Biologic Activity of CDG-IIb and Immunoglobulin

The levels of three abnormal N-linked high-mannose glycans (consisting of the following molecules: 3 glucose, 7 mannose, and 2 N-acetylglucosamine; 3 glucose, 8 mannose, and 2 N-acetylglucosamine; and 3 glucose, 9 mannose, and 2 N-acetylglucosamine) were significantly elevated in the purified plasma IgG from the patients, and the presence of N-linked glycans consisting of 3 glucose, 7 mannose, and 2 N-acetylglucosamine molecules at a mass-to-charge ratio of 2600.1 was unique to CDG-IIb (Figure 1B). Moreover, the number of normal N-linked glycans, particularly high-mannose glycans (e.g., glycans consisting of 6 mannose and 2 N-acetylglucosamine molecules), was reduced. This pattern of changes in protein glycosylation was also observed in the N-glycan profile for total glycoproteins of fibroblasts cultured from the skin of the patients. In addition to IgG, other plasma proteins also had abnormal N-glycosylation patterns (section S4 in the Supplementary Appendix).

Table 1. Table 1. Immunologic Characteristics of Two Siblings with Congenital Disorder of Glycosylation Type IIb (CDG-IIb).

The two siblings had normal or increased numbers of B cells in the peripheral blood, and examination of a bone marrow aspirate and biopsy specimen from Patient 1 showed normal numbers of plasma cells. No evidence of protein loss through the gastrointestinal or genitourinary tract was identified, since α 1 -antitrypsin clearance and a 24-hour urine specimen, respectively, were normal. Antibody levels to recall antigens were evaluated before and after the administration of booster doses of diphtheria–tetanus–acellular pertussis, conjugated Haemophilus influenzae type B, and hepatitis B vaccines, as well as 23-valent pneumococcal polysaccharide vaccine. Despite severe hypogammaglobulinemia, adequate immune responses to the four challenge vaccines developed. However, the results of tests for antibodies to measles, mumps, rubella, and varicella were negative or equivocal, despite adequate vaccination for age (each child had received two doses of the combined measles–mumps–rubella [MMR] and varicella vaccines, at approximately 1 and 5 years of age) (Table 1).

Two systems (unfolded-protein response and endoplasmic reticulum–associated degradation) that are responsible for the quality control of protein synthesis and for the degradation of misfolded N-glycosylated proteins1 were within the normal limits of activity, and the two systems responded similarly to controls when stimulated with tunicamycin (an inducer of unfolded-protein response) or MG-132 (an inducer of endoplasmic reticulum–associated degradation) (Figure 1C).

No reduction in the frequencies of antibody-secreting cells that were generated in vitro was detected, with the exception of IgA antibody–secreting cells in Patient 1. In addition, the amount of immunoglobulin secreted into the culture supernatants was similar for the patients and the healthy control, and the IgG from the patients did not undergo accelerated degradation after incubation at room temperature or at 4°C (Figure 1D).

As shown in Figure 1E, plasma IgG from patients with CDG-IIb cleared significantly more rapidly (half-life, 6 days) than did plasma IgG from healthy controls (half-life, 21 days) when injected into Rag1−/− mice. Surface-plasmon-resonance experiments showed that purified IgG from the patients with CDG-IIb and the healthy control had similar binding affinities to immobilized FcγRIa and FcγRIIIb receptors and the neonatal Fc receptor (FcRn), a receptor known to contribute to the half-life of immunoglobulins (Section S5 in the Supplementary Appendix). IgG derived from the patients bound FcγRIIa with significantly lower affinity than did IgG from healthy controls.

CDG-IIb and Susceptibility to Viral Infections

Figure 2. Figure 2. Viral Susceptibility Studies in the Patients. Panel A shows human immunodeficiency virus (HIV) infection experiments. The left graph shows entry of four HIV strains into activated, CD8-depleted peripheral-blood mononuclear cells (PBMCs) from a healthy control and from the two patients with CDG-IIb. Target cells were incubated with the various strains of HIV for 2 hours at 37°C. After extensive washing, the cells were lysed, and a real-time polymerase-chain-reaction (PCR) assay specific for HIV DNA was performed. T bars represent standard deviations. Data are representative of three independent experiments. ND denotes not done. The middle graph shows the level of HIV replication under the same conditions as those used to assess viral entry. Infected cells were maintained at 37°C, culture supernatants were harvested, and the HIV p24 protein level was determined by means of enzyme-linked immunosorbent assay (ELISA). Data shown represent the level of viral replication at day 3 or day 4 after infection. Data are representative of four independent experiments. The right graph shows viral entry of the ELI6 strain, recovered at day 4 from each cell culture performed for the assessment of HIV p24. Target cells from two healthy donors were prepared by activating their PBMCs for 2 days, followed by purification of their CD4+ T cells. The amount of virus from each of the three sources that was incubated with target cells was normalized according to the level of p24 measured by ELISA in the culture supernatant. A real-time PCR assay specific for HIV DNA was performed. T bars represent standard deviations. In Panel B, the top immunoblot shows primary fibroblast lysates from the patients and controls, which were transfected with an HIV glycoprotein 140 (gp140)-coding vector with or without a V5-tagged nonmutant mannosyl-oligosaccharide glucosidase (MOGS)–expression vector. As expected on the basis of their N-glycan trimming defect, the viral gp140 synthesized in the cells from the patients had a higher molecular weight than that synthesized in the control cells. When the cells from the patients were transfected with the nonmutant MOGS-V5 vector, the gp140 molecular weight reverted to the control molecular weight. The differences in molecular weight depended on the N-glycosylation pattern, as revealed after N-glycan removal by means of peptide-N-glycosidase F (PNGaseF) digestion. The bottom immunoblot shows the same distinctive HIV gp140 glycosylation pattern detected in cells from the controls and those from the patients, but in this case, the gp140 glycosylation pattern detected in the control cells was induced into the patients' pattern by the MOGS inhibitor castanospermine (CS). HIV-Ig denotes rabbit polyclonal antihuman HIV antibody, and V5 ab mouse monoclonal anti-V5 antibody. The graph shows the infectivity of HIV produced in fibroblasts from Patient 2, which was greater after cotransfection of fibroblasts with nonmutant MOGS than with empty expression vector. RLU denotes relative light units. T bars represent standard deviations. The P value is based on a two-way analysis of variance with repeated measures. Data are representative of three independent experiments. In Panel C, the upper chart shows viral titers after infection of cells from Patient 1, Patient 2, and a healthy control with the virus that caused the 2009 influenza A (H1N1) pandemic. Viral titers were assessed on the basis of hemagglutination (HA) and the 50% tissue-culture infective dose (TCID 50 ) of the virus produced by monocyte-derived macrophages (MDM) from the patients and the healthy control (three cultures per person). No virus was isolated from any of the MDM cultures from Patient 2. Only one of the cultures from Patient 1 replicated the virus, but the titer was 1.5 log lower than that in the control cells. The lower chart shows HA titers and evidence of a cytopathic effect (CPE) in Madin–Darby canine kidney (MDCK) cells that were infected with equivalent amounts of influenza virus produced by MDM cultures from Patient 1 and a control. Only one of three cultures incubated with virus from Patient 1 had a positive HA titer and minimal CPE (+/−). CPE and HA titers were negative (Neg) in the other two cultures. All three MDCK cultures that were infected with virus that was produced by the control cells showed marked CPE (+++) and positive HA titers. Panel D shows the results of a secondary-infection experiment in which adenovirus type 5 (AdV5) produced from fibroblasts obtained from patients and controls during primary-infection experiments was normalized to infect control fibroblasts. Cells were lysed, and virus was measured in triplicate by means of a quantitative PCR assay. T bars represent standard deviations. Panel E shows the results of a secondary-infection experiment in which poliovirus 1 (PV1, Mahoney strain) produced from fibroblasts obtained from patients and controls during primary-infection experiments was normalized to infect HeLa cells. The CPE was assessed 72 hours after inoculation, and titers were calculated with the use of the Reed and Muench method. T bars represent standard errors. Panel F shows the results of a secondary-infection experiment in which vaccinia virus (VV) produced from EBV-transformed B-cell lines obtained from the patients and a control during primary-infection experiments was normalized to infect control EBV-transformed B-cell lines. Cells were lysed, and serial dilutions of the cell lysates were tested for their ability to infect B cells from a healthy donor. MOI denotes multiplicity of infection. T bars represent standard deviations.

Viral entry into the cells of the two siblings with CDG-IIb and into the cells of unaffected controls was similar for the four strains of HIV tested (Figure 2A). However, productive HIV replication, as measured by the amount of virus released into the culture supernatant, was 3.6 to 89.0 times as high in control cells as in the patients' cells. No differences were observed among HIV strains that use CCR5 or CXCR4. The possibility that the patients' serum, purified IgG, or other secreted factors could be contributing to the reduced HIV replication (Figure 2A) was considered but ruled out (section S6 in the Supplementary Appendix). When we compared the infectivity of p24-normalized ELI6 virus produced in cells from the patients with CDG-IIb with that in cells from healthy controls, the virus that we recovered from the cells of the patients with CDG-IIb was 50 to 80% less infectious. Furthermore, binding of the glycan-dependent anti–HIV envelope antibody 2G12 to virus produced by cells from patients with CDG-IIb was reduced as compared with binding to virus produced by control cells, although no differences in binding were detected with the glycan-independent anti–HIV envelope antibody b12 (Section S7 in the Supplementary Appendix).

These differences in protein glycosylation were confirmed by the expression of HIV glycoprotein 140 in fibroblasts from the patients and healthy controls (Figure 2B). The glycosylation pattern detected in cells from the healthy controls was induced into cells from the patients by transfection with a nonmutant MOGS expression vector, and conversely, the glycosylation pattern detected in cells from the patients was generated in control cells after treatment with the MOGS inhibitor castanospermine. Finally, the infectivity of HIV produced in fibroblasts from the patients was significantly enhanced when normal glycosylation was provided by cotransfection with the nonmutant MOGS expression vector (Figure 2B).

Three different macrophage cultures were generated for each patient and infected with the virus that caused the 2009 influenza A (H1N1) pandemic. This resulted in one productive infection from one patient (Patient 1), whereas all three cultures from the control were efficiently infected. The virus collected from the culture from Patient 1 had lower hemagglutination titers and TCID 50 than did virus collected from the cultures from the healthy control. When equivalent amounts of virus collected from the macrophage cultures were used to infect influenza-susceptible MDCK cells, only one of three MDCK cell cultures infected with virus generated by macrophages from Patient 1 had evidence of a cytopathic effect and a positive result of a hemagglutination assay. In contrast, all three MDCK cell cultures infected with virus from the control macrophages developed obvious signs of infection, with a marked cytopathic effect and highly positive hemagglutination titers (Figure 2C, and Section S8 in the Supplementary Appendix).

A similar evaluation strategy was used to test primary infection, viral replication, and infectivity of virus recovered from cells infected with adenovirus type 5, PV1, and vaccinia virus. Adenovirus and PV1 infections of primary fibroblasts derived from patients and controls showed consistent results. Vaccinia virus infection of EBV-transformed B-cell lines from patients and controls also showed no marked differences in terms of sensitivity to infection, viral replication, or infectivity of progeny virus (Figure 2D, 2E, and 2F).