Stefano Buttò

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P)

Recent evidence suggests that a CD8-mediated cytotoxic T cell response against the Tat protein of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) controls primary infection after pathogenic virus challenge, and correlates with the status of long-term nonprogressor in humans. Due to the presence of unmethylated CpG sequences, DNA vaccination can boost the innate immunity driving more potent T cell-mediated immune responses. Therefore, cynomolgus monkeys were vaccinated with a tat-expressing vector containing defined unmethylated CpG sequences (pCV-tat). Here it is shown that the intramuscular inoculation of the pCV-tat contained primary infection with the highly pathogenic SHIV89.6P virus preventing the CD4(+) T cell decline in all the vaccinated monkeys. Undetectable virus replication and negative virus isolation correlated in all cases with the presence of anti-Tat CTLs. However, a CD8-mediated non cytolytic antiviral activity was also present in all protected animals. Of note, this activity was absent in the controls but was present in the monkey inoculated with the CpG-rich vector alone that was partially protected against viral challenge (i.e. no virus replication but positive virus isolation). These results suggest that a CTL response against Tat protects against primary infection by blocking virus replication at its early stage, in the absence of sterilizing immunity. Nevertheless, the boost of the innate immunity by CpG sequences can contribute to this protection both by driving more potent CTL responses and by inducing other CD8-mediated antiviral activities. Thus, the CpG-rich tat DNA vaccine may represent a promising candidate for preventive and therapeutic vaccination against AIDS.

Immune activation in Africa is environmentally-driven and is associated with upregulation of CCR5.

BackgroundHIV infection in Africa is associated with immune activation and a cytokine profile that stimulates CCR5 expression. We investigated whether this immune activation is environmentally driven; if a dominant expression of CCR5 could indeed be detected in African individuals; and if R5 HIV strains would be prevalent in this population. MethodsFreshly drawn peripheral blood mononuclear cells from HIV-uninfected African and Italian individuals living in rural Africa, from HIV-uninfected Africans and Italians living in Italy, and from HIV-infected African and Italian patients were analysed. Determinations of HIV coreceptor-specific mRNAs and immunophenotype analyses were performed in all samples. Virological analyses included virus isolation and characterization of plasma neutralizing activity. FindingsResults showed that: immune activation is detected both in Italian and African HIV-uninfected individuals living in Africa but not in African subjects living in Italy; CCR5-specific mRNA is augmented and the surface expression of CCR5 is increased in African compared with Italian residents (CXCR4-specific mRNA is comparable); R5-HIV strains are isolated prevalently from lymphocytes of African HIV-infected patients; and plasma neutralizing activity in HIV-infected African patients is mostly specific for R5 strains. ConclusionsImmune activation in African residents is environmentally driven and not genetically predetermined. This immune activation results in a skewing of the CCR5 : CXCR4 ratio which is associated with a prevalent isolation of R5 viruses. These data suggest that the selection of the predominant virus strain within the population could be influenced by an immunologically driven pattern of HIV co receptor expression.

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.

The Presence of Anti-Tat Antibodies Is Predictive of Long-Term Nonprogression to AIDS or Severe Immunodeficiency: Findings in a Cohort of HIV-1 Seroconverters

The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.

Efficient mucosal delivery of the HIV‐1 Tat protein using the synthetic lipopeptide MALP‐2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell‐mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV‐1 Tat protein as antigen and the synthetic lipopeptide, macrophage‐activating lipopeptide‐2 (MALP‐2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti‐Tat antibody responses, and Tat‐specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freunds adjuvant. Major linear B cell epitopes mapped within aa 1–20 and 46–60, whereas T cell epitopes were identified within aa 36–50 and 56–70. These epitopes have also been described in vaccinated primates and in HIV‐1‐infected individuals with better prognosis. Analysis of the anti‐Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP‐2, as opposed to the Th2 response observed with Tat plus incomplete Freunds adjuvant. Tat‐specific IFN‐γ‐producing cells were significantly increased only in response to Tat plus MALP‐2. These data suggest that Malp‐2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

HIV virology and pathogenetic mechanisms of infection: a brief overview

Studies on HIV virology and pathogenesis address the complex mechanisms that result in the HIV infection of the cell and destruction of the immune system. These studies are focused on both the structure and the replication characteristics of HIV and on the interaction of the virus with the host. Continuous updating of knowledge on structure, variability and replication of HIV, as well as the characteristics of the host immune response, are essential to refine virological and immunological mechanisms associated with the viral infection and allow us to identify key molecules in the virus life cycle that can be important for the design of new diagnostic assays and specific antiviral drugs and vaccines. In this article we review the characteristics of molecular structure, replication and pathogenesis of HIV, with a particular focus on those aspects that are important for the design of diagnostic assays.

SHIV89.6P pathogenicity in cynomolgus monkeys and control of viral replication and disease onset by human immunodeficiency virus type 1 Tat vaccine

The Tat protein of human immunodeficiency virus (HIV) is produced very early after infection, plays a key role in the virus life cycle and in acquired immunodeficiency syndrome (AIDS) pathogenesis, is immunogenic and well conserved among all virus clades. Notably, a Tat‐specific immune response correlates with non‐progression to AIDS. Here, we show that a vaccine based on the Tat protein of HIV blocks primary infection with the simian/human immunodeficiency virus (SHIV)89.6P and prevents the CD4 T cell decline and disease onset in cynomolgus monkeys. No signs of virus replication were found in five out of seven vaccinated macaques for almost 1 year of follow‐up. Since the inoculated virus (derived from rhesus or from cynomolgus macaques) is shown to be highly pathogenic in cynomolgus macaques, the results indicate efficacy of Tat vaccination in protection against highly pathogenic virus challenge. Finally, the studies of the Tat‐specific immunological responses indicate a correlation of protection with a cytotoxic T cell response. Thus, a Tat‐based vaccine is a promising candidate for preventive and therapeutic vaccination in humans.

A seroprevalence study of human herpesvirus type 8 (HHV8) in eastern and Central Africa and in the Mediterranean area

Human herpes virus type 8 (HHV8) is the major determinant of Kaposis sarcoma (KS), a neoplasm with wide geographic variations in incidence rates. To assess the prevalence of HHV8 infection among populations with differing rates of KS, we used sera from 1402 persons (Central Africa: Cameroon, n = 293, age range: 5–40; eastern Africa: Uganda, n = 315, age range: 1–64: Mediterranean area: Egypt, n = 236, age range: 13–19: Italy, blood donors n = 134, age range: 20–67: Italy. HIV seroconverters n = 424, age range: 16–65). Serum samples were tested for antibodies to lytic and latent antigens of HHV8 using two immunofluorescence assays. HHV8 prevalence was evaluated according to geographic area, gender and age groups. Overall, the highest prevalence of HHV8 lytic antigens (47.5%) was recorded among children and adults in Africa. Approximately 40% of children and adolescents from Egypt and of Italian HIV-positive persons (39.9%) were HHV8 seropositive. In eastern and Central Africa and in Egypt, no differences emerged between males and females for both types of HHV8 antibodies. Conversely, Italian females were at lower HHV8 risk than their male counterparts. Moreover the prevalence of HHV8 infection tended to increase with age. This investigation partially confirms that HHV8 infection mirrors incidence rates of KS. The high prevalence of HHV8 infection in newborns, children and adolescents in Egypt, in eastern and in Central Africa strongly suggests the existence of transmission modes other than sexual.

Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

ABSTRACT Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 106 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.