Leonardo Sernicola

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.

IRF-1 Is Required for Full NF-κB Transcriptional Activity at the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Enhancer

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-κB transcriptional activity. We provide evidence that IRF-1 and NF-κB form a functional complex at the long terminal repeat (LTR) κB sites, which is abolished by specific mutations in the two adjacent κB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-κB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone‐induced differentiation have been evaluated for the first time in 3T3‐L1 preadipocytes. Unlike control cells, oxLDL‐treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor‐1 (Pref‐1) mRNA expression at late times. By silencing Pref‐1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref‐1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref‐1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.

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.

HIV-1 Tat-Based Vaccines: From Basic Science to Clinical Trials

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.

Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.

The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4+ and CD8+ cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccinated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4+ and CD8+ cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-gamma and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.

Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes

The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator‐activated receptor γ‐independent CD36 overexpression, by up‐regulating nuclear factor erythroid 2 (NF‐E2)‐related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up‐regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin‐resistance, data herein presented highlight the significance of oxLDL‐induced CD36 overexpression within the cellular defence response.

HIV-1 Tat Promotes Integrin-Mediated HIV Transmission to Dendritic Cells by Binding Env Spikes and Competes Neutralization by Anti-HIV Antibodies

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study

HIV native Tat and V2 loop-deleted Env (EnvΔV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvΔV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID(50) (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIV(SF162P4cy)). Upon challenge, three of four macaques immunized with Tat and EnvΔV2, and two of three monkeys immunized with EnvΔV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvΔV2 or Tat/EnvΔV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvΔV2 alone, the Tat and EnvΔV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvΔV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvΔV2 approached the immune response assessed after two inocula with the Tat/EnvΔV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvΔV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvΔV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvΔV2 or Tat/EnvΔV2 vaccination groups in this pilot study, vaccination with Tat/EnvΔV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvΔV2 as compared to EnvΔV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvΔV2 vaccine may afford superior protection against infection.