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1 AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan
2 Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki 305-0843, Japan
3 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
4 Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
5 Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA
6 Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan
7 Center of Research Network for Infectious Diseases, Riken, Chiyoda-ku, Tokyo 100-0006, Japan
Correspondence
Kazuyasu Mori
mori{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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5G, derived from SIV239, a pathogenic clone of simian immunodeficiency virus (SIV), led to robust acute-phase viral replication followed by a chronic phase with undetectable viral load. This study examined whether humoral responses in 5G-infected animals played any role in the control of infection. Neutralizing antibodies (nAbs) were elicited more efficiently in 5G-infected animals than in SIV239-infected animals. However, functional nAb measured by 90 % neutralization was prominent in only two of the five 5G-infected animals, and only at 8 weeks post-infection (p.i.), when viral loads were already below 104 copies ml–1. These results suggest a minimal role for nAbs in the control of the primary infection. In contrast, whilst Ab responses to epitopes localized to the variable loops V1/V2 were detected in all 5G-infected animals at 3 weeks p.i., this response was associated with a concomitant reduction in Ab responses to epitopes in gp41 compared with those in SIV239-infected animals. These results suggest that the altered surface glycosylation and/or conformation of viral spikes induce a humoral response against SIV that is distinct from the response induced by SIV239. More interestingly, whereas V1/V2-specific Abs were induced in all animals, these Abs were associated with vigorous 5G-specific virion capture ability in only two 5G-infected animals that exhibited a functional nAb response. Thus, whereas the deglycosylation mutant infection elicited early virion capture and subsequent nAbs, the responses differed among animals, suggesting the existence of host factors that may influence the functional humoral responses against human immunodeficiency virus/SIV. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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) and antibody-dependent cellular lysis (Ahmad & Menezes, 1996; Forthal et al., 2001). Acquiring an understanding of these various mechanisms for their exploitation in the development of candidate vaccines has been a major challenge.
The envelope protein (Env) of HIV/simian immunodeficiency virus (SIV) comprises an exterior protein (gp120) and a transmembrane (TM) protein (gp41), and trimers of the gp120/gp41 complexes form viral spikes that promote binding to receptors and co-receptors on the cell membrane for entry into the target cells (Wyatt & Sodroski, 1998). The major viral receptors of HIV/SIV include CD4 and a variety of co-receptors such as CCR5 or CXCR4. One desirable target epitope for neutralizing antibody (nAb) that shows relative conservation across clades is the binding site for the co-receptor (Burton et al., 2004; Zolla-Pazner, 2004); however, this site is conformationally cryptic within the viral spike up until immediately after binding of the viral spike to CD4, providing an effective shielding mechanism to the virus. Another distinct feature of HIV/SIV Env is the extensive glycosylation that also effectively prevents access to antibodies directed at the epitopes (Chen et al., 2005; Wyatt & Sodroski, 1998; Wyatt et al., 1998). The gp120 protein possesses 18–33 Asn–X–Ser/Thr sequences, signals for the attachment of N-linked carbohydrate side chains (Leonard et al., 1990; Ohgimoto et al., 1998; Regier & Desrosiers, 1990; Zhang et al., 2004). As the carbohydrate moiety is generally weakly immunogenic and is recognized to a large extent as self by the host immune system, the massive glycans on the surface of viral spikes constitute an immunologically silent facade (Wyatt & Sodroski, 1998; Wyatt et al., 1998). As a result, mature viral spikes are protected from nAb and other host immune responses by a massive carbohydrate ‘glycan shield’ (Chen et al., 2005; Wyatt & Sodroski, 1998; Wyatt et al., 1998). In fact, a prominent role of carbohydrates of HIV/SIV in evasion from immune surveillance has been reported previously as follows. Variants of SIV that have evolved to acquire additional glycans in the variable regions of Env have increased neutralization resistance compared with the parental virus (Chackerian et al., 1997; Cheng-Mayer et al., 1999). Similarly, the appearance of neutralization escape mutants has been associated with altered glycosylation in HIV-1 evolved during the course of infection (Wei et al., 2003). Conversely, infection with SIV239 mutants with deglycosylated Env (lacking N-linked glycosylation sites) in the variable loops V1/V2 of gp120 elicited markedly increased titres of nAb (Reitter et al., 1998). We have reported that a deglycosylation mutant, 5G, lacking N-linked glycosylation sites at aa 79, 146, 171, 460 and 479 in gp120 of SIV239 displayed an attenuated phenotype when used to infect rhesus macaques (Mori et al., 2001; Ohgimoto et al., 1998). In addition, animals infected with 5G exhibited almost sterile protection against rechallenge with SIV239 (Mori et al., 2001).
Thus, we suggest that studies aimed at identifying the mechanisms underlying the early and potent immune control of deglycosylated SIV may provide knowledge for the formulation of effective HIV/SIV vaccines. Studies performed herein were therefore directed at attempts to define more precisely the early humoral responses (both virus-specific nAb and non-nAb) generated after infection with 5G in rhesus macaques and to compare these responses with those observed in macaques inoculated with wild-type SIV239, with the rationale that results from such studies may help to identify their potential contribution towards viral control of primary infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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) and its derived deglycosylated mutant, 5G, were used in this study. 5G was derived by mutagenesis of an SIV239 infectious DNA clone so that the asparagine residues for N-glycosylation at aa 79, 146, 171, 460 and 479 in gp120 were converted to glutamine residues (Fig. 1a) (Ohgimoto et al., 1998). Viral stocks of SIV239 and 5G were prepared as reported previously (Mori et al., 2001).
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5G sequence (see Fig. 5b). All peptides were dissolved in DMSO diluted in PBS.
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5G or SIV239 as described previously (Mori et al., 2001).
Plasma viral load measurements.
SIV infection was monitored by measuring the plasma viral RNA load using a highly sensitive quantitative real-time RT-PCR. Viral RNA was isolated from plasma samples from infected animals using a commercial viral RNA isolation kit (Roche Diagnostics). SIV gag RNA was amplified and quantified using a method originally developed by Hofmann-Lehmann et al. (2000) using a TaqMan EZ RT-PCR kit (Applied Biosystems). The detection sensitivity of plasma viral RNA by this method was 100 viral RNA copies per ml plasma (given as copies ml–1).
Neutralization assay.
SIV neutralization was tested according to a protocol using CEMx174/SIVLTR-SEAP cells, originally described by Means et al. (1997). To measure low levels of nAb, IgG was purified from plasma as described below and concentrated virus stocks were used.
Anti-gp120 Ab ELISA and anti-Env peptide ELISA.
Recombinant SIV239 gp120 and 5G gp120 were expressed utilizing a Sendai virus vector as described previously (Mori et al., 2005; Yu et al., 1997). Culture supernatant containing approximately 2 µg secreted SIV gp120 ml–1 was diluted with an equal amount of PBS, dispensed into each well of an ELISA plate and allowed to incubate at 4 °C overnight. For the peptide ELISA, each peptide was diluted to 0.5 µM with 50 mM carbonate buffer (pH 9.5) and captured on Nunc Immobilizer amino plates (Nalge Nunc) at 4 °C overnight. A 1 : 100 dilution (150 µl) of the plasma sample to be tested was dispensed into antigen-immobilized plates and incubated at 37 °C for 1 h. Ab responses were detected using peroxidase-conjugated goat anti-monkey IgG and o-phenylenediamine. Absorbance was measured at 490 nm.
Removal of linear V1/V2 epitope-specific Abs from IgG fractions.
A mixture of the peptides (V1V2-9, -10 and -11; see Fig. 5b
) was conjugated to a HiTrap NHS-activated HP column (GE Healthcare). IgGs from plasma samples were fractionated using a mAb trap kit (GE Healthcare) and applied to the peptide-conjugated column. The flow-through fractions devoid of anti-V1V2-9, -10 and -11 peptide-specific Abs were collected. The concentration of IgG was determined using a protein assay kit (Bio-Rad).
Virion capture assay.
The virion capture assay was modified using a method reported by Nyambi et al. (1998). ELISA plates were coated with the IgG samples described above at a concentration of 20 µg ml–1 in 50 mM carbonate buffer (pH 9.5) and incubated at 4 °C for 48 h. The plates were washed three times with PBS and blocked with 3 % BSA in PBS at 37 °C for 1 h. The plates were then washed three times with serum-free RPMI 1640. 5G or SIV239 virion solutions with a p27gag concentration of 15, 7.5 and 3.75 ng in 10 % fetal bovine serum/RPMI 1640 were added to each well of the IgG-coated plate and incubated at 37 °C for 3 h. The wells were washed five times with serum-free RPMI 1640 to remove unbound virus. The virus bound to IgG was lysed using MagNA Pure LC Lysis/Binding Buffer (Roche Diagnostics). The viral lysates were subjected to viral RNA purification using a MagNA Pure Compact nucleic acid purification kit (Roche Diagnostics). The copy number of the isolated SIV RNA was determined by real-time RT-PCR for SIV239 as described above.
Statistical analysis.
Correlation analysis was done using Spearman's non-parametric rank test and the Mann–Whitney test using GraphPad Prism 4.0 software. Correlations were considered to be statistically significant for values of P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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5G (n=5) or SIV239 (n=3) (Mori et al., 2001). Plasma viral RNA loads were assayed for up to 400 weeks p.i. and the data obtained in the 5G-infected (Fig. 1b) or SIV239-infected (Fig. 1c) animals were plotted. Both 5G and SIV239 replicated with similar kinetics during the early phase of primary infection for up to 4 weeks p.i. However, subsequent to this acute infection phase, virus replication was markedly different in the two groups of monkeys: SIV239-infected animals exhibited viral load set points around 105 copies ml–1 in two of three animals, with one animal (Mm13) having an undetectable viral load (<100 copies ml–1) by 30 weeks p.i. (Fig. 1c). In contrast, the 5G-infected animals showed uniformly controlled viraemia reaching undetectable levels by 12–16 weeks p.i. and maintained this control for more than 6 years p.i. (Fig. 1b).
nAb response in 5G-infected animals
Although failure to detect a nAb response is characteristic of SIV239-infected rhesus macaques (Johnson et al., 2003; Means et al., 1997), the rapid control of viraemia in 5G-infected animals prompted us to determine whether nAb played a role in this control of viraemia. We hypothesized that the deglycosylation might lead to the elicitation of a markedly more vigorous nAb response than infection with SIV239. To maximize the detection sensitivity of weak nAb responses at early time points p.i., an assay that measures neutralization titres based on 50 % inhibition of virus replication (IC50) in CD4+ T-cell lines was initially used. Consistent with the reported results in SIV239-infected animals, no appreciable nAb titre was detected in two animals (Mm13 and Mm25), despite the fact that viral load in Mm13 was distinctively decreased by 30 weeks p.i. However, we observed a rare animal (Mm20) that elicited a robust nAb response against SIV239 and a relatively delayed nAb response against 5G, despite the maintenance of a high viral load (Fig. 2a). These results indicated the lack of correlation of nAb response with viral load in SIV239-infected animals. In contrast, nAb was detected in two 5G-infected animals (Mm07 and Mm22) starting at 8 weeks p.i. and in two additional animals (Mm12 and Mm23) at 12 weeks p.i. (Fig. 2b, left panel). These titres peaked at either 12 or 18 weeks p.i., and the peak was followed by a decrease in titre that varied among animals. Mm12 and Mm23, which exhibited nAb induction at 12 weeks p.i., had essentially low titres, whilst Mm07 and Mm22, which exhibited nAb induction at an earlier time point, maintained vigorous nAb titres of >1 : 100. Of note, plasma from Mm26 did not contain detectable levels of nAb at any time p.i. In contrast, nAb against SIV239 was not induced in any of the 5G-infected animals (Fig. 2b, right panel). As low-level nAb may play a role in control of virus replication, purified IgG from the plasma samples was used to measure neutralizing activity. However, the results from the purified IgG corresponding to the plasma at a 1 : 3 dilution did not change the kinetics of nAb response in 5G-infected animals (data not shown).
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). In consideration of these results, data were recalculated based on a cut-off value of 90 % inhibition of virus replication (IC90) in CD4+ T-cell lines. As a result, nAb responses were detected in only two of the animals, Mm07 and Mm22, but with titres of 1 : 100 and 1 : 500, respectively (Fig. 2b, middle panel). Next, we examined the correlation between viral load and nAb titre at 8 and 12 weeks p.i. and found that the correlation was not statistically significant (Fig. 2c).
Anti-gp120 Ab response in 5G-infected animals
Next, we measured binding Ab responses against gp120. When the plasma samples were assayed for levels of Ab that bound to SIV239 gp120 or 5G gp120, essentially identical values were obtained. Fig. 3 shows the data obtained using SIV239 gp120. Remarkably, anti-gp120 responses during the early period p.i. between the two groups of monkeys were distinct. Whereas anti-gp120-specific Ab responses peaked at 3–4 weeks p.i. in 5G-infected animals (Fig. 3a), those in SIV239-infected animals remained generally lower and required longer periods of time to reach their peak (Fig. 3b). Of note, whilst anti-gp120 Ab responses did not correlate well with nAb titres in the chronic phase in 5G-infected animals, the hierarchy detected in nAb titres (Mm07, Mm22, Mm23, Mm12 and Mm26, in descending order) was similar to that observed for gp120-binding antibodies at 2 weeks p.i. (Fig. 3a).
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5G-infected animals differ from those detected in SIV239-infected animals and 3). We used 72 overlapping peptides encompassing the entire Env sequence of SIV239 for the detection of epitope-specific Ab in plasma samples from 5G-infected or SIV239-infected animals. As shown in Fig. 4 and Table 1, the plasma samples reacted with the peptides in six regions: two in gp120 and four in gp41. The regions in gp120 resided in the vicinity of V1/V2, designated region 1 (aa 109–193), and at the C terminus, designated region 2 (aa 493–529). Of note, only linear region 1 was directly affected by selected deglycosylation (aa 146 and 171). The regions in gp41 were located in the ectodomain for region 3 (aa 589–625) and region 4 (aa 660–685), and in the cytoplasmic domain for region 5 (aa 721–757) and region 6 (aa 841–879).
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5G-infected animals were similar to those in SIV239-infected animals, a few peptides were recognized by Abs only in samples from 5G-infected animals, and Ab reactivity to some peptides was significantly different between the two groups (Fig. 4b, c and Table 1). Firstly, in region 1, whereas five peptides (Env-10, -12, -13, -14 and -15) were recognized by Abs from 5G-infected animals, only three peptides (Env-10, -12 and -13) reacted with Abs from SIV239-infected animals (Fig. 4b and c). Peptide Env-10 was detected by Abs from four 5G-infected animals, but from only one of the SIV239-infected animals. Similarly, peptides Env-12 and -13 were detected by Abs from five 5G-infected animals and two SIV239-infected animals. In contrast, peptides Env-14 and -15 were detected by Abs from 5G-infected animals but not SIV239-infected animals. The specificity of 5G infection in the reactivity of peptide Env-14 was statistically significant (P=0.0149) (Table 1). Secondly, the reactivity of Ab from 5G-infected animals with the peptides in regions 2, 3 and 4 was lower than that recorded with Ab from SIV239-infected animals (Fig. 4b and c). As shown in Table 1, the reduction in Ab reactivity from 5G-infected animals to peptide Env-51 (region 3) and peptide Env-56 (region 4) was significant (P=0.014 and 0.0053, respectively); however, the reduction in Ab response in region 2 was not significant. In addition, there were no significant differences in the Ab responses to the peptides in regions 5 and 6 between 5G-infected and SIV239-infected monkeys (Fig. 4b, c and Table 1).
A 5G-specific linear epitope resides in the region containing the third deglycosylation site (aa 171) between V1 and V2
As region 1 also contained the site of two mutations introduced to limit glycosylation in the 5G mutant, we focused additional studies on this region. To identify the 5G-specific epitope(s) in region 1, peptide ELISA was performed with 12 newly synthesized shorter peptides based on the 5G sequence spanning the V1/V2 region (Fig. 5). Ab reactivity to peptide Env-14 was mapped to peptides V1V2-9–11 (Fig. 5a). Thus, three linear epitopes (encompassed in peptides Env-10, V1V2-3 and V1V2-9–11) were identified within the V1/V2 region (Figs 4 and 5). Whilst two epitopes contained in peptides Env-10 and V1V2-3 were recognized by Ab from both SIV239- and 5G-infected animals, the epitope(s) corresponding to peptides V1V2-9–11 was specific to 5G infection (Fig. 5a). As the latter contained the third deglycosylation mutation (Figs 1 and 5b, aa 171), 5G specificity was probably secondary to the removal of N-glycan at this site in SIV239 gp120 (Fig. 5).
5G-specific Ab responses to linear epitopes in Env elicited immediately following primary infection
In an effort to define the potential relevance of the linear epitope-specific Ab responses in the reduction of acute virus replication in 5G-infected animals, we examined the kinetics of Ab reactivity to 12 peptides: Env-10, V1V2-3 and V1V2-9, -10 and -11 for epitopes in region 1; Env-42 and -43 for epitopes in region 2; Env-50 and -51 for epitopes in region 3; Env-56 for epitopes in region 4; and Env-61 and -62 for epitopes in region 5 (Fig. 6). Whilst the induction kinetics of Ab to most peptides were variable in plasma from both groups of animals, Ab to V1V2-9, -10 and -11 was specific for 5G-infected animals, with rapid induction following primary infection. Ab responses to Env-61 and -62 were also induced rapidly in animals from the two groups; however, it has already been confirmed by SIV and HIV studies that a linear epitope covered by these peptides is the immunodominant epitope with no association with virus control (Eberle et al., 1997; Kent et al., 1992). In contrast to Ab responses to V1/V2 peptides, whilst Ab to peptides Env-51 and -56 in the gp41 ectodomain were detected in SIV239-infected animals, these reactions were low until at least 12 weeks p.i. in 5G-infected animals.
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5G-specific linear epitope5G-infected animals, these Abs were non-nAbs, as these binding Abs were detected in all 5G-infected animals, including a nAb-undetectable monkey (Mm26), and before nAb was detected. In addition, we attempted to inhibit neutralization by the addition of excess concentrations of V1V2-9, -10 and -11 to the neutralization assay performed with plasma from 5G-infected animals collected at 8 and 12 weeks p.i. The reduction of nAb by the addition of an excess amount of peptide was not detected in any samples, confirming that the epitopes targeted by nAb and V1V2-specific Ab were distinct (data not shown).
Next, we tested plasma IgG samples from SIV-infected animals for the quantitative capture of whole virions. IgG fractions of plasma samples from SIV-infected animals collected at 3–4 weeks p.i. were compared for their capacity to capture 5G or SIV239 virions. IgG fractions from two 5G-infected animals (Mm07 and Mm22) exhibited remarkably higher virion capture activity than those from other animals (Fig. 7a); however, this capture activity was 5G-specific, as no appreciable capture of SIV239 virion was detected with these samples. Furthermore, this activity was reduced to the level of control IgG (R374) after selective removal of IgG binding to V1V2-9, -10 and -11 peptides, suggesting that virion capture activity is associated with the 5G-specific linear epitope Ab (Fig. 7a). By contrast, IgG fractions from SIV239-infected animals collected at 3–4 weeks p.i. did not exhibit appreciable binding activity either to 5G virions or SIV239 virions (Fig. 7b). Thus, these results demonstrated that 5G infection elicited not only nAb after 8 weeks p.i., but also a much earlier humoral antiviral mechanism in the form of 5G-specific virion-binding Ab at 3–4 weeks p.i. in at least two monkeys (Mm07 and Mm22). To examine the relationship between the two antibody activities, we calculated the correlation of virion capture activity of IgG at 3 or 4 weeks p.i. with a peak nAb titre in 5G-infected animals (Fig. 2b) and found that this correlation was statistically significant (r=1, P=0.0167; Fig. 7c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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5G-infected animals; Fournillier et al., 2001; Haigwood & Stamatatos, 2003; Huso et al., 1988; Reitter et al., 1998). In support of these observations, the data presented here demonstrated that quintuple deglycosylation conferred live attenuated vaccine properties to an SIV239 mutant, 5G (Mori et al., 2001); however, a cellular but not humoral response was detected as an immune correlate of the protection of 5G-infected animals against SIV239 challenge infection. Therefore, we assumed that the complete control of robust acute virus replication in 5G-infected animals beyond the initial cell-mediated control would be due to the development of rapid and effective nAbs. This study indicated that, whereas 5G-infected animals clearly exhibited better nAb responses than SIV239-infected animals, the most stringent nAb assay, based on 90 % inhibition, provided evidence of nAb titres in only two of five 5G-infected animals and the appearance of these titres trailed the decline of acute viral loads by almost 4 weeks (Figs 1 and 2). Therefore, we concluded that, although deglycosylation did promote better development of nAbs in 5G-infection than SIV239 infection, it was still too late to control acute viraemia.
Zinkernagel and co-workers have categorized viruses into two types: ‘acutely cytopathic viruses’ and ‘poorly or non-cytopathic viruses’ (Hangartner et al., 2006b). The former contains viruses such as vesicular stomatitis virus in mice and influenza virus in humans, whose control depends primarily on a rapid and potent nAb response. The latter comprises viruses such as lymphocytic choriomenigitis virus in mice, and hepatitis B and C viruses and HIV in humans, against which a nAb response is apparent only following the reduction of primary viraemia, and which establish persistent chronic infections. Accordingly, although the viral loads in 5G infection resembled ‘acutely cytopathic virus’ infections, the kinetics of nAbs still conformed to the ‘non-cytopathic virus’ category. As the difference in nAb response between the two types of virus is determined by their surface glycoproteins (Pinschewer et al., 2004), this study suggests that the deglycosylation of 5G could not change this intrinsic property of SIV239.
Ab responses to Env peptides in 5G-infected animals
Aside from nAb, non-nAb responses to linear epitopes in V1/V2 were specifically induced by 3 weeks p.i. in all 5G-infected animals (Figs 4, 5 and 6). The heavy glycosylation of viral spikes clearly prevented access of B-cell receptors to the linear Ab epitopes located within limited regions of gp120 in SIV239, and the reduced glycosylation probably promoted better exposure of these linear epitopes in 5G (Fig. 4). Accordingly, the 5G-specific epitope in V1/V2 should be closely associated with the deglycosylation mutation at aa 171 in gp120 (Fig. 5). We speculate that this Ab induction might contribute to acute viral suppression in 5G infection because of the coincident decrease in peak viraemia (Figs 1 and 6). Non-neutralizing Abs can be divided into those that bind to the intact virion surface and debris-specific Ab. The former non-neutralizing Abs have occasional possibilities for antiviral activities such as antibody-dependent cell-mediated cytotoxicity and complement-mediated virus inactivation (Aasa-Chapman et al., 2005; Ahmad & Menezes, 1996; Forthal et al., 2001; Hangartner et al., 2006a). In fact, readily detectable virion capture Abs were induced in two of five 5G-infected animals (Fig. 7, Mm07 and Mm22). The importance of immediate-early suppression of SIV replication for the long-term containment of infection has been demonstrated by studies of post-exposure anti-retroviral therapy (Lifson et al., 2000; Mori et al., 2000). Thus, the early and complete control of viraemia in 5G-infected animals clearly suggests an antiviral mechanism(s) acting as early as 2–4 weeks p.i. Therefore, the early detection of IgG capable of virus capture in 5G-infected animals may provide mechanisms capable of contributing to undetectable viral load set points (Fig. 1b). The selective generation of such Ab directed to linear Env epitopes is expected.
Interestingly, deglycosylation in gp120 was also associated with a general reduction in the antigenicity of linear epitopes in gp41: the Ab response against the two epitopes that reside in the regions between the two heptad repeats (aa 601–625) and in the C-terminal heptad repeat (aa 660–685), respectively, was markedly reduced (Fig. 4, Table 1). The former corresponds to the highly conserved immunogenic epitope (Benichou et al., 1993; Gnann et al., 1987; Silvera et al., 1994), and the latter corresponds to an epitope identified in the chronic phase of SIVmac251 infection (Silvera et al., 1994) and corresponds to the nAb epitope of HIV-1 known as 2F5 (Muster et al., 1993), although this linear epitope has not been associated with SIV neutralization (Caffrey et al., 1998). Thus, these epitopes are probably exposed on the surface of viral spikes or their degraded fragments in most SIV and HIV-1 isolates with appropriate glycosylation and correct folding. We believe that the loss of glycosylation might induce a slight conformational change in the gp120 protein backbone, resulting in altered interaction of gp120 and gp41. In fact, the region encompassing the former epitope in gp41 was demonstrated to interact with gp120 (Cao et al., 1993; Maerz et al., 2001; York & Nunberg, 2004). As viral spikes determine virus properties such as viral receptor usage and cell tropism (Kolchinsky et al., 2001; Puffer et al., 2002), different cell populations might be infected in 5G-infected animals compared with SIV239 infection. More specifically, because of the distinct properties of the virus, vigorous 5G replication in the acute phase did not apparently impair immune function and thus established the control of chronic-phase infection and viral replication.
Host factors required for functional Ab responses against SIV infection
This study also demonstrated remarkable differences in humoral response with regard to nAb and virion capture Ab among 5G-infected animals. However, gp120-specific-binding Ab and the linear epitope-specific Ab were initially induced similarly in all animals. These findings imply that Abs measured by ELISA assay and Abs exhibiting antiviral activity are elicited by different pathways and that the properties associated with functional Abs depend largely on the host and underscore the importance of its genetic background. Rhesus macaques are present in various geographical locations within the Asian continent and are subdivided into many subspecies morphologically and genetically (Smith & McDonough, 2005). Some of the genetic differences among rhesus monkeys of different geographical origins, and especially those involving major histocompatibility complex (MHC) genotypes, probably influence the corresponding differences in immune responses, especially cellular response (Bontrop et al., 1996; O'Connor et al., 2003; Reimann et al., 2005). Schmitz et al. (2005) reported that Mamu-A*01-positive rhesus monkeys elicited a significantly higher cellular response and lower nAb titres than those in Mamu-A*01-negative animals at the time of challenge infection of animals vaccinated with live attenuated SIV. They suggested that both humoral and cellular immune responses contributed to the protection against the challenge infection and that the relative contribution of each of the responses may be genetically determined. We observed a similar relationship between nAb and cellular responses among 5G-infected animals: two animals (Mm07 and Mm22) elicited a lower cellular response while the other three animals (Mm12, Mm23 and Mm26) elicited a higher cellular response (data not shown). Notably two animals exhibiting highly functional Ab (Mm07 and Mm22) were the offspring of seed animals imported from Laos, whilst the others (Mm12, Mm23 and Mm26) were of Burmese origin, suggesting the potential association of such different humoral and cellular responses with host genetic factors. In clinical studies, considerable concordance of adaptive cellular and humoral responses and HIV evolution in monozygotic twins, but not in brothers, infected with the same virus has been reported (Draenert et al., 2006). HIV-1-exposed but uninfected status with significantly higher neutralizing IgA was linked to genotypes on chromosome 22 (Kanari et al., 2005). In the mouse Friend leukemia virus model, MHC II alleles were determined as host genetic factors required for effective nAb response (Miyazawa et al., 1992) and the host genetic factor was mapped to chromosome 15, which was associated with the clearance of viraemia by nAb (Hasenkrug et al., 1995; Kanari et al., 2005).
Taken together, we speculate that the functional humoral response is determined by host genetic properties similar to the cellular immune response. Thus, gaining knowledge of the genetic requirements for both humoral and cellular containment of viral infections will clearly be of primary importance for vaccine development and therapeutics against HIV and other infectious agents.
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ACKNOWLEDGEMENTS |
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Received 25 May 2007;
accepted 7 October 2007.
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Q substitutions at aa 79, 146, 171, 460 and 479 in Env (Ohgimoto et al., 1998
