Impact of viral dose and major histocompatibility complex class IB haplotype on viral outcome in mauritian cynomolgus monkeys vaccinated with Tat upon challenge with simian/human immunodeficiency virus SHIV89.6P.

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P(cy243.) In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID(50)]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID(50) (P < 0.0001), and contained acute CD4 loss at 15 MID(50) (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.

HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection.

Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.

Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy.

BACKGROUND: In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class. RESULTS: In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy. CONCLUSIONS: In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.

International multicenter study to assess a panel of reference materials for quantification of simian immunodeficiency virus RNA in plasma.

An international multicenter study was conducted to assess the performance of a panel of simian immunodeficiency virus (SIV) RNA reference materials for plasma viral load determinations. Reliable quantification was demonstrated across an approximately 6 log(10) dynamic range. Availability of external reference materials will enable independent calibration of SIV plasma viral load assays.

Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5alpha.

Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.

Mhc haplotype H6 is associated with sustained control of SIVmac251 infection in Mauritian cynomolgus macaques.

The restricted diversity of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques provides powerful opportunities for insight into host-viral interactions and cellular immune responses that restrict lentiviral infections. However, little is known about the effects of Mhc haplotypes on control of SIV in this species. Using microsatellite-based genotyping and allele-specific PCR, Mhc haplotypes were deduced for 35 macaques infected with the same stock of SIVmac251. Class I haplotype H6 was associated with a reduction in chronic phase viraemia (p = 0.0145) while a similar association was observed for H6 class II (p = 0.0063). An increase in chronic phase viraemia, albeit an insignificant trend, was observed in haplotype H5-positive animals. These results further emphasise the value of genetically defined populations of non-human primates in AIDS research and provide a foundation for detailed characterisation of MHC restricted cellular immune responses and the effects of host genetics on SIV replication in cynomolgus macaques.

Innovative approaches to develop prophylactic and therapeutic vaccines against HIV/AIDS.

The acquired immunodeficiency syndrome (AIDS) emerged in the human population in the summer of 1981. According to the latest United Nations estimates, worldwide over 33 million people are infected with human immunodeficiency virus (HIV) and the prevalence rates continue to rise globally. To control the alarming spread of HIV, an urgent need exists for developing a safe and effective vaccine that prevents individuals from becoming infected or progressing to disease. To be effective, an HIV/AIDS vaccine should induce broad and long-lasting humoral and cellular immune responses, at both mucosal and systemic level. However, the nature of protective immune responses remains largely elusive and this represents one of the major roadblocks preventing the development of an effective vaccine. Here we summarize our present understanding of the factors responsible for resistance to infection or control of progression to disease in human and monkey that may be relevant to vaccine development and briefly review recent approaches which are currently being tested in clinical trials. Finally, the rationale and the current status of novel strategies based on nonstructural HIV-1 proteins, such as Tat, Nef and Rev, used alone or in combination with modified structural HIV-1 Env proteins are discussed.

T cell receptor excision circles (TRECs) analysis during acute intrarectal infection of cynomolgus monkeys with pathogenic chimeric simian human immunodeficiency virus.

Several studies have shown the importance of evaluating Recent Thymic Emigrants (RTEs) by quantification of T cell receptor-rearrangement excision circles (TRECs), as a measure of de novo T cell generation during human immunodeficiency virus-1 (HIV-1) infection. To determine whether acute viral infection may have an impact on TRECs, cynomolgus monkeys (Macaca fascicularis) were infected intrarectally with simian human immunodeficiency virus (SHIV) 89.6P(cy11) and the number of signal-joint (sj) TRECs was determined in purified CD4+ and CD8+ populations for up to 28 weeks post-infection. Four weeks after infection, TRECs levels significantly decreased in both CD3+ CD4+ and in CD3+ CD8+ T lymphocytes of infected monkeys, whereas they remained unchanged in uninfected animals. This reduction was followed by a progressive TRECs number recovery in CD3+ CD4+ T lymphocytes that positively correlated with changes in the levels of circulating CD3+ CD4+ T cells. In the CD3+ CD8+ T cell subset, TRECs number remained significantly low and inversely correlated with the increase in the percentages of CD3+ CD8+ T cells. These data suggest that SHIV89.6P(cy11) intrarectal infection of cynomolgus monkeys differently affects TRECs content in CD3+ CD4+ and CD3+ CD8+ T cell subsets.

Problems and emerging approaches in HIV/AIDS vaccine development.

According to recent estimates, 39.5 million people have been infected with HIV and 2.9 million have already died. The effect of HIV infection on individuals and communities is socially and economically devastating. Although antiretroviral drugs have had a dramatically beneficial impact on HIV-infected individuals who have access to treatment, it has had a negligible impact on the global epidemic. Therefore, the need for an efficacious HIV/AIDS vaccine remains the highest priority of the world HIV/AIDS agenda. The generation of a vaccine against HIV/AIDS has turned out to be extremely challenging, as indicated by > 20 years of unsuccessful attempts. This review discusses the major challenges in the field and key experimental evidence providing a rationale for the use of non-structural HIV proteins, such as Rev, Tat and Nef, either in the native form or expressed by viral vectors such as a replicating adeno-vector. These non-structural proteins alone or in combination with modified structural HIV-1 Env proteins represent a novel strategy for both preventative and therapeutic HIV/AIDS vaccine development.

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.

Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env.

The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immunomodulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing antibodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env.

Development of a novel AIDS vaccine: the HIV-1 transactivator of transcription protein vaccine.

INTRODUCTION: Classical approaches aimed at targeting the HIV-1 envelope as well as other structural viral proteins have largely failed. The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. Notably, anti-Tat Abs are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven intervention. AREAS COVERED: Here, we review the rationale and the translational development of a therapeutic vaccine targeting the Tat protein. Preclinical and Phase I studies, Phase II trials with Tat in anti-Tat Ab-negative, virologically suppressed highly active antiretroviral therapy-treated subjects in Italy and South Africa were conducted. The results indicate that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Additionally, they may help in establishing key parameters for highly active antiretroviral therapy intensification and a functional cure. EXPERT OPINION: We propose the therapeutic setting as the most feasible to speed up the testing and comparison of preventative vaccine candidates, as the distinction lies in the use of the vaccine in uninfected versus infected subjects and not in the vaccine formulation.

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

Flow cytometry analysis of immune cell populations isolated from cervicovaginal secretions of cynomolgus monkeys.

Flow cytometric analysis was used in this study to characterize the lymphocyte population present in the vaginal mucosa of the cynomolgus monkey. Vaginal immune cells were obtained, using absorbent wicks, from 11 normal cycling female monkeys at different stages of the menstrual cycle and from three nursing monkeys (not cycling). Leucocytes, including lymphocytes and monocyte-macrophage cells, were present in the cervicovaginal secretions of healthy cynomolgous primates throughout the three phases of the menstrual cycle. We also found that even if immune cells were constant throughout the menstrual cycle, among the T cell subsets there were differences. CD8+ cells [14.5+/-9% (mean+/-S.D.); range 3-30%] were more numerous compared to the mean number of CD4+ cells [7.3+/-5% (mean+/-S.D.); range 2-15%]. Characterization of the vaginal cells during the nursing period showed that the monocyte-macrophage (CD14+, CD11c+) cells were abundant compared with the low number of both B (CD20+) and T cells (CD2+). Our results show that cytometric analysis by FACS can be used to identify the immune cell populations present at the local level. This technique may provide a useful tool by which the vaginal environment can be studied in order to correlate cell phenotype with immune function.

Infection of a simian B cell line by human and simian immunodeficiency viruses.

It is well known that HIV-1 does not establish infection in nonhuman primates, nor in cell lines derived from them, due to the existence of saturable resistance factors. In this study, we show that an in vitro established Macaca fascicularis-derived CD4(-) B cell line (F6) can be productively infected by the laboratory-adapted T-tropic HXBc2/HIV-1 strain at low multiplicity of infection, apparently because it does not express the restriction factor that has been detected in other simian cell lines. Moreover, efficient entry into F6 cells was obtained with pseudotyped recombinant HIV-1 viruses containing the laboratory-adapted T-tropic (HXBc2) or the dual-tropic (89.6) envelope glycoproteins, whereas entry of virus containing the envelope glycoproteins of the M-tropic Ba-L strain was less efficient. Virus containing primary T-tropic (Eli) envelope glycoproteins did not infect F6 cells. Furthermore, although CCR5 was not present on the cell surface and gpr15 and strl33 mRNAs were not expressed in the cells, a high level of infection of F6 cells by the M-tropic simian immunodeficiency virus SIVmac316 was observed. In contrast, F6 cells were poorly infected by T-tropic SIVmac239. Given the unique properties of the F6 cell line, i.e., that it is of simian origin yet is able to be infected by HIV-1 in a CD4-independent manner, F6 cells represent a useful model for studying cellular factors mediating resistance or permissivity to HIV-1 infection and may help to evaluate HIV-1 and SIV cell tropism.

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.

Effect of MHC haplotype on immune response upon experimental SHIVSF162P4cy infection of Mauritian cynomolgus macaques.

Little is known about the effects of Major Histocompatibility Complex (MHC) haplotypes on immunity to primate lentiviruses involving both acquired and innate immune responses. We present statistical evidence of the influence of MHC polymorphism on antiviral immunity of Mauritian cynomolgus macaques (MCM) following simian/human immunodeficiency virus SHIVSF162P4cy infection, involving the production of pro- and anti-inflammatory cytokines and α-defensins, which may modulate acquired immune responses. During the acute phase of infection, IL-10 correlated positively with viral load and negatively with CD4+T cell counts. Furthermore, α-defensins production was directly correlated with plasma viral RNA, particularly at peak of viral load. When the effects of the MHC were analyzed, a significant association between lower anti-Env binding and neutralizing antibody levels with class IB M4 haplotype and with class IA, IB M4 haplotype, respectively, was observed in the post-acute phase. Lower antibody responses may have resulted into a poor control of infection thus explaining the previously reported lower CD4 T cell counts in these monkeys. Class II M3 haplotype displayed significantly lower acute and post-acute IL-10 levels. In addition, significantly lower levels of α-defensins were detected in class IA M3 haplotype monkeys than in non-M3 macaques, in the post-acute phase of infection. These data indicate that the MHC could contribute to the delicate balance of pro-inflammatory mechanisms, particularly with regard to the association between IL-10 and α-defensins in lentivirus infection. Our results show that host genetic background, virological and immunological parameters should be considered for the design and interpretation of HIV-1 vaccine efficacy studies.

Biocompatible anionic polymeric microspheres as priming delivery system for effetive HIV/AIDS Tat-based vaccines.

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6Pcy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4+ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4+ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.

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.