Eric Saman

most env and gag subtype A Hiv-1 Viruses Circulating in West and West Central Africa Are Similar to the Prototype Ag Recombinant Virus Ibng

Summary: The genetic subtype was identified in gag and env of 219 HIV‐1‐positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG‐IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full‐length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG‐IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG‐IBNG‐like viruses is necessary.

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.

IDENTIFICATION AND HETEROLOGOUS EXPRESSION OF A NEW DENSE GRANULE PROTEIN (GRA7) FROM TOXOPLASMA GONDII

Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the dense granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.

Immunogenicity of recombinant BCG producing the GRA1 antigen from Toxoplasma gondii

Toxoplasmosis is a major parasitic disease, responsible for foetopathy in humans and domestic animals, especially sheep. Toxoplasma gondii infection generally protects immunocompetent hosts against subsequent reinfection, suggesting that efficacious vaccines can be developed against this disease. Excreted/secreted T. gondii antigens have previously been shown to provide immunoprotection in small rodents, and protective immunity is thought to be cell-mediated. Mycobacterium bovis BCG is known to be a good inducer of cellular immunity. In this study, we have developed a BCG strain which produces and secretes GRA1, one of the major excreted/secreted T. gondii antigens. This strain does not carry antibiotic-resistance determinants and is therefore safe for the environment. The intraperitoneal immunisation of OF1 outbred mice with this BCG strain failed to induce GRA1-specific humoral or cellular immune responses and only conferred a very limited degree of protection against challenge with virulent T. gondii. However, in sheep immunised subcutaneously and boosted intravenously, this recombinant BCG strain induced GRA1-specific cell-mediated responses, as evidenced by the proliferation of peripheral blood mononuclear cells and by the production of IFN-gamma, although it failed to elicit GRA1-specific antibody responses. Following oocyst challenge infection, sheep immunised with recombinant BCG exhibited an abbreviated temperature response compared with controls, suggesting partial protection.

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (κ value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animals life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

Sequence Note The Identification of a Complex A/G/I/J Recombinant HIV Type 1 Virus in Various West African Countries

In this sequence note we describe the full-length genome sequence of an HIV-1 isolate originating from the west African country of Mali. The phylogenetic tree analysis from the near full-length genome shows that the 95ML84 strain forms a separate cluster, supported by 100% of the bootstrap values, with the previously described A/G/J/? mosaic virus BFP90 from Burkina Faso. Additional analysis showed that throughout the genome the lowest diversity was seen between the 95ML84 and the BFP90 viruses, and bootscan analysis showed a similar complex genomic structure. In addition to the initial report describing the BFP90 virus as an A/G/J/? recombinant, our data show that for the BFP90 and 95ML84 strains the unclassified region corresponds to subtype I. The A/G/I/J BFP90 and 95ML84 strains represent the fifth and most complex circulating recombinant form of HIV-1 detected so far, and our data show its presence in various West African countries. Subtype I and J sequences, initially considered rare, seem to have broadened their geographical spread by way of these recombinant forms.

Redistribution of a murine humoral immune response following removal of an immunodominant B cell epitope from a recombinant fusion protein

Immunization of different mice strains with a recombinant fusion protein composed of the vector-encoded N-terminal leader peptide CroLac (containing lambda Cro and LacI fragments) and a part of the transmembrane protein of HIV-1 (gp41) led to a high anti-CroLac humoral immune response. A detailed analysis of this response revealed the presence of an immunodominant, linear B cell epitope localized near the C-terminus of the CroLac fragment. The immune response seemed to be biased towards this epitope since few or no monoclonal antibodies (mAb) could be generated against the remaining part of CroLac and the gp41 fragment. Upon removal of the immunodominant region from the fusion protein the immune response was redirected and spread over the previously non-immunogenic regions. Consequently, we report a model system in which an immunodominant B cell epitope biases the immune response away from less immunogenic epitopes on the same molecule.

Characterization and downstream processing of HIV-1 core and virus-like-particles produced in serum free medium

Abstract This work aimed at the characterisation and downstream processing of HIV-1 core and virus-like particles (CLPs and VLPs) produced in serum free medium. The produced core and virus-like particles have similar characteristics to those of the native immature HIV-1 virus in terms of protein composition, diameter, density and shape. The molecular mass (determined by gel filtration chromatography) and the diameter (determined by electron microscopy) were similar for CLPs and VLPs, this being correlated with the difference between the particle densities (1.14 g/cm 3 and 1.19 g/cm 3 , respectively, for CLPs and VLPs). After this characterization, several concentration and purification methods were tested to obtain enough material for further tests. For this purpose, a downstream processing strategy was developed, involving clarification by centrifugation and microfiltration, concentration and partial purification by ultrafiltration, and final purification by gel filtration chromatography. Quality analysis using Western immunoblot and gel filtration chromatography was performed to define the best operational conditions for each purification step. The particle recovery obtained was 87% with the main losses occurring in the ultrafiltration step. In addition, by evaluating the effect of operational conditions on product quality, it was possible to obtain a final product with high content of intact high molecular weight particles.

The identification of a complex A/G/I/J recombinant HIV-1 virus in different West African countries.

In this sequence note we describe the full-length genome sequence of an HIV-1 isolate originating from the west African country of Mali. The phylogenetic tree analysis from the near full-length genome shows that the 95ML84 strain forms a separate cluster, supported by 100% of the bootstrap values, with the previously described A/G/J/? mosaic virus BFP90 from Burkina Faso. Additional analysis showed that throughout the genome the lowest diversity was seen between the 95ML84 and the BFP90 viruses, and bootscan analysis showed a similar complex genomic structure. In addition to the initial report describing the BFP90 virus as an A/G/J/? recombinant, our data show that for the BFP90 and 95ML84 strains the unclassified region corresponds to subtype I. The A/G/I/J BFP90 and 95ML84 strains represent the fifth and most complex circulating recombinant form of HIV-1 detected so far, and our data show its presence in various West African countries. Subtype I and J sequences, initially considered rare, seem to have broadened their geographical spread by way of these recombinant forms.

PRESENCE OF HIV-1 GROUP O INFECTION IN WEST AFRICA

Two aberrant HIV-1 strains have been isolated from Cameroonian patients. The isolates had only 50% homology in the envelope region with other HIV-1 isolates and were thus classified as group O. Since then additional HIV-1 group O variants from Cameroonians living in France have been described and some of the group O sera have been shown to be unreactive in some commercial screening assays and can give indeterminate Western blot patterns. Little is known on the spread of HIV-1 group O viruses in Africa. Studying their spread is therefore very important in order to identify whether strategies for blood screening and serodiagnosis need to be modified. The authors present their serological evidence that group O infection is also present in Senegal Niger and Togo albeit to a very small degree.