High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A.

The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.

The uganda study on HPV variants and genital cancers.

BACKGROUND: Genital cancers in Uganda have been the most frequently diagnosed cancer in men as well as in women since the 1950s. Genetic studies have detected HPV-16 variants of Af1 class and identified a new sub-class designated Af1-u. OBJECTIVES: The main goal of this study is to analyze the prevalence of HPV strains and HPV variants in anogenital lesions of Ugandan male and female subjects in order to possibly determine their role in the pathogenesis of such lesions and to develop an Ugandan preventive HPV vaccine program. STUDY DESIGN: The study is planning to enroll male and female subjects affected by genital lesions, in particular to collect 200 scrapes/biopsies from women with normal ectocervical epithelium as well as with all different degrees of ectocervical lesions (from CIN 1/LSIL to cervical carcinoma). All samples are analyzed by PCR amplification of the L1 conserved region (nt 6584-7035) and the E6/E7 genes (nt 34-880), nucleotide sequence analysis, homology and phylogenetic studies. Variant distribution studies will be followed by serological studies of prevalence and incidence in 1000 women. PRELIMINARY RESULTS AND CONCLUSIONS: Penile cancers from the Kyadondo County have been analyzed for the presence of HPV sequences. More recently 16 ectocervical scrapes and three biopsies have been received from women attending the Nsambya Hospital and analyzed for the presence and type of HPVs. Our results, obtained by PCR and sequencing analysis, allowed the identification of HPV-16 Af1 sequences in 100% of tumor tissue and in 6.25% of scrapes. HPV 45 was identified only in one tumor together with HPV 16 infection. HPV 33 and HPV 58 were present in 20% and 40%, respectively of HPV positive benign samples. The results are showing a narrowing of the HPV pattern in more advanced lesions, suggesting that mainly HPV-16 Af1 patients are progressing to cancer.

The role of immunosuppression and immune-activation in classic Kaposi's sarcoma.

Immunodeficiency and elevated levels of cytokines have been associated with the development of Kaposi's sarcoma (KS) lesions in patients with AIDS and iatrogenic immunodeficiency. However, their role in classic KS (CKS) is unclear. We measured peripheral blood cell levels, including T-cell subsets, as well as neopterin and beta(2)-microglobulin in 91 HIV-negative Greek patients with histologically confirmed CKS and in 107 controls matched for age and sex. CKS cases had slightly lower leukocyte counts (p = 0.08) and lymphocyte counts (p = 0.02). Although the percentage of CD4 and CD8 T-lymphocytes were not significantly different from controls (p = 0.10 and p = 0.45, respectively), CD4 T-lymphocytes were lower in cases than controls (812 cells/microliter and 1,009 cells/microliter, respectively; p = 0.01); part of this difference resulted from the lower lymphocyte counts (p = 0.07 after adjusting for lymphocyte counts). However, neopterin and beta(2)-microglobulin were both considerably elevated [geometric mean (95% CI): 8.35 (7.27-9.73) nmol/L and 2,904 (2,479-3,401) microgram/L in cases and 5.86 (5.40-6. 35) nmol/L and 2,042 (1,880-2,218) microgram/L in controls, respectively]. We conclude that CKS patients are predominantly characterised by immune activation, although an element of minor immunosupression may also be present.

Sequence variations and viral genomic state of human papillomavirus type 16 in penile carcinomas from Ugandan patients.

Sequence variations in the E6/E7 (nt 34-880) and the L1-(nt 6584-7035) ORFs, and in the long control region (LCR) (nt 7289-93) of human papillomavirus type 16 (HPV-16) were analysed in five penile carcinoma biopsies obtained from Ugandan patients. Uganda is a country with a high incidence of genital cancers. All five isolates were classified as members of African-1 lineage (Af1) by phylogenetic analysis based on LCR sequences. The E6 gene phylogenetic analysis, however, showed that four isolates fell into a new subclass designated Af1-u. This subclass, characterized by three point mutations located at the 5' end of the E6 gene with resulting changes in amino acids at positions 10 and 14, is distinguishable from the Af1 class by the absence of synonymous mutations at nt 286 and 289. The nonsynonymous substitution at nt 335 was present in three out of five samples. The E6 Af1 mutation pattern was present in only a single Ugandan HPV-16 isolate. Nucleotide sequence analysis of the E7 and L1 regions did not allow any Af1 subclass identification. The physical state of the viral DNA in these samples was characterized by PCR and Southern blot analysis. Oligonucleotides which enable amplification of the full length E2 region (nt 2734-3872) failed to amplify the target sequence in four out of five samples, suggesting disruption of the E2 ORF and integration of the HPV genome into the human DNA. Southern blot analysis confirmed the virus integration status. Our results contribute to the characterization of the HPV-16 'African lineages' with the identification of the Af1-u subclass; furthermore, this is also the first report showing that in male genital cancers HPV-16 is integrated into the human genome with disruption of the E2 ORF.

Herpesvirus-like DNA sequences detected in endemic, classic, iatrogenic and epidemic Kaposi's sarcoma (KS) biopsies.

The identification of Kaposi's sarcoma (KS) clusters in sub-equatorial Africa (endemic KS, AKS) and the high frequency of KS in sexually transmitted AIDS (epidemic KS, EKS), have previously suggested a role for infectious agents in the etiopathogenesis of KS. The recent identification of herpesvirus (HHV)-like DNA sequences in one case of EKS and their detection in > 90% of all tested EKS, prompted us to determine the prevalence of these viral sequences in all types of KS, such as AKS, EKS, classic KS (CKS) and iatrogenic KS (IKS). The presence of herpesvirus(HHV)-like DNA sequences has been examined in 61 KS skin tumors obtained from Greece, Italy, USA, Uganda and Kenya. All KS types (100%) were positive by polymerase chain reaction (PCR) and Southern-blot analysis, while 5 out of 6 (83%) and 4 out of 7 (57%) uninvolved autologous skin biopsies from AKS and CKS patients, respectively, were positive for HHV-like sequences. All samples from non-KS patients were negative, i.e. 17 human biopsies from healthy individuals or patients affected by other pathologies, 5 human cell lines and 15 peripheral blood mononuclear cells (PBMC) from HIV-positive subjects. These results suggest that HHV-like sequences play a major role in the pathogenesis of this neoplasm.

Heteroduplex mobility assay and phylogenetic analysis of V3 region sequences of human immunodeficiency virus type 1 isolates from Gulu, northern Uganda. The Italian-Ugandan Cooperation AIDS Program.

We analyzed peripheral blood mononuclear cells from 19 asymptomatic seropositive pregnant women from the district of Gulu in northern Uganda. A 700-bp fragment of the human immunodeficiency virus type 1 (HIV-1) env gene, including the V3-V5 region, was successfully amplified by PCR from 10 samples (52.6%) and was subsequently subjected to both a heteroduplex mobility assay for genetic screening and subtyping and DNA sequence analysis (approximately 300 bp) for nucleotide comparison and phylogenetic studies. The results show the presence of HIV-1 A and D subtypes (or clades) in this rural area, with the prevalence of the A subtype (8 of 10) being greater than that of the D (2 of 10) subtype, which is unlike what was previously reported for Uganda. By pairwise comparison analysis, the percentage of sequence divergence among samples within each subtype is low (the average intrasubtype divergence is 15.8%), but it is significantly higher between the two subtypes (the average intersubtype divergence is 23%). At the amino acid level, the two HIV-1 subtypes show distinct tetramers at the apex of the V3 loop and, in particular, GPGQ in clade A and GPGR in clade D. In addition, 10 of the 19 viral samples (52.6%) have been isolated in vitro. Nine of the samples have been classified as rapid/high, which reflects a high in vitro replication capacity for the HIV-1 field isolates from this country, even for those obtained from seropositive asymptomatic individuals. These observations, despite being made on the basis of a limited sample size, show a modest degree of genetic divergence among samples isolated in the last 4 years in this country by comparison with those based on the 1990 data on HIV-1 isolates from Kampala. The results reported here are, therefore, extremely relevant for Uganda, which is one of the selected World Health Organization field sites for future HIV-1 vaccine evaluation programs.

Human immunodeficiency virus type 1 tat gene enhances human papillomavirus early gene expression.

To investigate the possible direct/indirect role of Human immunodeficiency virus (HIV) as a cofactor in human papillomavirus (HPV) oncogenesis, cotransfection experiments were carried out in which a recombinant plasmid containing the HPV16 long control region (LCR) linked to the chloramphenicol acetyltransferase (CAT) gene was cotransfected into cultured cells with a plasmid expressing HIV-1 Tat protein. Tat expression efficiency and transactivation activity were evaluated in different cell lines by cotransfecting plasmids containing the HIV tat gene and HIV LTR-driven CAT-coding sequences. HeLa and CaSki cell lines represented the most appropriate recipient cells for Tat-directed transactivation of both the HIV LTR and the HPV LCR promoters. Furthermore, HIV tat was transfected into HeLa cells (containing 10-20 copies per cell of HPV18), and HPV18 E7 protein expression was evaluated by a radioimmunoprecipitation assay using polyclonal antibodies against the E7 protein. Our results show that the Tat protein can transactivate the HPV LCR and increase HPV18 E7 expression in HeLa cells.

Human papillomavirus (HPV) DNA in penile carcinomas and in two cell lines from high-incidence areas for genital cancers in Africa.

Biopsies of 13 penile cancers (PC), from patients living in regions of Uganda with a high incidence of genital cancers, were studied for the presence, molecular characteristics and physical state of DNA related to that of human papillomavirus (HPV) types 6, 11, 16, 18, 31 and 33. HPV DNA sequences were detected in all PC specimens by dot/Southern blot analyses and by gene amplification of DNA sequences highly conserved among several HPVs. HPV 16 DNA sequences were found in one PC; DNA sequences with low homology to HPV16 or HPV18 were present in all other samples. Viral DNA is primarily integrated in the cellular DNA. To isolate and characterize a possible highly oncogenic HPV, a genomic library of the DNA extracted from the PC-8 biopsy has been constructed in the EcoRI arms of the EMBL4 phage. A single phage containing 8.30-kb HPV16-related sequences has been identified and the 3 segments of 0.45, 0.65 and 7.2 kb, released by EcoRI digestion, have been independently subcloned in pUC18 for further analysis.

Human papillomavirus (HPV) DNA in penile carcinomas and in two cell lines from high-incidence areas for genital cancers in Africa

Biopsies of 13 penile cancers (PC); from patients living in regions of Uganda with a high incidence of genital cancers; were studied for the presence; molecular characteristics and physical state of DNA related to that of human papillomavirus (HPV) types 6; 11; 16; 18; 31 and 33. HPV DNA sequences were detected in all PC specimens by dot/Southern blot analyses and by gene amplification of DNA sequences highly conserved among several HPVs. HPV 16 DNA sequences were found in one PC; DNA sequences with low homology to HPV16 or HPV18 were present in all other samples. Viral DNA is primarily integrated in the cellular DNA. To isolate and characterize a possible highly oncogenic HPV; a genomic library of the DNA extracted from the PC-8 biopsy has been constructed in the EcoRI arms of the EMBL4 phage. A single phage containing 8.30-kb HPV16-related sequences has been identified and the 3 segments of 0.45; 0.65 and 7.2 kb; released by EcoRI digestion; have been independently subcloned in pUC18 for further analysis

Neopterin and alpha-interferon in patients affected by Kaposi's sarcoma from Africa.

The presence of circulating alpha-interferon and neopterin was investigated in sera of 47 patients affected by African Kaposi's sarcoma, both HIV-seropositive (13 patients) and HIV-seronegative (34 patients). For comparison, analyses were also performed in 20 HIV-seropositive symptomatic African subjects as well as in 20 African and 20 Italian healthy individuals. Alpha-interferon and neopterin levels appeared significantly higher in comparison with healthy control groups (P less than 0.001) but not with HIV-seropositve African individuals without Kaposi's sarcoma. Moreover, alpha-interferon and neopterin levels were significantly higher in progressive Kaposi's sarcoma (27 patients) than in regressive Kaposi's sarcoma (20 patients) (P less than 0.001). A significant correlation between alpha-interferon and neopterin was observed (r = 0.57; P less than 0.01). Furthermore, alpha-interferon levels of HIV-seropositive Kaposi's sarcoma patients resulted significantly higher in comparison with the seronegative ones (P less than 0.05). It is concluded that alpha-interferon and neopterin may be reliable prognostic markers in Kaposi's sarcoma patients.

The role of opportunistic viruses in Kaposi's sarcoma (KS) evolution.

Etiopathogenetic mechanisms involved in the development of Kaposi's sarcoma and the possible role of viruses, such as CMV, BKV, HBV, HHV-6 and retroviruses are reviewed. An etiopathogenetic model is also presented.

Urinary neopterin and immunological features in patients with Kaposi's sarcoma.

We studied neopterin excretion levels and immunological features of 20 patients affected by Kaposi's sarcoma (KS), compared to 30 normal controls. Eighteen patients had the classic form of Kaposi's sarcoma (CKS), while two patients were anti-human immunodeficiency virus (HIV) seropositive and affected by the epidemic form associated with the acquired immunodeficiency syndrome (AIDS). In CKS patients, a trend of an increase of neopterin levels with more advanced stages appeared from our data whereas a significant reduction in CD3+ and CD4+ lymphocytes subsets was observed already at early stages (P less than 0.01). CD8+ cells did not show significant variations. A significant increase in serum IgA immunoglobulins (P less than 0.05) was also observed. Comparative analysis of the two patients affected by AIDS/KS showed the profound deficit in T-cell immunity but also the prognostic value of neopterin monitoring. Furthermore these findings seem to confirm Kaposi's sarcoma as an 'opportunistic neoplasia' and indicate neopterin as a useful prognostic marker.

Cytogenetic study of a cell line of human penile cancer.

A cell line of penile cancer from a 60-year-old Ugandan black patient has been studied by the authors. Transmission and scanning electron microscopy showed a large number of blebs and microvilli at cell surface; desmosomes were evident at TEM. Cytogenetic investigation (R-, C-, Nor-banding) showed the frequent presence of some markers: del(1p),del(1q),iso(3q),der(4),del(8p),11q+, t rob(13;14), 14p+, t rob(21;21). The epidemiology, geographical distribution, and aetiological role of human papilloma virus type 16 and herpes simplex type 2 are discussed.