Massimo Giuliani

Prevalence and determinants of anti‐lytic and anti‐latent antibodies to human herpesvirus‐8 among Italian individuals at risk of sexually and parenterally transmitted infections

Three hundred seventy‐nine individuals [137 non‐injecting drug using (non‐IDU) heterosexuals, 130 homosexual men and 112 IDU] attending the human immunodeficiency virus (HIV) testing program of a sexually transmitted disease (STD) clinic in Rome were studied to estimate the prevalence and to identify the modalities of transmission of human herpesvirus‐8 (HHV‐8) infection. Serological analysis was performed by using an immunofluorescence assay able to detect anti‐latent and anti‐lytic HHV‐8 antibodies. Twelve acquired immunodeficiency syndrome (AIDS)‐Kaposis sarcoma (KS) patients and 94 blood donors were tested as reference population groups. Anti‐lytic antibodies were detected in 185 (48.8%) individuals; 52 of them (13.7%) also had anti‐latent antibodies. Both anti‐lytic and anti‐latent antibody prevalence were higher among homosexual men (66.9% and 27.7%, respectively) than among IDU (49.1% and 8.0%, respectively) and non‐IDU heterosexuals (31.4% and 5.1%, respectively), and tended to increase with age. Anti‐lytic HHV‐8 antibodies were associated with syphilis [odds ratio (OR) = 3.81] but not with hepatitis C virus (HCV) seropositivity. HIV‐infected homosexual men were more likely to have HHV‐8 antibodies than those who were HIV‐negative. When using anti‐latent antibodies the direction of the OR remained the same, although the associations did not often reach statistical significance. Among AIDS‐KS patients, 83.3% had anti‐lytic and 66.6% had anti‐latent antibodies. Among blood donors, 28% had anti‐lytic antibodies and 2 of them (2.1%) also had anti‐latent antibodies. Our data indicate that HHV‐8 seroprevalence increases with age and is higher among homosexual men, particularly those infected with HIV. This is consistent with sexual transmission of HHV‐8 infection. In addition, the presence of HHV‐8 antibodies in HIV‐negative non‐IDU heterosexual contacts and in healthy blood donors is consistent with the high incidence of classic KS in Italy. Int. J. Cancer. 77:361–365, 1998.

Analysis of the ORFK1 hypervariable regions reveal distinct HHV-8 clustering in Kaposi’s sarcoma and non-Kaposi’s cases

BackgroundClassical Kaposi’s Sarcoma (cKS) is a rare vascular tumor, which develops in subjects infected with Human Herpesvirus-8 (HHV-8). Beside the host predisposing factors, viral genetic variants might possibly be related to disease development. The aim of this study was to identify HHV-8 variants in patients with cKS or in HHV-8 infected subjects either asymptomatic or with cKS-unrelated cutaneous lymphoproliferative disorders.MethodsThe VR1 and VR2 regions of the ORF K1 sequence were analyzed in samples (peripheral blood and/or lesional tissue) collected between 2000 and 2010 from 27 subjects with HHV-8 infection, established by the presence of anti-HHV-8 antibodies. On the basis of viral genotyping, a phylogenetic analysis and a time-scaled evaluation were performed.ResultsTwo main clades of HHV-8, corresponding to A and C subtypes, were identified. Moreover, for each subtype, two main clusters were found distinctively associated to cKS or non-cKS subjects. Selective pressure analysis showed twelve sites of the K1 coding gene (VR1 and VR2 regions) under positive selective pressure and one site under negative pressure.ConclusionThus, present data suggest that HHV-8 genetic variants may influence the susceptibility to cKS in individuals with HHV-8 infection.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposis sarcoma (KS), primary effusion lymphoma and Castelmans disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.

Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

Prevalence, genotype diversity and determinants of anal HPV infection in HIV-uninfected men having sex with men.

BACKGROUND Anal human papillomavirus (HPV) infection is very common in men having sex with men (MSM), but the available data on its burden and characteristics mainly concern HIV-infected individuals. OBJECTIVES This study aimed to assess the prevalence, spectrum of genotypes, and determinants of the anal HPV infection in metropolitan HIV-1 uninfected MSM. STUDY DESIGN A cohort of 258 MSM (median age 32 years, IQR 26-39) enrolled at an STI Clinic was screened for anal HPV infection using a highly sensitive PCR-based genotyping method. Medical history and behavioral data were collected. RESULTS Overall, 74.8% of the MSM were HPV-positive, with 56.2% of the participants being infected by high-risk (HR) types. A multiple infection was detected in 65.3% of the HPV-positive MSM, with up to 10 different HPV types detected in the same sample. A broad spectrum of infecting HPV types was observed, with 36 different types found overall and HPV16 representing the most common type (17.8%). The lifetime and recent number of sexual partners as well as having receptive anal sex were significantly associated with the anal HPV infection, confirming the role of sexual behavior in risk of HPV infection. However, neither younger age at first intercourse nor inconsistent use of condom was significantly associated with the infection. CONCLUSIONS The present findings highlight the need to create a more significant awareness about the risk of anal HPV infection among HIV-uninfected MSM and warrant the investigation of possible anal intraepithelial lesions, particularly in view of the increasing anal cancer incidence in high-risk populations.

The preventive phase I trial with the HIV-1 Tat-based vaccine.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

HIV infection increases the risk of squamous intra‐epithelial lesions in women with HPV infection: An analysis of HPV genotypes

We assessed the association between different HPV genotypes, HIV infection, and cervical squamous intra‐epithelial lesions (SIL) in 236 women with known HIV serostatus enrolled in a longitudinal multicentric study in Italy. Of these women, 135 were HIV‐infected, and were not markedly different from HIV‐negative women with regard to demographic characteristics, sexual practices, smoking, or intravenous drug use. We obtained 232 cervical smears suitable for cytological examination and HPV‐genotype analysis (134 from HIV‐positive women and 98 from HIV‐negative women). For 86 HIV‐positive and 89 HIV‐negative women, the smears appeared normal at cytomorphological analysis. Cytological dysplasia of varying degrees was detected in 48 smears from HIV‐positive women and in 9 from HIV‐negative women. HPV prevalence, assessed using polymerase‐chain‐reaction analysis, did not significantly differ between HIV‐positive and HIV‐negative women. The prevalence of HPV‐associated SIL was much greater among HIV‐infected women. The most frequently detected genotypes in both groups were HPV 16 and HPV 18. The prevalence of HPV 16 among HIV‐positive women was similar to that for HIV‐negative women; this was also true for HPV 18. However, in the HIV‐positive group, most of these genotypes were associated with SIL. HIV‐positive women showed a wider spectrum of genotypes, including non‐oncogenic and rare types. An association between SIL and HIV infection was confirmed for all HPV genotype classes. Int. J. Cancer 72:982–986, 1997.

p16/Ki-67 dual staining in cervico-vaginal cytology: Correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy

OBJECTIVES To evaluate the CINtec PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16(INK4a) and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities. METHODS 140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec PLUS kit and the morphology assessment. RESULTS Cytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84-31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39-47.77 vs COR=4.20, 95% CI: 0.99-20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16-29.12). CONCLUSIONS This preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion.

Population dynamics of HIV-1 subtype B in a cohort of men-having-sex-with- men in Rome, Italy

A recent increase in HIV diagnoses among men-having-sex-with-men (MSM) has been shown by surveillance data from Europe and Italy, and new approaches to inferring viral population dynamics from heterochronously sampled gene sequences have been developed. The aim of this study was to reconstruct the epidemiological history of HIV-1 subtype B in a homogeneous group of Italian MSM using a coalescent-based Bayesian framework. A total of 125 HIV-1 subtype B pol sequences were analyzed using Bayesian methods and a relaxed molecular clock to reconstruct their dated phylogeny and estimate population dynamics. At least 10 epidemiological clusters of 3-9 isolates were identified: half including the largest clades originated in the early 1990s and the other half radiated from 1999. Demographic analysis showed that the HIV epidemic grew in accordance with a logistic model characterized by a rapid exponential increase in the effective number of infections (r = 1.54 year−1) starting from the early 1980s and reaching a plateau 10 years later. Our data suggest that the HIV B epidemic entered our MSM population through multiple transmission chains about 20 years later than in other Western European country. Epidemiological clusters originating in the early 2000s suggest a recent re-emergence of HIV in Italian MSM.

Ageing with HIV: newly diagnosed older adults in Italy

Abstract The prevalence of HIV/AIDS among people in midlife and late adulthood has been increasing in Western countries over the last decade. We analyzed data from a prospective, observational multi-centre study on individuals newly diagnosed with HIV between January 2004 and March 2007 in 10 public counselling and testing sites in Latium, Italy. At diagnosis, routine demographic, epidemiological, clinical and laboratory data are recorded, and patients are asked to complete a questionnaire investigating socio-demographic and psycho-behavioural aspects. To analyze the association of individual characteristics with age, we compared older adults (≥50 years) with their younger counterpart (18–49 years). To adjust for potential confounding effect of the epidemiological, clinical and behavioural characteristics, to identify factors associated with older age at HIV diagnosis, multivariate logistic regression analysis was performed. Overall, 1073 individuals were identified, 125 of whom (11.6%) were aged 50 years or above. The questionnaire was completed by 41% (440/1073). Compared with their younger counterparts, a higher proportion of older patients were males, born in Italy, reported heterosexual or unknown HIV risk exposure, were never tested for HIV before and were in a more advanced stage of HIV infection at diagnosis. In addition, older adults had a lower educational level and were more frequently living with their partners or children. With respect to psycho-behavioural characteristics, older patients were more likely to have paid money for sex and have never used recreational drugs. Interestingly, no differences were found regarding condom use, which was poor in both age groups. These findings may have important implications for the management of older adults with HIV, who should be targeted by appropriate public health actions, such as opportunistic screening and easier access to healthcare. Moreover, strategies including information on HIV and prevention of risk behaviours are needed.