Biomedical subjects

P Coursaget

Publications and source records attributed to P Coursaget.

At least 19 recordsLinked to original sources

PubMed2000

The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity.

Four C-terminal deletion mutants of the human papillomavirus 16 L1 protein were expressed in the baculovirus expression system. They consist of the deletion of amino acids 497-505, 477-505, 403-505 and 302-505 (delta C9, delta C31, delta C103 and delta C204 respectively). Only two of the C-terminally deleted proteins, delta C9 and delta C31, retained the ability to form virus-like particles (VLPs) resembling those obtained with the full length L1 protein. Analysis of deleted L1 proteins and corresponding VLPs indicated that the C-terminus was necessary both for DNA binding and DNA packaging. These results were corroborated by the loss of the gene transfer capacities of C-terminal deleted VLPs.

Amino Acid Sequence↗

PubMed2000

DNA-containing and empty hepatitis B virus core particles bind similarly to envelope protein domains.

DNA synthesis within the hepatitis B virus (HBV) nucleocapsid appears to be coupled to nucleocapsid envelopment. The nature of the envelopment signal is unknown, but is thought to involve a conformational change at the surface of the capsid that facilitates interaction with HBV envelope proteins. In binding assays in vitro, it was found that empty HBV core particles bound synthetic peptides corresponding to HBV envelope protein domains with the same affinity as did HBV DNA-containing core particles. This suggests that the selection of replication-competent nucleocapsids for envelopment is not related to the capacity of DNA-containing core particles to bind specifically to HBV envelope proteins, and that there must be an alternative mechanism.

Capsid↗

PubMed2000

Identification of a novel hepatitis E virus in Nigeria.

Sporadic cases of acute hepatitis E among ten native Nigerian adults were reported in Port-Harcourt (Nigeria). Hepatitis E virus (HEV) was detected in serum and/or faecal samples of seven patients by RT-PCR of the open reading frame (ORF)-1 polymerase region and the 3'-end of ORF2. Restriction analysis widely used to distinguish genotypes I and III showed that all Nigerian strains have a pattern similar to the Mexican strain (NotI, nt 286; SmaI, nt 397; no KpnI restriction site) but displayed a BsmI restriction site at nt 213 as do most African HEV strains sequenced so far. Sequence analysis performed from internal ORF1 and ORF2 PCR products displayed strong homogeneity between the HEV isolates, determining a regional cluster. Phylogenetic analysis of nucleotide sequences revealed that these strains were more related to the Mexican prototype genotype III (87% homology in ORF1, 80% homology in ORF2) than to either the African strain genotype I (74% homology in ORF1, 77% homology in ORF2) or the USA strain genotype II (75% homology in ORF1, 77% homology in ORF2). Genetic divergence up to 15% in ORF2 with the Mexican genotype clearly defined a new subgenotype within genotype III. At the amino acid level, Nigerian strains showed more homology with genotype III (96%) than with genotype I (92%). This study clearly determined the co-existence of genotypes I and III in Africa. These Nigerian HEV strains belonging to genotype III, but sharing some properties with genotype I, could be one of the missing links between African and Latin American HEV and could help us to determine the phylogenetic evolution of HEV from the ancestral virus.

Adult↗

PubMed2000

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16.

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.

Animals↗

PubMed1999

Comparison of the humoral and cellular immune responses to two preparations of Cryptosporidium parvum CP15/60 recombinant protein.

This study compares the immune responses produced by immunising mice and rabbits with two preparations of the recombinant 15/60 kDa protein of Cryptosporidium parvum. Genomic C. parvum DNA was amplified and the recombinant protein was synthesized as a fusion protein with glutathione-S-transferase in Escherichia coli and in the eukaryotic system of baculovirus/insect cells. Both recombinant proteins induced similar levels of serum antibodies against the fusion recombinant protein, but the eukaryotic recombinant protein triggered a stronger humoral response to C. parvum. Similarly, increased lymphoproliferation occurred only after stimulation of spleen cells from mice immunised with the eukaryotic recombinant protein. This suggests that the eukaryotic protein is a better candidate for immunological studies on cryptosporidiosis.

Animals↗

PubMed1999

Human papillomavirus vaccines.

Genital human papillomavirus (HPV) infections are the viral sexually transmitted diseases most frequently diagnosed that include anogenital condylomas and squamous intra-$bepithelial lesions, among which the precursors of invasive carcinomas of the uterine cervix. In animal PV models, vaccination against L1 and/or L2 viral capsid proteins provides an efficient protection against infection, involving virus type-specific neutralizing antibodies. Vaccination against non-structural E1, E2, E6 or E7 viral proteins does not prevent infection, unless administered altogether, but tends to stimulate regression, warranting the design of therapeutic vaccines. Prophylactic vaccines based on the use of virus-like particles (VLPs) obtained by auto-assembly of L1 or L1 and L2 proteins produced by recombinant DNA technology are under phase I/II clinical trials for HPV6/11 associated with condylomas and for HPV16, the most frequent oncogenic genotype. Second generation vaccines are chimeric proteins or VLPs incorporating one of the structural proteins (L1 or L2) fused to a non-structural protein (E6, E7 or E2), which should induce both humoral and cellular immunity. Vaccine valency (number of genotypes), route of administration (humoral versus local immunity), vaccinees (children, young adults, gender) and forms of vaccines (recombinant $LSalmonella typhimurium*I$L, edible plants expressing L1 and L2 proteins, DNA vaccines, synthetic antigenic peptides) are under study. End points to evaluate vaccine efficacy in phase III trials should include viral DNA detection and typing, and screening for low or high grade intraepithelial lesions. Therapeutic vaccines based on recombinant HPV E6 and/or E7 vaccinia virus, L2-E7 fusion proteins or E7 peptides corresponding to cytotoxic T cell epitopes are currently tested (phase I/II trials) in patients with cervical carcinomas of advanced clinical stages or high grade intraepithelial lesions. Animal studies, phase I/II clinical trials and implementation of the community support that HPV vaccines will constitute an efficient means to prevent carcinoma of the uterine cervix.

Animals↗

PubMed1999

Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia. I. Differential T-helper and IgG responses in relation to HPV infection and disease outcome.

T-helper (Th) cell-dependent IL-2 production and plasma IgG responses to virus-like particles consisting of the human papillomavirus type 16 (HPV-16) major capsid protein L1 (L1-VLP) were determined in patients with cytological evidence of cervical intraepithelial neoplasia (CIN) participating in a non-intervention prospective cohort study. IgG responses were associated with HPV-16 persistence and high-grade CIN lesions, while high frequencies of Th responses were observed in patients with both virus clearance and virus persistence, irrespective of CIN grade. The IgG response was found in conjunction with an IL-2 response to L1-VLP in 87% of the patients. Recognition of the HPV-16 L1 Th epitope (amino acids 311-335) was found to be more closely associated than recognition of L1-VLP as a whole to HPV exposure and CIN development. Among the HPV-16+ patients included in this study, those showing a Th response to amino acids 311-335 were more likely to carry the HLA DRB1*11/DQB1*0301 haplotype, while those showing an IgG response to L1-VLP were more likely to carry DRB1*0101/DQB1*0501. However, neither cell-mediated nor humoral immune responses against HPV-16 L1 appear to be sufficient for the natural control of HPV infection and CIN development.

Adult↗

PubMed1999

Baculovirus expression of chimeric hepatitis B virus core particles with hepatitis E virus epitopes and their use in a hepatitis E immunoassay.

Two hepatitis B core proteins bearing the immunodominant region of the hepatitis E virus (HEV) capsid protein, one at the C terminus of hepatitis B virus core antigen (HBcAg) and the other within the HBcAg immunodominant loop, were constructed. Both chimeric proteins exhibited HEV reactivity, but only the first construct retained HBcAg reactivity. The second construct was used to develop an anti-HEV test which is equivalent to a commercial test for the detection of anti-HEV immunoglobulin G (IgG) but is more sensitive for the detection of anti-HEV IgM.

Baculoviridae↗

PubMed1999

Nasal immunization of mice with human papillomavirus type 16 (HPV-16) virus-like particles or with the HPV-16 L1 gene elicits specific cytotoxic T lymphocytes in vaginal draining lymph nodes.

Human papillomavirus type 16 (HPV-16) infects the genital tract and is closely associated with the development of cervical cancer. HPV-16 initiates infection at the genital mucosal surface; thus, mucosal immune responses are likely to contribute to defense against HPV-16 infection. However, little information is available regarding the induction of immune responses in the genital tract mucosa. In this study, we evaluated the potential of intranasally administered papillomavirus vaccines to elicit both systemic and vaginal immune responses. HPV-16 virus-like particles (VLPs) produced by self-assembly of L1 protein and the HPV-16 L1 gene cloned into a mammalian expression vector were used as vaccines. Intranasally administered VLPs induced serum immunoglobulin G (IgG) and vaginal IgA secretory antibodies. Very weak serum IgG and vaginal IgA responses were found after DNA immunization. Both splenic and vaginal lymphocytes could be activated by intranasal immunization with VLPs and the HPV-16 L1 gene. Activated CD4(+) Th1-like T cells were shown to synthesize gamma interferon, and activated CD8(+) T cells were demonstrated to be cytotoxic.

Administration, Intranasal↗

PubMed1998

In vitro gene transfer using human papillomavirus-like particles.

Recombinant papillomavirus-like particles have recently been shown to be highly effective for the prevention of papillomavirus infections and associated tumors, and a virus-like particle-based vaccine against the most prevalent HPV causing genital infection in humans will be developed in the near future. Another use of these virus-like particles may lie in gene therapy and DNA immunization. We report here that human papillomavirus-like particles composed of the major capsid protein (L1) of HPV-16 are able to package unrelated plasmid DNA in vitro and then to deliver this foreign DNA to eukaryotic cells with the subsequent expression of the encoded gene. The results indicate higher gene transfer than with DNA alone or with liposome. Virus-like particles are a very promising vehicle for delivering genetic material into target cells. Moreover, the preparation of the gene transfer vehicle is relatively easy.

Animals↗

PubMed1998

Production of recombinant virus-like particles from human papillomavirus types 6 and 11, and study of serological reactivities between HPV 6, 11, 16 and 45 by ELISA: implications for papillomavirus prevention and detection.

The L1 major capsid proteins of human papillomaviruses types 6 and 11 were expressed in insect cells using recombinant baculoviruses. These L1 proteins were shown to self-assemble into virus-like particles resembling papillomavirus virions as previously observed for HPV 16 and 45. However, we observed variations in the yield of virus-like particles among the four genotypes investigated. This suggests that more than one strain of each genotype has to be investigated to obtain the high level of virus-like particle production necessary to develop HPV vaccines or serological tests. Cross-reactivities between HPV 6, 11, 16 and 45 were studied using polyclonal and monoclonal antibodies to virus-like particles, L1 proteins and synthetic peptides. Although antisera react strongly against homologous virus-like particles, there is evidence of some cross-reactivity. This could be one of the explanations for the fact that antibodies to one genotype are detected in individuals infected with another genotype. This study also identified a linear epitope recognized by anti-HPV 16 virus-like particle sera.

Amino Acid Sequence↗

PubMed1998

Outbreak of enterically-transmitted hepatitis due to hepatitis A and hepatitis E viruses.

BACKGROUND/AIMS: One hundred and eleven patients with acute hepatitis and 61 controls were investigated for hepatitis serological markers in order to determine the viral etiology of cases involved in a waterborne epidemic of hepatitis observed in 1993 in Djibouti, Republic of Djibouti (East Africa). These cases occurred both in indigenous Djiboutians, and in French soldiers and their families in Djibouti. A retrospective study of the viral etiology of acute hepatitis cases observed in French soldiers and relatives living in Djibouti during the 3-year period preceding the epidemic was also undertaken. METHODS: HAV, HBV and HCV infections were investigated using commercial ELISA tests. HEV infections were investigated by testing IgG and IgM-specific antibodies by means of three different ELISA tests using recombinant proteins or synthetic peptides. RESULTS: Hepatitis A was observed in 37 (33%) and hepatitis E in 43 (39%) of the 111 cases of acute hepatitis recorded during this epidemic. Hepatitis B represented only 6% of the indigenous cases and hepatitis C was not observed among the cases investigated. Anti-HEV IgG antibodies were also detected in 19% of the indigenous control group. CONCLUSION: These results suggest that both HAV and HEV were responsible for this waterborne epidemic of acute hepatitis. However, HAV and HEV infections were not equally distributed between French expatriates and Djibouti residents. Whereas HAV infections were mainly observed in French patients, HEV was almost exclusively found in indigenous patients. This study reports for the first time a waterborne outbreak of acute hepatitis simultaneously due to HAV and HEV.

Adolescent↗

PubMed1998

The L1 major capsid protein of human papillomavirus type 16 variants affects yield of virus-like particles produced in an insect cell expression system.

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.

Amino Acid Sequence↗

PubMed1998

Role of hepatitis E virus in sporadic cases of acute and fulminant hepatitis in an endemic area (Chad).

Forty-one patients with acute or fulminant hepatitis and 86 control patients were entered into a study of sporadic, acute, and fulminant hepatitis in the N'Djamena area of Chad in 1993. Acute hepatitis B was diagnosed in nine (22%) patients and acute hepatitis E in 27 (66%) patients. No acute hepatitis A was observed and 10% of the patients had serologic markers of hepatitis C virus (HCV) infection. Dual acute hepatitis B and E were observed in four patients (10%) and acute HEV infection was associated with chronic hepatitis B surface antigen carriage in 16 (39%). Epidemiologic findings concerning HBV from Chad suggest that these patients had undiagnosed chronic liver disease due to HBV, with acute deterioration caused by superimposed HEV replication. Moreover, it is obvious that in developing countries only the most severe cases of hepatitis are seen in hospital settings and a large proportion of them are related to superinfection with HBV and HEV. Antibody to HEV was observed in 22% of the control patients. This observation and the fact that epidemic and sporadic cases of HEV are observed in Chad indicates that HEV is highly endemic in this country.

Acute Disease↗

PubMed1997

Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence.

The purpose of this study was to analyze partial nucleotide sequences and derived peptide sequences of hepatitis E virus (HEV) from two outbreaks of hepatitis E in Africa (Chad 1983-1984; Algeria 1978-1980). A portion of ORF3 and the major portion of ORF2 were amplified by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The PCR products were sequenced directly or after cloning into the pCRII vector. Sequences were then compared to the corresponding regions of reported full length HEV sequences. In the ORF2 and ORF3 regions, the homology between the Algerian and the Chad isolates at the nucleic acid level was 92 and 95%, respectively. At the peptide level the homology was 98% in both regions. In these regions, both strains are more related to Asian strains at the nucleic acid level (89 to 95%) and at the amino acid level (95 to 100%) than to the Mexico strain. At the peptide level the differences are less apparent. Both African isolates have amino acid changes in common with some reference strains although the Chad isolate has three unique changes. These African strains of HEV, based on the ORF2 and ORF3 phylogenetic trees, appear to be a distinct phylogenetic group, separate from the Mexican and Asian strains.

Algeria↗

PubMed1997

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles.

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-gamma and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8+ T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.

Animals↗

PubMed1997

Detection of antibodies to hepatitis E virus in patients with autoimmune chronic active hepatitis and primary biliary cirrhosis.

BACKGROUND: Viral hepatitis E (HIV) frequently causes epidemic outbreaks in many developing countries. It is also present in developed countries as imported cases. The role of hepatitis A virus as trigger for autoimmune chronic hepatitis has been demonstrated, and it has been suggested that this may also apply to HEV. METHODS: The presence of anti-HEV antibodies in serum samples from patients with autoimmune chronic active hepatitis (n = 52) and with primary biliary cirrhosis (n = 25) was investigated using an Abbott assay and a peptide-based test. RESULTS: Anti-HEV antibodies were detected with the Abbott test in 13% (7 of 52) of the patients with autoimmune chronic active hepatitis, but not of these were positive in the synthetic peptide-based test. CONCLUSION: These findings indicate the HEV is not associated with primary biliary cirrhosis but may be implicated in some cases of autoimmune chronic active hepatitis.

Autoimmune Diseases↗

PubMed1997

African strains of hepatitis E virus that are distinct from Asian strains.

Partial genomic sequences of four hepatitis E virus (HEV) strains from Africa (Morocco and Tunisia) and one from Central Asia (Tashkent, Uzbekistan) were obtained. The reverse transcriptase-polymerase chain reaction was used to amplify 5' and hypervariable regions of open reading frame 1 (ORF1) and a region overlapping all 3 ORFs. Sequence analysis of these regions revealed the African strains to be quite distinct from all known Asian strains but more similar to them than to the Mexican strain. Sequence analysis of the Tashkent strain revealed almost complete identity with another central Asian strain from Osh, Kirgizia. These results thus further confirm the geographical origin of HEV strain divergence.

Animals↗