HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus

Early treatment of acute HIV-1 infection followed by treatment interruptions has shown promise for enhancing immune control of infection. A subsequent loss of control, however, allows the correlates of protective immunity to be assessed. Here we show that sudden breakthrough of plasma viraemia occurred after prolonged immune containment in an individual infected with HIV-1 at a time when 25 distinct CD8+ T-cell epitopes in the viral proteins Gag, RT, Integrase, Env, Nef, Vpr, Vif and Rev were being targeted. Sequencing of the virus in plasma and cells showed that superinfection with a second clade-B virus was coincident with the loss of immune control. This sudden increase in viraemia was associated with a decline in half of the CD8+ T-cell responses. The declining CD8+ T-cell responses were coupled with sequence changes relative to the initial virus that resulted in impaired recognition. Our data show that HIV-1 superinfection can occur in the setting of a strong and broadly directed virus-specific CD8+ T-cell response. The lack of cross-protective immunity for closely related HIV-1 strains, despite persistent recognition of multiple CD8 epitopes, has important implications for public health and vaccine development.     

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.     

Challenges in the development of an HIV-1 vaccine

The development of a safe and effective human immunodeficiency virus (HIV)-1 vaccine is a critically important global health priority. Despite recent advances in our understanding of HIV-1 pathogenesis and immunology, however, major scientific obstacles remain. Prototype HIV-1 vaccine candidates aimed at eliciting humoral and cellular immune responses have so far failed to protect against HIV-1 infection or to reduce viral loads after infection in clinical efficacy studies. A renewed and coordinated commitment to basic discovery research, preclinical studies and clinical trials will therefore be required to overcome the hurdles currently facing the field. Here I review key challenges and future prospects in the quest to develop a prophylactic HIV-1 vaccine.     

A novel nuclear export activity in HIV-1 matrix protein required for viral replication

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.     

Antibody neutralization and escape by HIV-1

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving 'glycan shield' mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.     

Extensive variation of human immunodeficiency virus type-1 in vivo

Genotypic variation among independent isolates of human immunodeficiency virus type-1 (HIV-1) is well known, but its molecular basis and biological consequences are poorly understood. We examined the genesis of molecular variation in HIV-1 by sequential virus isolations from two chronically infected individuals and analysis of recombinant HIV-1 genomic clones. In three different virus isolates full-length HIV-1 clones were identified and found to consist, respectively, of 17, 9 and 13 distinguishable, but highly-related, viral genotypes. Thirty-five viral clones derived from two HIV-1 isolates obtained from the same individual but 16 months apart showed progressive change, yet were clearly related. Similar changes in the HIV-1 genome did not occur in vitro during virus isolation and amplification. The results indicate that HIV-1 variation in vivo is rapid, that a remarkably large number of related but distinguishable genotypic variants evolve in parallel and coexist during chronic infection, and that 'isolates' of HIV-1, unless molecularly or biologically cloned, generally consist of complex mixtures of genotypically distinguishable viruses.     

HIV-specific cytotoxic T lymphocytes in seropositive individuals

Virus-specific cytotoxic T lymphocytes (CTL) which kill virus-infected cells are thought to be a major host defence against viral infections. Here we report the existence of human immunodeficiency virus (HIV)-specific CTL in persons infected with this virus, the aetiological agent of AIDS (acquired immunodeficiency syndrome). Recombinant HIV-vaccinia viruses were used to express HIV antigens in B-cell lines established from subjects seropositive for HIV and seronegative controls. Circulating lymphocytes capable of killing HIV env-expressing autologous B cells were detected in eight of eight seropositive subjects; in addition, at least three seropositive subjects demonstrated gag-specific cytotoxic responses. No HIV-specific cytotoxicity was observed in seronegative subjects. Selective inhibition of the env-specific cytotoxicity by a CD3-specific monoclonal antibody indicates that the effectors are T cells. This demonstration of a cytotoxic T-cell immune response to HIV in infected individuals should prove useful in investigating the immunopathogenesis of HIV infection further and in evaluating AIDS vaccine strategies.     

Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection

It has recently been established that both acute human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are accompanied by a dramatic and selective loss of memory CD4+ T cells predominantly from the mucosal surfaces. The mechanism underlying this depletion of memory CD4+ T cells (that is, T-helper cells specific to previously encountered pathogens) has not been defined. Using highly sensitive, quantitative polymerase chain reaction together with precise sorting of different subsets of CD4+ T cells in various tissues, we show that this loss is explained by a massive infection of memory CD4+ T cells by the virus. Specifically, 30-60% of CD4+ memory T cells throughout the body are infected by SIV at the peak of infection, and most of these infected cells disappear within four days. Furthermore, our data demonstrate that the depletion of memory CD4+ T cells occurs to a similar extent in all tissues. As a consequence, over one-half of all memory CD4+ T cells in SIV-infected macaques are destroyed directly by viral infection during the acute phase-an insult that certainly heralds subsequent immunodeficiency. Our findings point to the importance of reducing the cell-associated viral load during acute infection through therapeutic or vaccination strategies.     

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.     

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (T(EM)) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIV(MAC239) infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4(+) memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8(+) T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8(+) or CD4(+) lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated T(EM) responses might significantly contribute to an efficacious HIV/AIDS vaccine.     

Biologically diverse molecular variants within a single HIV-1 isolate

AIDS is a disorder characterized by a slow progressive impairment of immune function and by infection of human immunodeficiency viruses (HIV-1, HIV-2). Our knowledge of how these viruses cause disease in man, or how the related lentiviruses (visna and equine infectious anaemia virus) cause disease in animals, is still fragmentary. In particular, the significance of genetic variation in HIV-1, occurring within populations, within individuals and over periods of time, and the mechanisms of viral persistence remain unclear. To address these issues we prepared a series of proviral clones of HIV-1 originating from a single patient and compared their biological properties. Here we show that hybrid genomes (in which the envelope region of six viral clones were separately substituted into a prototype HIV-1 genome) generated viruses with widely differing capacity to grow in human T cells, cell lines and monocytoid cultures. These data suggest that extensive biological variation exists in vivo within an infected individual and is in part determined at the level of the viral envelope.     

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.     

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.     

Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy

Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.     

HIV-1感染与树突状细胞相互作用的研究进展(综述)

树突状细胞(dendritic cells，DC)是目前所知的机体内功能最强的抗原呈递细胞，在免疫应答中发挥重要作用。在AIDS发病机制中有关人类免疫缺陷病毒(HIV)与宿主细胞之间的作用已经有很多报道，但对树突状细胞与HIV感染之间的作用了解较少。近年来的研究发现HIV-1能诱导树突状细胞分化，改编细胞基因表达，上调细胞因子和趋化因子的产生活化T细胞，而不同的DC亚群表达不同的甘露糖C-型凝集素受体(MCLRs)结合HIVgp120，从而促进病毒感染复制和扩散。     

Modelling how ribavirin improves interferon response rates in hepatitis C virus infection

Nearly 200 million individuals worldwide are currently infected with hepatitis C virus (HCV). Combination therapy with pegylated interferon and ribavirin, the latest treatment for HCV infection, elicits long-term responses in only about 50% of patients treated. No effective alternative treatments exist for non-responders. Consequently, significant efforts are continuing to maximize response to combination therapy. However, rational therapy optimization is precluded by the poor understanding of the mechanism(s) of ribavirin action against HCV. Ribavirin alone induces either a transient early decline or no decrease in HCV viral load, but in combination with interferon it significantly improves long-term response rates. Here we present a model of HCV dynamics in which, on the basis of growing evidence, we assume that ribavirin decreases HCV infectivity in an infected individual in a dose-dependent manner. The model quantitatively predicts long-term response rates to interferon monotherapy and combination therapy, fits observed patterns of HCV RNA decline in patients undergoing therapy, reconciles conflicting observations of the influence of ribavirin on HCV RNA decline, provides key insights into the mechanism of ribavirin action against HCV, and establishes a framework for rational therapy optimization.     

分支DNA探针法检测21例丙型肝炎血清病毒RNA

目前主要依赖检测丙型肝炎抗体来确定对丙型肝炎病毒(Hepatitis C virus,HCV)感染的诊断,但它不能反应机体是否有活动的病毒血症。分支DNA探针法应用合成的DNA分子与靶HCV—RNA特异性的杂交,形成RNA—DNA杂交体,用dioxetane作为底物与化学发光物结合,通过测定其发光强度可直接检测血清HCV—RNA的含量。本文测定21例慢性活动性丙型肝炎血清。HCV—RNA最低值为0.66Meg/ml;最高值为58Meg/ml,21例中86％的数值分布在0.66Meg/m-12Meg/ml的范围内。此方法cutoff值为0.5Meg//ml。我们所测定的21例均高于cutoff值。此方法操作简便,特异性强。为临床丙型肝炎治疗的监测及疗效的判断提供了重要的依据。