A weak neutralizing antibody response to hepatitis C virus envelope glycoprotein enhances virus infection

We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately 25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1 glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV envelope glycoproteins.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

Binding characteristics of Rauscher leukemia virus envelope glycoprotein gp71 to murine lymphoid cells.

The major envelope glycoprotein (gp71) purified from Rauscher leukemia virus (R-MuLV) binds efficiently to murine lymphoid cells but not to either murine nonlymphoid cells or lymphoid cells from other species. Binding of 125I-labeled R-MuLV gp71 was competitively inhibited by unlabeled glycoprotein, as well as by whole R-MuLV, but not by murine xenotropic viruses, R-MuLV p30, and several unrelated proteins. Polyacrylamide gel electrophoresis profiles of iodinated gp71 after binding to lymphoid cells were similar to prebound profiles. Antibody to R-MuLV gp71 prevented binding, whereas normal serum had no effect. Adsorption of the glycoprotein to murine lymphoid cells occurs rapidly and is time and temperature dependent. The procedure described is sensitive for detecting the binding activity of approximately 10(4) cells. Binding was proportional up to 2.5 X 10(5) cells per ml and plateaued above 10(7) cells per ml. In the presence of excess R-MuLV gp71, BALB/c thymocytes bound approximately 2.4 X 10(4) molecules per cell.     

Envelope glycoproteins of Rauscher murine leukemia virus: isolation and chemical characterization.

The envelope glycoproteins (designated gp70 and gp45) of the Rauscher strain of murine leukemia virus were solubilized by osmotic shock and freeze-thawing in chaotropic solutions. The viral glycoproteins were then purified by phosphocellulose chromatography and gel permeation chromatography on Bio-Gel A-1.5m. Yields by this procedure were 6.2% for gp70 and 1.3% for gp45 on a protein input basis. The apparent molecular weights were respectively 67 500 and 47 500 with a polypeptide chain molecular weight of approximately 45 000 for both glycoproteins. Amino acid analysis showed a high degree of similarity for both components, with some differences subject to further evaluation. The total carbohydrate content was approximately 32% for gp70 and 6-9% for gp45. In keeping with the amino acid compositional similarity suggesting relationships, alanine was found to ba the amino-terminal amino acid of both glycoproteins, and cross-reactivity was demonstrated by immunologic tests. The data suggest that the chief difference between gp70 and gp45 lies in the carbohydrate content.     

A single mutation turns a non-binding germline-like predecessor of broadly neutralizing antibody into a binding antibody to HIV-1 envelope glycoproteins

Broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV)-1 are rare in natural infection and elicitation of HIV-1 bnAbs has not been achieved by any vaccine candidates. We and others have reported that HIV-1 bnAbs are highly diversified from their germline-like predecessors, and the germline-like predecessors of bnAbs lack measurable binding to HIV-1 envelope (Env) glycoproteins, suggesting that Env structures containing the epitopes of bnAbs may not initiate somatic maturation pathway, which may partially explain the rarity of HIV-1 bnAbs. To determine the minimum mutations required for converting non-binding germline-like predecessors to Env-binding antibodies, we started with the bnAb b12 as a prototype and generated six “chimeric” scFv b12 variants by sequentially replacing the heavy chain V-segment (HV), D(J)-segment [HD(J)] in the heavy chain variable region (VH), and the whole light chain variable region (VL) in b12 germline-like predecessor with the mature counterparts. We tested the recombinant scFv variants for binding and neutralizing activities. Results showed that a single point mutation in germline D-segment was enough to convert nonbinding germline-like b12 to an Env-binding antibody. Replacement with either mature HV or mature VL also made the germline-like b12 bind to Env, but none of single segment replacements conferred neutralization ability to the germline antibody. Mature VL in combination with mature HD(J), or mature HV, or both conferred increasing neutralization activity to the germline antibody. However, hybrid scFv, mature VH / germline VL did not neutralize the virus, suggesting the importance of mature VL in neutralizing the virus. These results may have implications for vaccine development.     

A single mutation turns a non-binding germline-like predecessor of broadly neutralizing antibody into a binding antibody to HIV-1 envelope glycoproteins

Broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV)-1 are rare in natural infection and elicitation of HIV-1 bnAbs has not been achieved by any vaccine candidates. We and others have reported that HIV-1 bnAbs are highly diversified from their germline-like predecessors and the germline-like predecessors of bnAbs lack measurable binding to HIV-1 envelope (Env) glycoproteins, suggesting that Env structures containing the epitopes of bnAbs may not initiate somatic maturation pathway, which may partially explain the rarity of HIV-1 bnAbs. To determine the minimum mutations required for converting non-binding germline-like predecessors to Env-binding antibodies, we started with the bnAb b12 as a prototype and generated six “chimeric” scFv b12 variants by sequentially replacing the heavy chain V-segment (HV), D(J)-segment [HD(J)] in the heavy chain variable region (VH), and the whole light chain variable region (VL) in b12 germline-like predecessor with the mature counterparts. We tested the recombinant scFv variants for binding and neutralizing activities. Results showed that a single point mutation in germline D-segment was enough to convert nonbinding germline-like b12 to an Env-binding antibody. Replacement with either mature HV or mature VL also made the germline-like b12 bind to Env, but none of single segment replacements conferred neutralization ability to the germline antibody. Mature VL in combination with mature HD(J) or mature HV, or both conferred increasing neutralization activity to the germline antibody. However, hybrid scFv, mature VH/germline VL, did not neutralize HIV-1, suggesting the importance of mature VL in neutralizing the virus. These results may have implications for vaccine development.Key words: germline, antibody, immune responses, HIV, vaccine     

Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.     

Characterization of Rauscher murine leukemia virus envelope glycoprotein receptor in membranes from murine fibroblasts.

Plasma membrane preparations from KA31 (mouse) cells contained receptors for the binding of Rauscher murine leukemia virus (R-MuLV) envelope glycoprotein, gp70. This binding was demonstrated by gel filtration of a mixture of the microsomal fraction of the cells and 125I-labeled gp70. A rapid and convenient assay was developed to measure the complex formation between the membrane receptors and gp70 involving specific precipitation of the complex by 3 to 4% polyethylene glycol. The complex formation was responsive to the concentrations of both the receptor and gp70 and also to changes in temperature and pH. The gp70 binding was a noncooperative, saturable process, and an association constant of 3.5 X 10(8) M-1 was estimated from the binding data. The complex formation was reversible and a near-total exchange of 125I-labeled gp70 in the complex was achieved by incubation with excess of unlabeled gp70. The complex formation was inhibited by protein denaturing agents, guanidine-hydrochloride and urea. Pretreatment of the membrane fractions with either chymotrypsin or phospholipase C led to a loss of the membrane-associated receptor activity, indicating that a lipoprotein structure was important for the receptor function, consistent with the observation that nonionic detergents strongly inhibited the complex formation.     

Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1

Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man₁₇-BSA and mannan, by -d-GlcNAc₄₇-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human ₁-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal₁₇-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc₄₇-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures.     

Primary virus envelope cross-reactivity of the broadening neutralizing antibody response during early chronic human immunodeficiency virus type 1 infection

To test the hypothesis that changing neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1) during chronic infection were a response to emergence of neutralization escape mutants, we cloned expressed and characterized envelope clones from patients in the Multicenter AIDS Cohort Study (MACS). Pseudotyped HIV-1 envelope clones obtained from differing time points were assessed for sensitivity to neutralization by using sera from different times from the same and different patients. Clones from early and late time points during chronic infection had similar neutralization sensitivity, and neutralizing antibody responses cross-reacted with early, late, and heterologous envelopes. The potential for broadly effective HIV-1 immunization is supported.     

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.     

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.     

Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response

The envelope (Env) glycoprotein of human immunodeficiency virus (HIV) contains 24 N-glycosylation sites covering much of the protein surface. It has been proposed that one role of these carbohydrates is to form a shield that protects the virus from immune recognition. Strong evidence for such a role for glycosylation has been reported for simian immunodeficiency virus (SIV) mutants lacking glycans in the V1 region of Env (J. N. Reitter, R. E. Means, and R. C. Desrosiers, Nat. Med. 4:679-684, 1998). Here we used recombinant vesicular stomatitis viruses (VSVs) expressing HIV Env glycosylation mutants to determine if removal of carbohydrates in the V1 and V2 domains affected protein function and the generation of neutralizing antibodies in mice. Mutations that eliminated one to six of the sites for N-linked glycosylation in the V1 and V2 loops were introduced into a gene encoding the HIV type 1 primary isolate 89.6 envelope glycoprotein with its cytoplasmic domain replaced by that of the VSV G glycoprotein. The membrane fusion activities of the mutant proteins were studied in a syncytium induction assay. The transport and processing of the mutant proteins were studied with recombinant VSVs expressing mutant Env G proteins. We found that HIV Env V1 and V2 glycosylation mutants were no better than wild-type envelope at inducing antibodies neutralizing wild-type Env, although an Env mutant lacking glycans appeared somewhat more sensitive to neutralization by antibodies raised to mutant or wild-type Env. These results indicate significant differences between SIV and HIV with regard to the roles of glycans in the V1 and V2 domains.     

Factor-dependent binding of Rauscher leukemia virus RNA to ribosomes

Rauscher leukemia virus (RLV)-³H-RNA was shown to bind to ribosomes with the sedimentation properties of polyribosomes when incubated in a cell-free amino acid incorporation system derived from RLV-infected JLS-V5 cells. The binding of RLV-³H-RNA to ribosomes requires factors present in the high-salt wash of JLS-V5 polyribosomes, and is inhibited by aurintricarboxylic acid (ATA). The RLV-³H-RNA-ribosome complexes are sensitive to ribonucleases (RNase), ethylenediaminetetraacetate (EDTA), and puromycin.     

Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene.

Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M. Hummel, D. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417). These RNAs have the same 5' and 3' ends. The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame. The smaller RNA has one internal splice which maintains the same open reading frame. Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J. Virol. 49:413-417). The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties. Antisera raised in rabbits to the protein expressed in E. coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame. The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement. This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody. We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.     

Antiidiotypic antibody mimicry of a bluetongue virus neutralizing antigen.

Syngeneic antiidiotypic (Anti-Id) mAb were produced to the Bluetongue virus (BTV) serotype 17 neutralizing mAb. Three anti-Id mAb had the characteristics of an internal image of the Id as demonstrated by 1) the internal image anti-Id mAb were capable of mimicking BTV by blocking the neutralizing activity of the idiotypic neutralizing mAb and 2) the anti-Id mAb bound specifically to the surface of BTV susceptible cells in indirect binding experiments as determined by immunocytochemistry and flow cytometric analysis. The potential application of these internal image anti-Id mAb in this arbovirus system is discussed.     

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.     

Stability of Rauscher leukemia virus under certain laboratory conditions.

The stability of Rauscher leukemia virus (RLV) was investigated under certain laboratory conditions. The half life of the virus at 37 degrees was 7 hr, and considerably longer at lower temperatures. RNA dependent DNA polymerase activity was more stable than infectivity at all temperatures. Air dried virus had a half life of approximately 1 hr, but was rapidly inactivated by uv light or 70% alcohol.     

Human respiratory syncytial virus glycoproteins are not required for apical targeting and release from polarized epithelial cells

Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.