due to a change in a conformational neutralizing antibody epitope that involved the V1 region [16]. V1/V2 loop glycosylation changes were also correlated with neutralization resistance in a SHIV model of infection. In this study, a pathogenic strain of SHIV was derived through serial passaging of the parental virus in vivo [39, 40]. One noted feature of the newly derived pathogenic strain was that it was more efficiently transmitted via a mucosal route than the parental strain [39]. When comparing N-linked glycosylation between the parental and more pathogenic variant, two additional sites were noted in the latter: one within the V1 domain and one within the N-terminal base of the V2 loop (amino acid 158) [68]. Further study revealed that a single addition of a glycosylation site at residue 158 enhanced mucosal transmissability through increased binding to DC-SIGN and at the same time conferred increased resistance to autologous serum neutralization [68]. Likewise, detection of a neutralization-resistant variant with increased pathogenicity has been reported in an infected macaque 16 months after inoculation with SHIV SF33 [67] [17]. Sequence analysis revealed multiple amino acid changes within Env, including the addition of an N-linked glycosylation site within the N-terminus of the V3 loop and loss of an N-linked glycosylation site in V1 [17]. Addition of a N-linked glycosylation site within the N-terminus of the V3 loop (amino acid 301) or elimination of the site within V1 was directly correlated with increased neutralization resistance to a pool of sera from HIV-1 infected individuals. Both glycosylation changes occurring simultaneously had an additive effect on neutralization resistance. The sole effect of this N-terminal V3 loop glycosylation site on neutralization sensitivity was further evaluated in a study by Malenbaum, et al. [70]. Using clade A and B viruses in which this N- linked site was variably present, neutralization sensitivity to CD4BS and CD4i monoclonal antibodies and to IgGCD4 was directly correlated with the absence of this glycan. In a similar study, an analysis of the V3 loop of 4 wellcharacterized molecular clones of HIV-1 demonstrated that the presence of this N-terminal V3 loop glycosylation site increased resistance to neutralization by V3-loop and CD4BS monoclonal antibodies [6]. Studies evaluating this same glycan from primary isolates also point to an important correlation with neutralization resistance. Polzer et al. [90] inserted the V3 loop of primary isolates either lacking or possessing this glycan into laboratory adapted HIV-1 strains and neutralization was determined using pooled, immune sera from HIV-infected individuals. The presence of this glycan appeared to confer a moderate to significant enhancement of resistance to neutralization on 3 of the 4 strains examined. In a vaccination study attempting to take advantage of enhanced neutralization sensitivity of HIV-1 strains lacking this glycosylation site, mice were primed with DNA of HIV BRU modified at this site to prevent glycosylation or with DNA of unmodified HIV BRU and then boosted with the same immunogens [9]. Although significantly higher titers of antibody were generated by the mice primed and boosted with the strain of HIV BRU lacking the N-terminal V3 glycan, sera pooled from these mice did

5 Glycosylation of the ENV Spike of Primate Immunodeficiency Current HIV Research, 2004, Vol. 2, No Fig. (2) Secondary structure of gp120 of SIV239 and HIV-1 HXBc2 adapted from [20, 63]. Circles connected with a line represent intrachain disulfide bridges. The prototype is for that of HIV-1, therefore, some regions of SIV (such as the V1/V2 loop) are considerably longer. The asparagine residues used in the canonical N-linked glycosylation sequence, NXT/S are highlighted in a manner designed to distinguish between those that changed naturally during infection with either SIV, HIV-1 or SHIV or those that were experimentally removed in SIV and HIV-1. The exact locations of the N s within the NXT/S of SIV within V1/V2 were estimated within this structure based upon their proximity to the conserved cysteines. A question mark indicates an inability to specifically locate the N of the glycosylation site, however, the site lies within the structure indicated. not demonstrate enhanced neutralizing antibody titers to either autologous or wild type HIV BRU compared with those that were primed and boosted with the parental HIV BRU strain. Perhaps, in this setting, elimination of an increased number of glycosylation sites is required in order to elicit a discernible increase in neutralizing antibody titers. Additionally, the antigenic specificities of antibodies raised in mouse models of immunization against HIV Env protein may not closely mimic that of humans.

6 248 Current HIV Research, 2004, Vol. 2, No. 3 Cheryl A. Pikora EVALUATION OF THE EFFECTS OF ARTIFICIALLY INTRODUCED GLYCOSYLATION CHANGES ON ANTIBODY NEUTRALIZATION IN VITRO AND IN VIVO To further understand the correlation of glycosylation changes with neutralization escape many investigators have created strains of HIV and SIV that lack selected glycosylation sites. The variable loops, V1/V2 and V3, have been most commonly targeted for mutation of glycosylation sites, as these have been noted most often in natural infection to acquire glycosylation changes that increase viral neutralization resistance. V1/V2 Loop Glycosylation Changes The role played in immune evasion by one of the most variable of the hypervariable regions [3, 12], V1/V2, is of particular interest. This structure is well-exposed on the surface of gp120 and is composed of two (HIV) or more (SIV) looped regions that form from one disulfide linkage and that share a stem consisting of two antiparallel strands linked together at both ends by disulfide bonds [63]. The proximity of this structure to the V3 loop [30, 118] and the observation that some well-conserved residues of the V1/V2 stem contribute to CD4 binding [57] put this structure in a unique position to impact on gp120 immunogenicity. Mutagenic studies and structural data support the hypothesis that this region is brought into proximity with the V3 loop following CD4 binding [57, 118, 119]. The conformational changes that result from CD4 binding apparently include repositioning of the V1/V2 loop, which then allows for the formation of an antiparallel β sheet that contributes to CD4i antibody and second receptor binding [57]. Removal of the V1/V2 loop does not abrogate viral replication [15, 45, 119], supporting the notion that the V1/V2 domain has evolved mainly to protect the virus from the host immune response. Although dispensable for viral replication, changes in the V1/V2 loop have been shown to impact on cellular tropism [11, 52, 53, 105, 117], syncytium formation [4, 37, 108] and virus infectivity [108, 114]. Given the position and hypothesized protective function of the V1/V2 loop, one would expect that addition of carbohydrate to this structure would expand upon its ability to shield underlying residues, as well as residues within its structure, from neutralizing antibodies. This, indeed, appears to be the case. The V1/V2 loop and stem of HIV-1 and SIV contains disproportionally more N-linked glycosylation sites (5-11) than any of the other hypervariable regions. Elimination of any of these relatively well-conserved glycans either singly or in certain double combinations is welltolerated by the virus [54, 69, 95], which is not surprising in view of the fact that elimination of this entire loop is well-tolerated. The extensive N-linked glycosylation in this region is thought to play an important role in occlusion of neutralizing antibody epitopes rather than maintenance of viral replicative capacity [46, 55, 69, 92, 95, 96]. However, there are some N-linked glycosylation sites that cannot be eliminated from the V1/V2 domain without a significant compromise in viral infectivity. For instance, Wang et al. [115] showed that the infectivity of a HIV-1 mutant strain, in which half the N-linked glycosylation sites in V1/V2 were removed, was severely impaired. Interestingly, when this virus reverted, it did not restore glycosylation sites within V1/V2, but rather substituted amino acids within C1 and C4 [115]. This raises the possibility that the Env trimer accomodates extensive glycosylation in V1/V2 by changes in adjacent structures in such a way that, upon removal of a significant fraction of these glycans, accomodative changes have a deleterious effect on infectivity. Amino acid substitutions in C1 and C4 may allow for structural readjustment to the missing glycans. The observation that the virus did not require the addition of N- linked glycosylation sites in V1/V2 in order to revert provides further experimental evidence that N-linked glycosylation in V1/V2 does not play a direct role in viral replication. Studies directed at evaluating the role of particular glycans in vitro within the V1/V2 loop and stem in neutralization sensitivity of SIV have shown that loss of particular glycans results in increased neutralization sensitivity (Table 2) (Fig. (2)) without significant loss of infectivity. One such study describes an ability to raise high neutralizing antibody titers to SIV239 in rhesus macaques inoculated with mutant strains of SIV239 either lacking 2 N- linked glycosylation sites in the V1/V2 domain [95, 96] or with the M5 strain of SIV239 which lacks 3 N-linked sites within V1/V2 and 2 adjacent sites within C2 [46, 95]. One of the striking findings of this study was that inoculation of the mutant strains not only resulted in strong, high-titered neutralizing antibody responses to autologous virus, but also in higher neutralizing antibody titers to the parental SIV239 strain when compared to animals inoculated with WT SIV239. The array of neutralizing antibody specificities raised in the mutant infected animals was not directly addressed in this study, although high titers of antibodies to V1/V2 peptides were present. A broadly-reactive neutralizing antibody response was likely in these animals, based upon the finding that the mutant viruses (M5, in particular) were markedly sensitive to an extensive panel of SIV239-Env specific monoclonal antibodies [46]. This included linear and conformational epitopes that represented at least 10 distinct epitope competition groups spanning the entire Env sequence from the NH-terminal end of gp120 to gp41 [19, 27, 48, 49]. This finding may be explained by a resultant global conformational change in SIV239 wrought by elimination of these N-linked sites that renders the virus more sensitive to neutralization through alteration of receptor-binding kinetics or by revealing epitopes that are adjacent to V1/V2 in the heterotrimeric Env structure. The increase in autologous neutralizing antibody titers was accompanied by tighter control of viral replication in the mutant infected animals. Although acute viremic peak titers of the mutants and wild type SIV239 were similar, the viral loads at setpoint of macaques infected with the mutants (particularly with M5) were considerably lower. A separate analysis of the reactivity of serum antibodies from mutant infected animals to overlapping peptides spanning V1/V2 and the V3 loops [20] showed a moderate increase in immunoreactivity to V1 peptides. Immunoreactivity targeted to the V1 region was further substantiated with the selective recognition of chimeric HIV-1 Env in which the V1 sequence of HIV was exchanged for that of SIV. The modest reactivity to V3 peptides observed in these animals implies

7 Glycosylation of the ENV Spike of Primate Immunodeficiency Current HIV Research, 2004, Vol. 2, No Table 2. Studies Evaluating the Elimination of Glycosylation Sites Within V1/V2 and V3 Loops to Enhance Neutralization Sensitivity Study Viral Strain Locations of N-linked glycosylation mutations in V1/V2 and V3 loops Neutralizing Reagent Increased neutralization susceptibility Reitter, et al. [96] SIV , 146, 156 Autologous and pooled sera from SIV+ macaques Kolchinsky, et al. [55] HIV-1 ADA 197 CD4BS, CD4i, V3 loop, gp41 antibodies + + Ly, et al. [69] HIV-1 SF62 154, 186, 195 CD4BS, V3 loop antibodies + Gram, et al. [36] HIV-1 BRU 116 V3 loop antibodies + Quinones-Koch, et al. [93] VSV chimeric for HIV 89.6 Env 130, 135, 139, 158, 190, 200 Sera from immunized mice _ Schonning, et al. [103] HIV LAI 301 (V3) V3 loop antibodies + Bolmstedt, et al. [10] HIV-1 BRU 308 (V3) Autologous sera from immunized guinea pigs + that removal of glycans within V1 might also reveal regions within V3. The observation of changes in disease pathogenesis and the demonstration of increases in neutralizing antibody titers following infection using mutant strains of SIV239 lacking particular glycosylation sites was the first of its kind to lend credence to the theory of glycan shielding of neutralizing antibody sites through in vivo studies. As further evidence for the ability of neutralizing antibody responses to influence viral load setpoint, M5-infected macaques were depleted of B cells using a monoclonal antibody against human CD20 at days 7, 0 and 7 relative to infection [44]. The B-cell depleted animals demonstrated an increase in viral load that did not influence the height of the peak of primary viremia, but resulted in much higher viral loads later in infection after week 10 - compared to monkeys infected with M5 that received an isotype-matched control antibody [44]. Evaluation of the effects of glycosylation changes in HIV-1 on neutralization sensitivity is facilitated by the availability of a number of well-characterized monoclonal antibodies capable of neutralizing a broad range of viral strains [42, 91, ]. Mutagenesis of Env glycosylation sites within the V1/V2 loop of HIV-1 has been inspired by naturally occuring changes observed during infection or generated under particular tissue culture conditions [16, 67]. For example, Kolchinsky et al. [56] derived a CD4- independent variant of the primary HIV-1 isolate, ADA, by repeated passage of virus in a CD4 negative cell line. Cloning and analysis revealed that CD4-independence resulted from changes in the V1/V2 stem [54]. Further analysis revealed that elimination of a single glycosylation site at position 197 within the V1/V2 stem could solely mediate CD4-independent gp120 binding to CCR5. The explanation given for the CD4-independent phenotype was that the carbohydrate at position 197, located at a pivotal position in the V1/V2 stem, probably inhibits the movement of the V1/V2 loops from their native positions prior to binding CD4. Elimination of this glycan likely frees up the movement of V1/V2 and allows gp120 to achieve the conformation it requires to bind the second receptor, even in the absence of CD4 binding. Not surprisingly, loss of this glycan also enhanced neutralization sensitivity to CD4i neutralizing antibodies [55]. The CD4-independent phenotype was conferred solely by the loss of this N-linked glycosylation site as changes in nearby glycosylation sites (at positions 188 and 195) had no effect on CD4- independent CCR5 binding. Interestingly, follow up studies demonstrated that the sensitivity of the ADA variant strain lacking glycan 197 to neutralization by a number of neutralizing antibodies specific to various regions within gp120 (CD4i sites, CD4BS, V3 loop and gp41) was signicantly enhanced [55] reminiscent of the global increases in neutralization sensitivity seen in SIV239 lacking particular combinations of glycosylation sites in V1/V2 [46]. The hypothesis put forth to explain this observation relates to gp120 conformational changes and their impact on ligand binding and cell entry. The authors propose that the structure of the gp120 Env trimer may, upon loss of the glycosylation site at 197, exist in a metastable conformational state that increases its vulnerability to the binding of a number of ligands which then could deactivate the virus likely in a time-dependent fashion. CD4i neutralizing antibody sensitivity and CD4 independent viral replication seem, therefore, to be linked specifically to the elimination of N-linked glycosylation at position 197 in the V1/V2 loop stem. Interestingly, the three SIV239 glycosylation mutants described earlier, in which a combination of two out of three N-linked sites within V1V2 were mutagenized in each mutant, also demonstrated moderate CD4-independent replication [92]. Effects of N-linked glycosylation changes within V1/V2 upon CD4-independence and sensitivity to neutralization mediated by a wide range of ligands may rely upon the specific location of the N-linked site or the context in which the glycosylation change is made. For instance, elimination of an N-linked glycosylation site in the V1/V2 loop of HIV- 1 SF62 (a neutralization-resistant, primary isolate of HIV-1) at a location near to 197 (at position 186) resulted in a ten fold decrease in the 90% inhibitory concentration (IC 90 ) of the monoclonal CD4BS antibody IgGb12 [69]. However, this mutant strain did not demonstrate the global sensitivity seen in the mutant ADA strains lacking glycan 197, and was not

8 250 Current HIV Research, 2004, Vol. 2, No. 3 Cheryl A. Pikora rendered CD4-independent. Two other glycosylation changes were made independently within the V1/V2 stem in the context of the HIV-1 SF162 background: at position 154 and 195. These two mutant strains demonstrated enhanced neutralization sensitivity to a number of monoclonal antibodies specific for either the V3 loop or CD4BS but not to CD4i monoclonals and did not display CD4- independence. Similar results were obtained in a study evaluating the neutralization sensitivity of HIV-1 BRU lacking one glycosylation site at position 116 within V1 [36]. This mutant strain was moderately more sensitive to monoclonal antibodies directed to the V3 loop, but was no more sensitive than the parental strain to neutralization by 2G12 (a monoclonal antibody directed to a glycosylated, conformational epitope) or scd4. Therefore, it appears that sensitivity to neutralization mediated by specific ligands and loss of CD4 dependence depends not only upon which N- linked glycosylation site within V1/V2 is eliminated but also upon the viral context in which the change is made. Another example of this is provided by a study in which mice were inoculated with VSV expressing HIV-1 Env containing glycosylation site mutations within V1/V2 for the induction of antibody with potent neutralizing activity against HIV-1 [93]. The mice mounted measureable antibody responses to the mutated HIV-1 Env; however their neutralizing titers were relatively low and similar to mice inoculated with VSV expressing WT HIV-1 Env. The authors hypothesize that structural differences between HIV and SIV Env lacking the same glycans in V1/V2 may have accounted for their inability to elicit strong neutralizing antibody titers such as those induced in monkeys infected with SIV239 glycosylation mutants [96]. It should also be kept in mind that murine immune responses to HIV-1 Env in the context of VSV could be quite different from immune responses of macaques infected with whole virus. V3 Loop Glycosylation Changes Acquisition of N-linked glycosylation within the N- terminal region of the V3 loop during infection, or the natural occurrance of this glycan within certain HIV-1 strains, has been shown in multiple in vivo studies to mediate neutralization resistance (Table 2) (Fig. (2)) [6, 17, 70, 90]. To directly demonstrate this and to evaluate the rapidity with which this resistance is acquired, a neutralization-sensitive strain of HIV-1 (HIV LAI ), modified by elimination of the V3 loop N-terminal glycan, was subjected to passage in the presence of monoclonal antibodies directed to the V3 loop [104]. Passaging in the presence of V3-specific mabs rapidly selected for variants whose only change in the V3 sequence was the addition of the N-terminal glycosylation site. This same group further analyzed whether there were conformational requirements that related to the decreased interaction of the V3 monoclonal antibody, NEA-9205, with gp120. [103]. Interestingly, NEA-9205, directed at a linear epitope within V3, bound equally well to gp120, whether it contained the N-linked glycosylation site within V3 or not. Differential binding was only seen in the context of intact virions HIV-1 with a glycosylated V3 loop was resistant to NEA- 9205, whereas, its nonglycosylated counterpart displayed a 100-fold increase in sensitivity to V3-mediated neutralization. This study supports the notion that monomeric gp120 is much more permissive to antibody binding than gp120 in the more conformationally constrained context of the trimer. Monomeric gp120 likely exists in a higher entropic state and therefore possesses a greater amount of flexibility in binding than the oligomer [77]. Miscellaneous Regions Within gp120 Evaluation of the contributions made by N-linked glycosylation sites to neutralization sensitivity has mostly focused on the V1/V2 and V3 loops. However, glycosylation changes within other regions of HIV and SIV have also been correlated with a change in neutralization sensitivity. The previously mentioned study by Wei, et al. [116], in which escape mutations of autologous viral isolates from neutralization largely mapped to the silent face of gp120, is an example. Another study identified a glycan in the V4 loop that also conferred marked sensitivity to neutralization [28]. This was discovered in the context of a CD4-independent mutant of HIV-1 HXBc2, that contained a substitution of asparagine with lysine at position 386 within V4 [43, 59]. Elimination of Multiple Glycosylation Sites in Discontinuous Regions of gp120 Several studies have addressed the possibility of enhancing neutralization sensitivity through elimination of multiple, N-linked glycosylation sites distributed throughout gp120 (Table 3) (Fig. (2)). Ohgimoto, et al. [81] created a replication-competent, variant of SIV239 that lacked N-linked sites at five positions (79, 146, 171, 460 and 479); spanning regions from the N- to C-terminus of gp120. Rhesus macaques were inoculated with this variant to evaluate whether attenuation was present in vivo and, if so, whether the attenuated phenotype could be explained by more potent immune responses [75]. Viral loads in the animals peaked at similar levels, but then fell off to set points much lower than animals inoculated with wild type SIV239. However, neutralizing antibody titers to the variant strain appeared to be elevated significantly in only one of the two monkeys inoculated. Neutralizing antibody also appeared relatively late at 20 weeks post infection in one of the two animals at a time point 10 weeks beyond the establishment of the viral load setpoint. Additionally, the animals were also not protected against challenge with wild type SIV239 virus. This is consistent with the observation that neither of the two animals inoculated with the variant strain possessed measurable neutralizing antibody titers to wild type SIV239. The three vaccinated animals did have slightly earlier cytotoxic T lymphocyte (CTL) responses than the vaccine-naïve animals following challenge. It is possible that the deglycosylation of env at these sites enhanced HIV-specific cellular immune responses in the same manner in which deglycosylation enhances SIVspecific CD4 help in SIV-infected macaques [100, 101] and CTL responses in HIV-1-immunized mice [25]. Tight viral setpoint control, therefore, might have been mediated by stronger SIV-specific cellular immunity in these animals. Koch et al. [51] approached the issue of neutralization sensitivity and glycosylation changes using the crystal

9 Glycosylation of the ENV Spike of Primate Immunodeficiency Current HIV Research, 2004, Vol. 2, No Table 3. Studies Evaluating the Elimination of Multiple, Nonadjacent Glycosylation Sites to Enhance Neutralization Sensitivity Study Viral strain Locations of N- linked glycosylation mutations Neutralizing Reagent Increased neutralization susceptibility Reitter, et al. [96] SIV , 146, 156, 185, 244,247 Multiple monoclonal antibodies to SIV239 covering at least 10 epitopes Mori, et al. [81] SIV239 79, 146,171, 460, 479 Autologous serum + a Koch, et al. [51] HIV-1 JR-FL, YU-2 and HXBc2 197, 276, 301, 386 CD4BS, V3 loop, CD4i + Bolmstedt, et al. [10] HIV-1 BRU 406, 448, 463 Autologous sera from immunized guinea pigs a 1 out of 2 monkeys had measureable neutralizing antibody titers to autologous virus + + structure of HIV-1 gp120 as a guide. This group hypothesized that elimination of N-linked glycans lying proximal to receptor binding sites might increase neutralization sensitivity by exposing underlying or adjacent neutralizing epitopes. Two difficult-to-neutralize primary strains of HIV-1 (JR-FL and YU2) and a lab adapted isolate, HXBc2 were mutagenized to eliminate glycosylation both singly and in combination at asparagines 197, 276 (proximal to the CD4 binding site), 301 and 386 (relatively close to the putative chemokine receptor binding site). As in previous studies [10, 103, 104], elimination of the V3 loop N-terminal glycosylation site (301) in all three strains resulted in enhanced neutralization sensitivity to CD4BS antibodies and a V3 loop antibody. The observed influence that the V3 loop N-terminal glycan had on neutralization sensitivity in both lab adapted and primary strains is consistent with previously reported studies [6, 17, 70, 90, 103, 104] although, unlike the findings of Malenbaum et al. [70], no observed increased neutralization to CD4i antibodies was observed in strains solely lacking the V3 loop N-terminal glycosylation site. The enhanced neutralization appeared to correlate with increased binding of CD4BS and V3 loop antibodies to Env trimers on the surface of cells transfected with the glycan-deficient variants compared to the parental strain. As expected, differential binding of gp120 monomers was not detected. Interestingly, although proximal to receptor binding sites, loss of glycosylation sites 276 and 386 had no effect on neutralization sensitivity unless combined with the elimination of glycan 301. This is not unlike the findings of Bolmstedt et al. [10] who evaluated whether eliminating glycans lying proximal to the CD4 binding domain would enhance immunogenicity of HIV-1 Env. Guinea pigs were immunized with vaccinia virus recombinant for HIV-1 BRU lacking 3 N-linked glycosylation sites proximal to the CD4 binding domain (sites 406, 448 and 463). Animals were able to raise neutralizing antibodies to autologous virus at levels similar to WT Env-inoculated animals but were unable to raise broadly-reactive neutralizing antibody responses. In addition to the findings described above, Koch, et al. described enhancement of CD4i antibody neutralization only when multiple glycosylation sites were eliminated [51]. The authors suggest that larger holes created by removal of multiple receptor-proximal glycans are required in order to enhance CD4i mediated neutralization. Based upon the location of the glycan at the base of the V3 loop, the authors propose that loss of this glycan is unlikely to uncover underlying CD4BS epitopes, but may have resulted in repositioning of structures on the same or adjacent monomers, thus allowing access to the CD4 binding site. Addition of N-Linked Glycosylation Sites that Redirect Neutralizing Antibody Responses Most studies described in this review thus far involved removal of N-linked glycosylation sites in order to enhance neutralization sensitivity of SIV and HIV Env. However, few have examined the effect of adding additional N-glycan attachment sites. Hyperglycosylation of the immunodominant variable loops of the spike might shield these sites from immune recognition and redirect antibody responses to better conserved gp120 regions. The desired outcome of creating such an immunogen would be to elicit more broadly reactive antibody responses to the Env spike. To address this issue, Pantophlet et al. [84] created a mutated and hyperglycosylated form of the HIV-1 JR-FL gp120 monomer. An initial study done by this group [83] demonstrated that substitution of four residues on the perimeter of the Phe-43 cavity of HIV-1 JR-FL with alanine resulted in deficient or absent binding of weakly neutralizing or non-neutralizing monoclonal antibodies to the CD4 binding site. However, the binding of b12 was unaffected. Addition of a number of glycosylation sites within gp120 of this HIV-1 JR-FL mutant resulted in a monomeric gp120 molecule which maintained the desirable b12 epitope but was unable to bind antibodies to variable loop epitopes or weakly neutralizing antibodies to the CD4 binding site. These findings suggest that such an immunogen may prove useful in masking epitopes that elicit either type-specific, non-neutralizing or weakly neutralizing antibodies, while focusing the antibody response on epitopes that elicit strongly neutralizing, broadly-reactive antibodies. Along the same lines, Garrity et al. 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