Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by surface plasmon resonance

We report here that sera of children and adults infected with Schistosoma mansoni, S. haematobium, or S. japonicum contain antibodies against GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) and to a lesser extent to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)) and GalNAcbeta1-4GlcNAc (LDN). Surface plasmon resonance (SPR) spectroscopy was used to monitor the presence of serum antibodies to neoglycoconjugates containing these carbohydrate epitopes and to define the immunoglobulin M (IgM) and IgG subclass distribution of the antibodies. The serum levels of antibodies to LDN-DF are high related to LDN and Lewis(x) for all examined groups of Schistosoma-infected individuals. A higher antibody response to the LDN-DF epitope was found in sera of infected children than in sera of infected adults regardless of the schistosome species. With respect to the subclasses, we found surprisingly that individuals infected with S. japonicum have predominantly IgG antibodies, while individuals infected with S. mansoni mainly show an IgM response; high levels of both isotypes were measured in sera of individuals infected with S. haematobium. These data provide new insights in the human humoral immune response to schistosome-derived glycans.     

Deficiency in immunoglobulin G2 antibodies against staphylococcal enterotoxin C1 defines a subgroup of patients with atopic dermatitis

Background and aim Atopic dermatitis (AD) is a common chronic inflammatory skin disease often accompanied by cutaneous Staphylococcus aureus colonization and, in this regard, especially complicated by the presence of superantigen‐producing strains. Because IgG antibodies comprise an important defence mechanism of the adaptive immune system against bacteria, it was investigated whether AD patients have an abnormal pattern or distribution of superantigen‐specific IgG subclass antibodies in association with disease severity and activity. Methods Staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin C1 (SEC1) specific IgG antibody subclasses were assessed in n=89 adult AD patients with mild to severe disease activity as determined by the SCORAD score and in n=28 healthy age‐matched controls. Results were correlated with the current status of bacterial skin colonization and severity score. Results Thirty‐eight per cent of the AD patients showed a selective deficiency in IgG2 antibodies against SEC1 compared with only 14% in the control group. The absence of these antibodies was found in both currently colonized and non‐colonized AD patients and was associated with a severe phenotype (SCORAD more than 40 points in two‐thirds of the deficient patients). However, these patients had normal production levels of IgG2 antibodies against pneumococcal capsular polysaccharide (PCP) and SEB, but higher IgG1 and IgG4 titres against SEC1. Except for elevated total IgG1, total IgG subclass levels were normal in this AD subgroup. Yet, peripheral blood mononuclear cells (PBMCs) derived from these patients clearly produced IL‐4 and IL‐5 upon SEC1 re‐stimulation whereas PBMCs from those providing SEC1‐specific IgG2 antibodies failed in the production of these cytokines. Conclusion A subgroup of AD patients suffers from a selective deficiency to produce anti‐SEC1 IgG2 antibodies. This patient group is characterized by a severe AD phenotype.     

Broadly reactive immunofluorescence test for measurement of immunoglobulin M and G antibodies to Ureaplasma urealyticum in infant and adult sera.

The indirect immunofluorescence test was used to measure immunoglobulin M (IgM) and IgG antibodies to acetone-fixed Ureaplasma urealyticum organisms in sera from 128 adults with genital infections and from 713 symptomatic newborns and babies 1 day to 18 months old. Thirty-four percent of the adults had demonstrable IgG antibody to ureaplasma. IgM antibody was detected in 2 of the adult sera and in 17 of the infant sera. These babies were divided into two distinct groups. Ten of the infants presented at birth with various physical findings, whereas the onset of symptoms for the other 7 occurred 3 to 13 weeks after birth, and the major clinical finding in 6 of the 7 was respiratory distress. The results of this study suggested that U. urealyticum infection may be associated with fetal damage and infant pneumonia, and if this is substantiated, the indirect immunofluorescence test employing acetone-fixed antigen to measure IgM antibody to U. urealyticum may be an important diagnostic tool.     

Construction and functional activities of chimeric mouse-human immunoglobulin G and immunoglobulin M antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 epitopes

We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease.     

Anti-RNA polymerase I antibodies in the sera of MRL lupus mice at the initial stages of disease are directed primarily against phosphorylation-dependent epitopes.

Anti-RNA polymerase I (RPI) antibodies in the sera of MRL/Mp-lpr/lpr and MRL/Mp(-)+/+ mice, which develop an autoimmune disease similar to human systemic lupus erythematosus, were screened for reactivity with purified RPI or RPI which had been dephosphorylated. In every case (n = 10), dephosphorylation of RPI resulted in a significant decrease (33-95%) in antibody binding. The anti-RPI antibodies in the sera of the same mice approximately 6 weeks later also reacted better with untreated as compared to dephosphorylated RPI but, in every case, the decrease in antibody (0-30%) caused by dephosphorylation was substantially diminished. That the proportion of anti-RPI antibodies in the sera of MRL mice decreased with progression of lupus-like disease was confirmed by closely monitoring the antibodies over the course of disease. Anti-RPI antibodies produced at the earliest stages appeared to be directed almost exclusively against phosphorylation-dependent determinants since dephosphorylation of RPI essentially abolished antibody binding. Subsequently, the percentage of the total anti-RPI antibodies in the sera of these mice directed towards phosphorylation-independent epitopes increased linearly with time. The importance of phosphorylation-dependent epitopes on RPI for the development of the anti-RPI autoimmune response was supported by the observation that treatment of mice with alkaline phosphatase partially attenuated anti-RPI antibody production.     

Changes in avidity and level of immunoglobulin G antibodies to Mycobacterium tuberculosis in sera of patients undergoing treatment for pulmonary tuberculosis

Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens.     

Detection of antibodies directed against the cytoplasmic region of the human acetylcholine receptor in sera from myasthenia gravis patients

The nicotinic acetylcholine receptor (AChR) is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore the determination of their specificities requires the use of native AChR. Antibody competition studies suggest that most MG antibodies are directed against the extracellular part of the molecule, whereas antibodies directed against the cytoplasmic region of the AChR have not been detected. To determine whether even small quantities of such antibodies exist in MG sera, we performed competition experiments based on the inhibition by MG sera of the binding of MoAbs to the human AChR, rather than inhibition by MoAbs of the binding of MG sera performed earlier. When MoAbs directed against cytoplasmic epitopes on the alpha or beta subunits (alpha 373-380 and beta 354-360) were used as test MoAbs, 17% or 9% of MG sera inhibited the binding of the anti-alpha or anti-beta subunit MoAbs, respectively, by > or = 50%. Non-specific inhibition was excluded. These results suggest the presence, in several MG sera, of antibodies directed against cytoplasmic regions of the AChR; yet these antibodies seemed to represent a relatively small proportion of the total anti-AChR antibodies. The corresponding epitopes may be involved in the inducing mechanisms in certain MG cases, and knowledge of the presence of such antibodies may be useful in understanding the autoimmune mechanism involved in MG.     

Detection of antibodies directed against human herpesvirus 6 U94/REP in sera of patients affected by multiple sclerosis

The association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P > 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.     

Existence of an immunoglobulin G component of naturally occurring HLA class I antibodies that are not directed against self-antigens in human serum

We compared the frequency of immunoglobulin G (IgG) type of human leukocyte antigen (HLA) class I antibodies between patients with systemic lupus erythematosus (SLE) and healthy controls using a highly sensitive FlowPRA method. Sixteen of 130 normal healthy males and 2 of 10 normal females without a history of pregnancy (none had ever been transfused) possessed HLA class I antibodies. In SLE, male, but not female patients, showed a significant increase in the frequency of the antibodies compared with the corresponding controls. The antibodies did not appear to be involved in the development of SLE because of no substantial relationship to the incidence of cytopenia or SLE disease activity index score. Each individual had 1-31 types of HLA class I antibodies. Interestingly, HLA class I antibodies did not correspond to the individual's own HLA antigens. Eight of 32 types of HLA class I antigens detected were rare in the Japanese population. These results suggest that an IgG component of naturally occurring HLA class I antibodies exists in human serum and that these antibodies are not antibodies against self-antigens.     

Epitopes,immunoglobulin classes and immunoglobulin G subclasses of calsequestrin antibodies in patients with thyroid eye disease

A number of serum autoantibodies are associated with thyroid eye disease (TED), including those reactive against the calcium binding protein calsequestrin (CASQ). There are two isoforms of CASQ namely; CASQ1, found in skeletal, including extra ocular, muscle, and CASQ2, found in cardiac muscle. We determined (i) the reactivity profiles of CASQ1 and CASQ2 antibodies and (ii) the immunoglobulin (Ig) classes and IgG subclasses of CASQ1 antibodies, using enzyme-linked immunosorbent assay. Of the 20 patients with TED tested, 35% were positive for CASQ1 antibodies, 25% for CASQ2 antibodies and two patients (10%) were positive for both antibodies. Of the 12 patients with Hashimoto's thyroiditis and ophthalmopathy tested, 25% were positive for CASQ1 antibodies, 42% for CASQ2 antibodies and two patients (17%) were positive for both antibodies. CASQ1 antibodies were mainly of the IgG class and IgG1 and IgG3 subclasses. These results suggest that CASQ1 and CASQ2 do not share major epitopes. Because antibodies of the IgG1 and IgG3 subclasses are cytotoxic, CASQ1 antibodies may contribute to the eye muscle damage in patients with TED. Because CASQ1 antibodies were positive in only a third of patients with active TED we are unable to draw conclusions about their role in its pathogenesis. On the other hand, a possible role of CASQ2 antibodies in the aetiology of the cardiac complications of Graves' disease is a new avenue for research and appears worthy of further investigation.     

中国HIV/AIDS患者保守表位的2F5抗体与疾病进展关系

目的 研究中国HIV/AIDS患者gp41保守表位中和抗体2F5与疾病进程的关系.方法 应用合成4(ELDKWA)-C24肽及gp41抗原肽包被96孔酶标板,ELISA方法检测特异性抗体水平.提取正常人外周血单个核细胞(PBMC),应用HIV-1 SF33毒株进行病毒多轮感染PBMC中和试验.结果 缓慢进展者(slow progressor,SP)的2F5样中和抗体水平显著高于典型进展者(typical progressor,TP)(P<0.05);2F5样中和抗体水平与gp41特异性抗体水平正相关(r=0.406,P<0.05).结论 中国HIV.-缓慢进展者体内具有高水平的2F5样抗体,2F5抗体可能是疾病不进展的保护性因素之一.     

Negative venom skin test results in patients with histories of systemic reaction to a sting

For more than 20 years venom immunotherapy has been the preferred treatment for Hymenoptera allergy and venom skin testing the preferred diagnostic test. Most allergists consider venom skin tests to be highly accurate and interpret a negative venom skin test result to indicate the absence of insect allergy. Furthermore, current practice guidelines do not adequately address the question of how best to manage the patient with a convincing history of insect allergy but negative skin test results. Recent case reports and published studies have forced us to reexamine this important management issue and to consider what role in vitro venom testing might have in the management of insect allergy. We reviewed the current status of what is known about the management of individuals with a history of insect allergy but negative venom skin test results and suggested modifications of current working guidelines.     

Increased concanavalin A-binding capacity of immunoglobulin G purified from sera of patients with rheumatoid arthritis.

A solid phase radioimmunoassay was set up for direct measurement of the binding capacity of human IgG to three lectins recognizing different carbohydrates of the Fc domain, i.e. peanut agglutinin (PNA), Concanavalin A (Con A) and pokeweed mitogen (PWM) which mainly bind to beta-galactose, alpha-mannose and dimers of N-acetyl-beta-glucosamine respectively. The mean specific binding of the 96 normal IgG tested to PNA and to PWM was statistically higher (P less than 0.001) than that to Con A, whereas no significant differences were observed between the mean specific bindings to PNA and to PWM. A statistically significant linear negative correlation could be established only between the relative bindings (expressed in percentage of the total binding to the three lectins) to PNA and to PWM (r = -0.65, P less than 0.001). The mean specific binding of IgG purified from 34 patients suffering from rheumatoid arthritis (RA) to PNA and to Con A was statistically higher (P less than 0.001) than that reached with PWM, whereas no significant differences were noted between their mean binding capacities to PNA and to Con A. When compared to normal IgG, only four out of 34 RA IgG exhibited a significantly higher binding capacity to PNA, whereas all but one RA IgG possessed a significantly higher binding capacity to Con A. Accordingly, the mean specific binding of RA IgG to Con A was significantly higher than that of normal IgG (P less than 0.001). Besides (and contrary to normal IgG), a statistically significant negative linear correlation was noted between the relative bindings of RA IgG to PNA and to Con A (r = -0.89, P less than 0.001). All the five RA IgG tested exhibited an abnormal circular dichroism. Our data suggest that, by altered steric conformation and glycosylation, mannosyl-residues of RA IgG become prominent or terminal or both, and are therefore able to react more effectively with Con A than normal IgG do.     

Distinct contributions of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to protective immunity against influenza

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.     

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels

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Time course of anti-SL-IV immunoglobulin G antibodies in patients with tuberculosis and tuberculosis-associated AIDS.

Immunoglobulin G (IgG) and IgM antibodies against the SL-IV antigen of Mycobacterium tuberculosis in the sera of patients with tuberculosis with negative serology for human immunodeficiency virus (HIV) infection (TB group; n = 97), patients with tuberculosis with positive serology for HIV infection (TB-HIV group; n = 59), and healthy controls (n = 289) were determined by enzyme-linked immunosorbent assay. All sera were obtained at the onset of tuberculosis, i.e., when clinical symptoms appeared. Clinical specimens were collected and cultured for the isolation of M. tuberculosis, and treatment with antituberculous drugs was started. Sera were also obtained from patients in the TB group at fixed intervals during treatment; sera were available from 13 patients in the TB-HIV group before the onset of tuberculosis. The best specificity and positive predictive values were obtained with the IgG assays. In the IgG assays at specificities above 96.0%, the sensitivities of the tests were 45.3 and 72.8% for the TB and TB-HIV groups, respectively, and the sensitivity was 51.9% when data from both groups were combined for analysis. For the TB group, results of this study indicated that the levels of IgG antibodies remain high during treatment. Thus, repetitive serological assays may not be useful for treatment follow-up. In the TB-HIV group, 12 of 13 patients had IgG-specific antibodies against the SL-IV antigen between 1 and 30 months before the onset of tuberculosis, so we suggest that the IgG antibody assay against SL-IV may be helpful for identifying tuberculosis in patients infected with HIV.     

Levels of immunoglobulin G antibodies against defined epitopes of the L1 and L2 capsid proteins of human papillomavirus type 6 are elevated in men with a history of condylomata acuminata.

Sera from 159 men attending the sexually transmitted disease clinic at Karolinska Hospital, Stockholm, Sweden, were analyzed for the presence of immunoglobulin A (IgA) and IgG antibodies to a panel of synthetic peptides derived from the E2, L1, and L2 regions of the human papillomavirus types 1 (HPV 1), 6, 8, 11, 16, 18, 31, and 33. The study subjects were divided into three groups: (i) asymptomatic men with no history of genital warts who served as controls, (ii) men with visible condylomata, and (iii) men who had previously been afflicted with condylomata. There were no significant differences in antibody titers for any of the HPV 6- or 11-derived peptides among patients with current condylomata and the controls. For the peptide from L1 of HPV 6, there was an increase in the IgG titers among men with previous condylomata compared with the titers for the controls (52% versus 27% seropositivity; P less than 0.05). Also, for the peptide from L2 of HPV 6, there was an increase in the IgG titers among men who had been afflicted with condylomata previously (P less than 0.05). Increased IgA antibody titers against an HPV 16-derived peptide and an HPV 18-derived peptide were also detected. For the peptides from L1 and L2 of HPV 6, the study was extended to an additional group of 127 males attending the sexually transmitted disease clinic at Huddinge Hospital in southern Stockholm. Again, significantly increased antibody levels were detected only for IgG and only among asymptomatic men with a history of condylomata (P < 0.01 for the L1 peptide and P < 0.05 for the L2 peptide). The results suggest that the IgG response against the late proteins of HPV 6 reflects mainly previous exposure to the virus rather than ongoing viral disease.     

Comparison of quantitative and semiquantitative enzyme-linked immunosorbent assays for immunoglobulin G against Chlamydophila pneumoniae to a microimmunofluorescence test for use with patients with respiratory tract infections

We previously reported a high degree of variation in the sensitivities of serodiagnostic kits for the detection of Chlamydophila pneumoniae in sera from healthy donors. Since a low predictive value of a test can impair its diagnostic value, we have extended our studies to samples from patients with pneumonia. We focused on the most promising enzyme-linked immunosorbent assays (ELISAs) (SeroCP and SeroCP Quant; Savyon) identified in our previous study and included a new ELISA (sELISA; Medac). The agreement between all ELISAs for immunoglobulin G (IgG) and a reference microimmunofluorescence (MIF) test for IgG (SeroFIA; Savyon) was > or = 90% for a collective of 80 patients. The positive predictive values were all > or = 93%. The negative predictive values ranged from 68 to 83%. False-negative results were obtained only for samples that had low titers in the MIF test. The correlation of the IgG antibody titers determined by the MIF and SeroCP Quant tests was high (r(sp) = 0.9). Since the semiquantitative SeroCP and quantitative SeroCP Quant ELISAs achieved the highest sensitivities, they were evaluated further by using a second batch of sera from 50 patients with predominantly medium and low antibody titers in the MIF test and a control collection of sera from 80 children with negative MIF results. Again, the tests showed a high concordance with the MIF results (96%), and the antibody titers in the SeroCP Quant and MIF tests correlated well (r(sp) = 0.8). The specificities determined with the negative sera were > or = 99% for the SeroCP Quant test and 86% for the SeroCP test. These results show that ELISAs that are fast and objective deliver seroprevalence results, sensitivities, and specificities that are very similar to those of the MIF test.     

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

Summary It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1_BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp120.