Diagnosis of HIV infection and laboratory monitoring of its therapy.

BACKGROUND: Serological diagnosis of human immunodeficiency virus (HIV) infection became available in 1985, with the rapid increase in sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) and the supplement tests. Molecular tests for detection of HIV in the diagnosis of HIV infection in special settings and monitoring of HIV-1 infection followed this. OBJECTIVE AND DESIGN: In this review it is intended to give a brief overview of the diagnosis and monitoring of HIV infection. Results and conclusion: Serological methods and molecular methods for the detection and quantitation of HIV are discussed.     

Viro-immunopathogenesis of HIV disease: implications for therapy

Advances in defining the roles of immune system activation and T-cell apoptosis in the viro-immunopathogenesis of HIV disease were discussed at a recent symposium*.     

The influence of HIV on CD127 expression and its potential implications for IL-7 therapy

Interleukin-7 (IL-7) is critical for early T-cell development and plays an important role in T-cell homeostasis, differentiation and function. Signalling via the IL-7 receptor is dependent on the expression of its components, IL-7Rα (CD127) and IL-2Rγ (CD132) and is mediated in part by alterations in CD127 expression levels in different cell subsets. Naïve and memory T-cells express high levels of CD127, while effector cells are CD127^lo and retention of the receptor is thought to influence the development of memory cells. Reduced expression of CD127 has been associated with markers of disease severity in HIV infection and other chronic viral infections as well as in various cancers. In HIV infection, decreased CD127 expression on T-cells is correlated with reduced CD4⁺ T-cell counts, increased viral replication and immune activation. The loss of IL-7 activity, due to decreased CD127 expression, may contribute to the observed loss of CD8⁺ cytotoxic T lymphocyte (CTL) activity in HIV infection. The downregulation of CD127 expression in HIV infection may be due to host (e.g. IL-7, IL-4, immune activation) and/or viral (e.g. HIV-tat) factors and mechanisms of receptor regulation may differ by cell type. In addition, the expression of a soluble form of CD127 (sCD127) has been shown to be increased in HIV infection. This protein may affect IL-7 activity in vivo and therefore may have implications for IL-7-based therapies which are currently being tested in clinical trials. Understanding how CD127 is regulated during HIV infection will provide insight for the development of novel therapeutics to improve immune function and anti-viral T-cell activity.     

A consensus statement on the renal monitoring of Australian patients receiving tenofovir based antiviral therapy for HIV/HBV infection

A number of antiviral agents used against Human Immunodeficiency Virus (HIV) infection and hepatitis B virus (HBV) mono or co-infection have been associated with real nephrotoxicity (including tenofovir disoproxil fumarate (TDF), atazanavir, indinavir and lopinavir) or apparent changes in renal function (e.g. cobicistat, ritonavir, rilpivirine and dolutegravir). Patients with HIV are at higher risk of acute and chronic renal dysfunction, so baseline assessment and ongoing monitoring of renal function is an important part of routine management of patients with HIV.Given the paucity of evidence in this area, we sought to establish a consensus view on how routine monitoring could be performed in Australian patients on ART regimens, especially those involving TDF. A group of nephrologists and prescribers (an HIV physician and a hepatologist) were assembled by Gilead to discuss practical and reasonable renal management strategies for patients particularly those on TDF-based combination regimens (in the case of those with HIV-infection) or on TDF-monotherapy (in the case of HBV-mono infection). The group considered which investigations should be performed as part of routine practice, their frequency, and when specialist renal referral is warranted. The algorithm presented suggests testing for serum creatinine along with plasma phosphate and an assessment of urinary protein (rather than albumin) and glucose.Here we advocate baseline tests of renal function at initiation of therapy. If creatinine excretion inhibitors (e.g. cobicistat or rilpivirine) are used as part of the ART regimen, we suggest creatinine is rechecked at 4 weeks and this value used as the new baseline. Repeat testing is suggested at 3-monthly intervals for a year and then at least yearly thereafter if no abnormalities are detected. In patients with abnormal baseline results, renal function assessment should be performed at least 6 monthly. In HBV mono-infected patients advocate that a similar testing protocol may be logical.     

Pathogenesis of idiopathic pulmonary fibrosis and its clinical implications

Idiopathic pulmonary fibrosis (IPF) is the most common and lethal form of idiopathic interstitial pneumonia. The disease is thought to arise following an aberrant reparative response to recurrent alveolar epithelial cell injury leading to progressive loss of function. The median survival time is 3–5 years from diagnosis. Cigarette smoking, exposure to organic and inorganic dust and genetic factors have been shown to increase the risk of disease, although the cause of IPF remains elusive and its pathogenesis incompletely understood. In the last decade, several clinical trials evaluating novel therapies for IPF have been conducted but the results have been mostly disappointing. Conversely, compounds that target anti-fibrotic and growth factor pathways have been proven effective in slowing functional decline and disease progression. These promising results notwithstanding, truly effective therapeutic strategies will likely require combinations of drugs in order to target the multitude of pathways involved in disease pathogenesis.     

Hepatitis a virus infection: Pathogenesis and serodiagnosis of acute disease

The early development of immune electron microscopic (IEM) methods for the detection of HAV in acute-phase stool suspensions and antibody to HAV (anti-HAV) in serum made it possible to serologically identify cases of hepatitis A using paired acute and convalescent phase sera. Introduction of less cumbersome and time-consuming serologic test methods, including complement fixation (CF) and immune adherence hemagglutination (IAHA), made it feasible to rapidly assay larger numbers of specimens for HAV or anti-HAV. Subsequent development of sensitive immunofluorescence (IF) assays, solid-phase radioimmunoassays (RIA), and enzyme immunoassays (EIA) for HAV and anti-HAV heralded intensive laboratory studies of the biophysical and biochemical properties of the virus as well as efforts to define the pathogenesis and clinical course of disease. Results of the latter studies showed that the bulk of HAV was usually excreted in stool before the onset of clinical symptoms. Other serologic studies demonstrated that all acutely ill patients had circulating anti-HAV IgM, while all convalescent patients were positive for anti-HAV IgG. The development of sensitive serologic tests (RIA and EIA) that could differentiate between anti-HAV IgM and IgG made it possible to serodiagnose an acute case of hepatitis A using a single-phase serum specimen.     

The natural history of recurrent herpes simplex labialis: implications for antiviral therapy.

We performed daily examination of 80 patients with recurrent herpes simplex labialis to define the course of the disease and to identify quantitative and objective measurements for use in monitoring the efficacy of antiviral chemotherapy. Pain, lesion size, mean virus titers from lesion swabs (10(5) plaque-forming units [PFU]) and frequency of virus-positive lesions (89 per cent) were maximal during the first 24 hours and decreased thereafter. Lesion punch-biopsy virus titers increased from a mean of less than 10(1) PFU in the prodromal and erythema stages to a mean of 10(4.7) in the vesicle stage. MEasurements potentially useful in monitoring antiviral efficacy include: time to loss of crust, time to complete healing, intensity and duration of lesion pain, area defined by lesion virus titer and duration of lesion virus excretion, and maximum lesion virus titer after the first visit. Early application of topical antiviral therapy should theoretically be able to alter the course of this disease.     

Pathogenesis of HIV encephalitis

A wide spectrum of infectious agents attack the central nervous system (CNS) of acquired immunodeficiency syndrome (AIDS) patients. Human immunodeficiency virus (HIV) itself, infects the CNS of a subgroup of these patients. The mechanism behind why HIV enters the CNS is unclear. We have observed an interesting association between HIV and opportunistic viral infections that may explain why HIV enters the brain. Infection of the CNS by opportunistic agents results in recruitment of latently HIV-infected monocytes. Upon differentiation into macrophages these cells produce abundant HIV. Latent HIV-infection of monocytes/macrophages provides a unique opportunity for cooperativity between opportunistic infections and HIV in mediating CNS damage.     

Pathogenesis of HIV Encephalitis

A wide spectrum of infectious agents attack the central nervous system (CNS) of acquired immunodeficiency syndrome (AIDS) patients. Human immunodeficiency virus (HIV) itself, infects the CNS of a subgroup of these patients. The mechanism behind why HIV enters the CNS is unclear. We have observed an interesting association between HIV and opportunistic viral infections that may explain why HIV enters the brain. Infection of the CNS by opportunistic agents results in recruitment of latently HIV infected monocytes. Upon differentiation into macrophages these cells produce abundant HIV. Latent HIV-infection of monocytes/macrophages provides a unique opportunity for cooperativity between opportunistic infections and HIV in mediating CNS damage.     

Pathogenesis of HIV and the Lung

Antiretroviral therapy has improved longevity for HIV-infected persons, but long-term HIV infection is now complicated by increased rates of chronic medical conditions including pulmonary disorders. Chronic obstructive pulmonary disease, lung cancer, asthma, and pulmonary hypertension are becoming common comorbidities of HIV infection, and these diseases may develop as a result of HIV-related risk factors, such as antiretroviral drug toxicities, colonization by infectious organisms, HIV viremia, immune activation, or immune dysfunction. It also appears that the ability to control HIV infection does not completely eliminate the risk for infectious complications, such as bacterial pneumonia and tuberculosis. The effect of HIV infection on lung-specific immune responses is being elucidated to help develop better prevention and treatment strategies in HIV-infected persons.     

Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.     

Pathogenesis and therapy for transthyretin related amyloidosis

Transthyretin (TTR) is a beta-sheet rich protein whose plasma half life is 1.9 days. It behaves as a tetramer and binds to retinol binding protein (RBP) and thyroxin in plasma. Since TTR is a tryptophan-rich-protein, the protein is used as a useful marker protein for nutrition supporting team (NST). However, TTR is also an anti-acute phase protein, and its concentration is influenced by various conditions, such as inflammation and infection. Mutated forms of TTR are the precursor protein of familial amyloidotic polyneuropathy (FAP). Since plasma TTR is predominantly synthesized by the liver, liver transplantation has been performed as an effective therapy for FAP. However, the surgery has several problems, so we must develop novel essential therapies for FAP. Single stranded oligonucleotides (SSOs) or short interference RNA (SiRNA) is a promising option for an essential therapy for FAP. In mutated TTR, instability of tetrameric form of TTR occurs, resulting in misfolding of TTR molecule, which lead to amyloid fibril formation. Since mutated TTR exposes criptic epitopes on the surface of TTR molecule, induction of an antibody for the epitopes was thought to be effective. We synthesized ATTR Y78P, a spontaneously misfolded TTR, and injected it to amyloid laden transgenic mice having human ATTR V30M to induce the antibody for amyloid fibrils. As we expected, amyloid deposition was significantly reduced by the injection of ATTR Y78P to the mice. These therapies may become novel strategies for essential FAP therapy instead of liver transplantation.     

Comparison of real-time PCR assays for monitoring serum hepatitis B virus DNA levels during antiviral therapy

The performance characteristics of the RealART and Molecular Beacons assays were compared with those of the Digene Hybrid Capture II assay (ultrasensitive). The results of the RealART and Digene Hybrid assays were related (r = 0.94; P < 0.001) and diverged by 2 orders of magnitude. The RealART assay can be used to effectively monitor serum hepatitis B virus DNA levels.     

HIV integrase as a target for antiviral chemotherapy

One of the three key enzymes encoded by the pol gene of HIV is a M(r) 32 000 protein called HIV integrase. This viral enzyme is involved in the integration of HIV DNA into host chromosomal DNA. There appears to be no functional equivalent of the enzyme in human cells. The biochemical mechanism of integration of HIV DNA into the host cell genome involves a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. Thus, new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. The focus of this review will be on several classes of compounds, including nucleotides, dinucleotides, oligonucleotides and miscellaneous small molecules such as heterocyclic systems, natural products, diketo acids and sulfones, that have been discovered as inhibitors of HIV integrase. Special emphasis in the review will be placed on discoveries from my laboratory on HIV integrase inhibitors that are non-natural, nuclease-resistant dinucleotides. Comments on future directions and the prospects for developing integrase inhibitors as therapeutic antiviral agents are discussed.