Putative Envelope Glycoprotein Research Articles

DNA Vaccination against Channel Catfish Virus Results in Minimal Immune Response and is Not Efficacious against Challenge

AbstractWe evaluated DNA vaccination against channel catfish virus (CCV) by comparing 11 encoded genes, multiple doses, codelivery of DNA vaccines, and the resultant immune responses, as well as making a direct comparison with previously published DNA vaccines for CCV. The genes selected included immediate early genes, membrane genes, the major capsid gene, and the putative major envelope glycoprotein gene. The open reading frame (ORF) for each gene was amplified by using the polymerase chain reaction, cloned into pcDNA3.1/V5/His‐TOPO, and expression of the predicted molecular weight proteins was confirmed in cell culture. Each vaccine was injected intramuscularly in doses of 50, 25, 5, or 1 μg and evaluated by immersion challenge. Detection of mRNA specific for ORF 59 and ORF 6 at the injection site indicated that the DNA vaccines were transcriptionally active in situ. Serum‐neutralizing titers, enzyme‐linked immunosorbant assay (ELISA) antibody titers, and Mx transcription were measured in response to vaccination. Significant protection was not observed in any experiment, including groups receiving multiple constructs. Published DNA vaccines encoding one of the outer membrane protein genes and the putative major envelope glycoprotein gene also failed to protect against disease in direct comparison with those constructed here, singly or in combination. Serum‐neutralizing and ELISA antibody titers were generally low and not significantly different between groups. Mx transcription was induced in response to vaccination with both single and multiple DNA constructs. Although both innate and adaptive immune responses were detected in response to DNA vaccination for CCV, vaccination was not protective for any of the constructs tested.

Analysis of the Drosophila gypsy endogenous retrovirus envelope glycoprotein.

gypsy is the only endogenous retrovirus of Drosophila whose infectious properties have been reported. Previous studies have shown an unexpected relationship between the gene encoding the putative envelope glycoprotein (Env) of gypsy and genes encoding the fusion protein of several baculoviruses. The fact that fusion proteins mediate membrane fusion suggests that Env of insect retroviruses might also have fusogenic properties. The results reported here indicate that gypsy Env mediates cell-to-cell fusion. Cleavage of the Env precursor was also studied; it is shown that this polypeptide is cleaved at a furin-like cleavage site. This is the first report that the env-like gene of insect retroviruses encodes a fusion protein.

Prevention of hepatitis C virus infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1

We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.

Comparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis

A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.

Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus.

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5 degrees C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.

Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells

A putative second envelope glycoprotein (E2) of hepatitis C virus (HCV) was constitutively produced in a Chinese hamster ovary cell line stably transformed with a plasmid expressing E2 protein under the control of an exogenous promoter and a signal sequence. E2 protein that lacked part of the C-terminal hydrophobic region was glycosylated with high-mannose type oligosaccharides and retained in the cells. On the other hand, E2 protein lacking the entire C-terminal hydrophobic region was glycosylated with complex type oligosaccharides (complex form) and excreted into the culture medium. Immunoreactivity of the high-mannose and complex forms of E2 proteins against sera from HCV infected patients were analyzed. We found that the antigenicity of the complex form of E2 protein was greater than that of the high-mannose form of E2 protein. This result indicated that the complex form of the E2 protein is superior for use in diagnosing HCV infection.

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni 2+-nitrilotriacetic acid (Ni 2+-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni 2+-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase A isolated from bovine heart and γ-[ 32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Characterization of the Putative E2 Envelope Glycoprotein of Hepatitis C Virus Expressed in Stably Transformed Chinese Hamster Ovary Cells

Characterization of the Putative E2 Envelope Glycoprotein of Hepatitis C Virus Expressed in Stably Transformed Chinese Hamster Ovary Cells

Humoral response to linear B cell epitopes in the amino terminus of the hepatitis C virus envelope glycoprotein gp72 (E2): Role in protective immunity still unknown

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.

Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients

The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed.

Prevalence of hepatitis C virus sequence variants in South-East Asia.

The nature and distribution of hepatitis C virus (HCV) genotypic variants present in south-east Asia have not been extensively investigated. We analysed HCV RNA obtained from 67 clinical serum samples from Singapore, Thailand, Indonesia, the Philippines and South Korea. All samples were amplified by semi-nested RT-PCR and the nucleotide sequence determined for four regions within the E1, E2/NS1, NS4 and NS5 genes. Each isolate had a unique nucleotide and deduced amino acid sequence, consistent with the genetic heterogeneity of this virus. There was remarkably little amino acid sequence variation between isolates of the same genotype, apart from variable domains within putative envelope glycoproteins that are likely to be under immune pressure. All isolates could be classified according to the currently recognized genotypes of HCV, with the exception of one Singapore isolate that defined a new group 3 subtype. The 1b genotype, which predominates in Japan, was the most widely distributed genotype and accounted for 58% of all isolates sequenced. Regional variations in HCV genotype distribution were observed, with type 3a being found almost exclusively in Thailand. By contrast, the 1a genotype, which predominates in the USA was the most prevalent genotype in the Philippines. Genotype 1a was found less commonly among the Thai isolates, presumably having been introduced from the West in stored blood products or by sporadic transmission. The significant prevalence of HCV types 2 and 3 restates the need for variant genotypes to be included in immunodiagnostic and vaccine development strategies. This study reveals that the 1b genotype of HCV, previously found to be the major variant present in east Asia, also predominantes in the south-east Asian region, and may be the major HCV type found worldwide.

Virus isolate-specific antibodies against hypervariable region 1 of the hepatitis C virus second envelope protein, gp70.

Hypervariable region 1 (HVR1), located in the N‐terminal region of a putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV), contains immunological B‐cell epitopes which might be neutralizing epitopes. To clarify whether B‐cell epitopes within HVR1 are common among virus isolates or specific for the homologous virus isolate, we examined the reactivities of sera from 53 patients with chronic hepatitis or hepatocellular carcinoma/liver cirrhosis against two different HVR1 peptides (HVR11‐1 and HVR1 Y‐l) derived from patient I with sporadic acute hepatitis and an asymptomatic carrier Y, respectively, using our original assay system for the detection of anti‐HVR1 antibody. All patients examined had a history of blood transfusion. Most sera showed no reactivity with either HVR1 1‐1 or HVR1 Y‐l peptide. Only seven and fourteen serum samples reacted significantly, although weakly, with HVR1 1‐1 and HVR1 Y‐l peptides, respectively, compared with the serum from patient I or asymptomatic carrier Y. The blood transfusions of most reactive cases had occurred more than thirty years earlier. Six cases reacted with both HVR1 1‐1 and HVR1 Y‐l peptides, but further analysis revealed that only three cases reacted weakly with the peptide for either epitope I or II, identified within HVR11‐1, These results indicate that the B‐cell epitopes within HVR1 are fairly specific for the homologous virus isolate, and this may represent a serious difficulty in the development of a vaccine against HCV.

Vaccination Against Hepatitis C Virus Infection: Miles to Go Before We Sleep

A high incidence of community–acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatocellular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees ( Pan troglodytes ) have been immunized with both putative envelope glycoproteins [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells infected with a recombinant vaccinia virus expression vector. Despite the induction of a weak humoral immune response to these viral glycoproteins in experimentally infected chimpanzees, a strong humoral immune response was obtained in all vaccines. The five highest responders showed complete protection against an i.v. challenge with homologous hepatitis C virus 1. The remaining two vaccinees became infected, but both infection and disease may have been ameliorated in comparison with four similarly challenged control chimpanzees, all of which developed acute hepatitis and chronic infections. These results provide considerable encouragement for the eventual control of hepatitis C virus infection by vaccination.

Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection.

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.

Peptide immunogen mimicry of putative E1 glycoprotein-specific epitopes in hepatitis C virus.

Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B hepatitis with serious consequences that may lead to hepatocellular carcinoma. The putative envelope glycoproteins (E1 and E2) of HCV probably play a role in the pathophysiology of the virus. In order to map the immunodominant domains of the E1 glycoprotein, two epitopes from amino acid residues 210 to 223 (P1) and 315 to 327 (P2) were predicted from the HCV sequence. Immunization of mice with the synthetic peptides conjugated to bovine serum albumin induced an antibody response, and the antisera immunoprecipitated the E1 glycoprotein (approximately 33 kDa) of HCV expressed by recombinant vaccinia virus. A panel of HCV-infected human sera was also tested with the synthetic peptides by enzyme-linked immunosorbent assay for epitope-specific responses. Of 38 infected serum samples, 35 (92.1%) demonstrated a spectrum of reactivity to the P2 peptide. On the other hand, only 17 of 38 (44.7%) serum samples were reactive to the P1 peptide. Strains of HCV exhibit a striking genomic diversity. The predicted P1 epitope showed localization in the sequence-variable region, and the P2 epitope localized in a highly conserved domain. Results from this study suggest that the E1 glycoprotein of HCV contains at least two potential antigenic epitopes. Synthetic peptides corresponding to these epitopes and antisera to these peptides may serve as the monospecific immunological reagents to further determine the role of E1 glycoprotein in HCV infection.

Genetic alterations of the putative envelope proteins encoding region of the hepatitis C virus in the progression to relapsed phase from acute hepatitis: Humoral immune response to hypervariable region 1

Hypervariable region I (HVRI) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) undergoes sequential alterations at intervals of several months during the chronic phase of hepatitis. To evaluate the implications of sequence variability in HVRI of HCV, we investigated the sequence variability of the whole envelope-protein(gp35 and gp70)-coding regions of HCV genome derived from patient M in acute and relapsed phases (8-month interval) of hepatitis. From this analysis, we found that a Leu (position 405) in HVRI substituted to Pro, and that 4 additional substitutions could be detected in gp70 during the relapsed phase. Sequence-specific antibody against HVRI derived from patient M was first detected in the serum at 8 months after the onset of hepatitis, but no other specific antibodies against peptides containing amino-acid position(s) substituted in regions other than HVRI could be detected. Epitope mapping using the sequence of HVRI derived from the acute phase of hepatitis was also performed, and a B-cell epitope (positions 397 to 407) of 11 amino acids was identified. However, the Pro variant at position 405 did not display an escape pattern from the antibody produced at 8 months after the onset. In addition, we demonstrated the existence of important amino-acid residue positions which are recognized by the anti-HVRI antibody produced in patient M using introduction point mutations within HVRI.

Vaccination of chimpanzees against infection by the hepatitis C virus.

A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatocellular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells infected with a recombinant vaccinia virus expression vector. Despite the induction of a weak humoral immune response to these viral glycoproteins in experimentally infected chimpanzees, a strong humoral immune response was obtained in all vaccines. The five highest responders showed complete protection against an i.v. challenge with homologous hepatitis C virus 1. The remaining two vaccines became infected, but both infection and disease may have been ameliorated in comparison with four similarly challenged control chimpanzees, all of which developed acute hepatitis and chronic infections. These results provide considerable encouragement for the eventual control of hepatitis C virus infection by vaccination.

Genetic alteration of the hepatitis C virus hypervariable region obtained from an asymptomatic carrier.

Hepatitis C virus (HCV) genome shows extensive sequence diversity at 2 hypervariable regions (HVR1 and HVR2) of the putative envelope glycoprotein (gp70). We recently reported that the amino-acid sequence of HVR1, but not of HVR2, underwent a striking mutation or mutated sequentially over a period of several months in patients with chronic hepatitis (CH). Here, we examined whether these genetic alterations in HVR1 occurred in an asymptomatic HCV carrier. The level of HCV RNA in serum was almost the same throughout the 4 time points sampled over 16 months. However, we found that the amino-acid sequence of the HCV HVR1 from this asymptomatic carrier altered with time, as seen in patients with CH. Alterations of amino acids in the HVR1 were correlated with persistent HCV infection rather than with clinical symptoms. Sequence heterogeneity of HVR1 was not correlated with alanine aminotransferase (ALT) values or liver histological findings. The necessity of clinical follow-up of HCV asymptomatic carriers is discussed.

Immunogens of bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle that induces economically important diseases affecting multiple organ systems. In the United States, over 150 biological products are licensed for control of BVDV. These products contain live or killed BVDV, and many products contain other viruses or bacteria. Potency tests for these vaccines are based on animal inoculation and serology. For live virus vaccines, titration of viral infectivity in cell culture is an accepted alternative to animal inoculation. The immunogens in a killed virus vaccine may be measured by enzyme linked immunoabsorbent assay. Immunogens of BVDV that stimulate a protective immune response have not been conclusively identified. Epitopes on a putative viral envelope glycoprotein, gp53, are involved in viral neutralization. Other viral glycoproteins, gp48 and gp25, are immunogenic but epitopes on these proteins do not stimulate production of antibodies that efficiently neutralize virus. Progress in developing meaningful in vitro assays for quantitation of BVDV immunogens awaits identification of viral proteins that stimulate a protective immunity.

Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus

We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occured sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.