Anders Bolmstedt

Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response

One of the functions of N-linked glycans of viral glycoproteins is protecting otherwise accessible neutralization epitopes of the viral envelope from neutralizing antibodies. The aim of the present study was to explore the possibility to obtain a more broadly neutralizing immune response by immunizing guinea pigs with gp160 depleted of three N-linked glycans in the CD4-binding domain by site-directed mutagenesis. Mutant and wild type gp160 were formulated into immunostimulating complexes and injected s.c. into guinea pigs. Both preparations induced high serum antibody response to native gp120 and V3 peptides. Both preparations also induced antibodies that bound equally well to the V3 loop or the CD4-binding region, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The sera from animals, immunized with mutated glycoprotein, did not neutralize nonrelated HIV strains better than did sera from animals, immunized with wild type glycoprotein. Instead, a pattern of preferred homologous neutralization was observed, i.e., sera from animals, immunized with mutant gp160, neutralized mutant virus better than wild type virus, and vice versa. These data indicated that elimination of the three N-linked glycans from gp160 resulted in an altered local antigenic conformation but did not uncover hidden neutralization epitopes, broadening the immune response.

Dendritic cells mediate herpes simplex virus infection and transmission through the C-type lectin DC-SIGN

Dendritic cells (DCs) are essential for the induction of specific immune responses against invading pathogens. Herpes simplex virus (HSV) is a common human pathogen that causes painful but mild infections of the skin and mucosa, and which results in latency and recurrent infections. Of the two HSV subtypes described, HSV-1 causes mainly oral-facial lesions, whilst HSV-2 is associated with genital herpes. DCs are involved in HSV-induced immune suppression, but little is known about the molecular interactions between DCs and HSV. This study demonstrated that HSV-1 and -2 both interact with the DC-specific C-type lectin DC-SIGN. Further analyses demonstrated that DC-SIGN interacts with the HSV glycoproteins gB and gC. Binding of HSV-1 to immature DCs depended on both DC-SIGN and heparan sulfate proteoglycans. Strikingly, HSV-1 infection of DCs was almost completely inhibited by blocking antibodies against DC-SIGN. Thus, DC-SIGN is an important attachment receptor for HSV-1 on immature DCs and enhances infection of DCs in cis. In addition, DC-SIGN captures HSV-1 for transmission to permissive target cells. These data strongly suggest that DC-SIGN is a potential target to prevent HSV infection and virus dissemination. Further studies will show whether these interactions are involved in HSV-induced immune suppression.

Protection of Neutralization Epitopes in the V3 Loop of Oligomeric Human Immunodeficiency Virus Type 1 Glycoprotein 120 by N-Linked Oligosaccharides in the V1 Region

The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.

Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals : Effects of elimination of the V3 N306 glycan

DNA encoding HIV-1 env is a poorly efficient B-cell immunogen and one probable explanation is that the numerous gp120 N-linked glycans gp120 may interfere with B-cell epitope presentation. The N306 glycan in gp120 shields HIV-1 from neutralizing antibodies. A DNA immunogen lacking the N306 glycosylation signal (T308A) was constructed to determine whether this glycan affected the immune response. Mice were immunized intranasally twice with DNA containing either the wild type or the mutant env. Two additional groups were primed with wild type or mutant env and boosted with rgp160 protein, containing the complete set of N-linked glycans. Immunization with DNA alone resulted in priming of B-cell clones but was not sufficient to induce a complete antibody response. Animals primed with the N306 mutant and subsequently boosted with rgp160 protein displayed higher serum IgG-binding titers to gp120 than animals primed with wild type env DNA. The manipulation of the glycosylation sites of the env DNA strongly primes antibody responses (but non-neutralizing) as well as T-cell responses to the wild type strain gp160. However, priming with mutant plasmid did not result in higher neutralization titers to wild type or T308A-mutated virus than did the wild type plasmid. With the N306 mutant DNA we thus immunized a non-neutralization epitope, but obtained strong env-binding IgG after rgp160 boosting.

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4.

SummaryGlycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.

Carbohydrate determinant NeuAc-Galβ(1-4) of N-linked glycans modulates the antigenic activity of human immunodeficiency virus type 1 glycoprotein gp120

In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gp120). Recombinant gp120 was purified from GMK cells infected with a recombinant vaccinia virus expressing gp120. Purified gp120 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gp120 was then incubated with monoclonal antibodies recognizing specific epitopes of gp120 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Gal beta (1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Gal beta (1-4), whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp120.

The N-linked glycan of the V3 region of HIV-1 gp120 and CXCR4-dependent multiplication of a human immunodeficiency virus type 1 lymphocyte-tropic variant

We have previously shown that an N‐glycosylation site of N306 of HIV‐1 gp120 is not necessary for the HIV‐1 infectivity but protects HIV‐1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367–372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T‐tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re‐examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co‐receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.

Simplified procedure for fractionation and structural characterisation of complex mixtures of N-linked glycans, released from HIV-1 gp120 and other highly glycosylated viral proteins.

HIV-1 gp120 is heavily glycosylated containing 24 N-glycosylation sites, and this makes elucidation of the significance of glycans at individual glycosylation sites a difficult task. A procedure is described where a complex mixture of biologically radiolabelled glycans of gp120, derived from a relatively small number of virus-infected cells may be characterized by a combination of N-glycanase release, single lectin separation, and normal phase HPLC (NP-HPLC). The method was applied in analysis of three N-linked glycosylation sites essential for the in vivo priming of T-cells, specific for an epitope in their vicinity (Sjölander, S., Bolmstedt, A., Akerblom, 1996. Virology 215, 124-133.). The carbohydrate compositions of wild type gp120 and of mutant variants gp120 lacking one, two, or all of these three active N-linked glycans were analysed. Cells were infected with r-vaccinia virus expressing wild-type gp120 or mutated gp120, or were infected with HIV-1BRU (wild type) or mutant virus variants. HIV-1 glycoproteins were purified by immunosorbent affinity chromatography and released glycans were separated on lectins, then analysed with NP-HPLC. Our data showed that the structural composition of glycans occupying two of the three glycosylation sites was heterogeneous but the site located adjacent to the T-cell epitope was equipped with one large, high mannose-type structure (> 11 units) with the capacity to cover a substantial part of the gp120 surface.

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

SummaryWe have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wildtype virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.

Identification of three N-linked glycans in the V4-V5 region of HIV-1 gp 120, dispensable for CD4-binding and fusion activity of gp 120.

SummarySite-directed mutagenesis was used to study the biological significance of three N-linked glycans (linked to Asn 406, Asn 448, and Asn 463), situated in the CD4-binding region of gp120. Mutagenesis was carried out in a phage M13 system, and the mutatedenv genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the level of expression affected the biological phenotype of mutant gp 120, we expressed the envelope glycoproteins using either a weak (7.5 K) or a strong (11 K) promoter of vaccinia virus. The expression of mutatedenv proteins was analyzed after infecting CD4-expressing HeLa cells with the r-vaccinia virus, by monitoring the ability of the infected cells to generate CD4-dependent syncytia.Env gene products lacking all three glycans as well asenv gene products lacking different permutations of one or two glycans were analyzed. All mutated gp 120 species had the expected electrophoretical mobility as anticipated from elimination of one, two, and three N-linked glycans, respectively. Moreover, all mutantenv gene products demonstrated the same capacity to induce formation of syncytia, irrespective of using the weak or strong promoter for expression. These data indicate that the three N-linked glycans studied are dispensable for HIVenv gene products to function in CD4-binding and the subsequent fusion step.