[show abstract][hide abstract] ABSTRACT: Recent studies have highlighted the importance of eradication of human immunodeficiency virus (HIV) and cure of acquired immunodeficiency syndrome (AIDS). However, a pivotal point that the patient immunity controls HIV reactivation after highly active anti-retroviral therapy [HAART or combination anti-retroviral therapy (cART)] remains less well addressed. In spite of the fact that both innate and adaptive immunities are indispensable and numerous cells participate in the anti-HIV immunity, memory CD4 T-cells are indisputably the key cells organizing all immune actions against HIV while being the targets of HIV. Here we present a view and multidisciplinary approaches to HIV/AIDS eradication and cure. We aim at memory CD4 T-cells, utilizing the stem cell properties of these cells to reprogram an anti-HIV memory repertoire to eliminate the viral reservoir, toward achieving an AIDS-free world.
[show abstract][hide abstract] ABSTRACT: Genital infection with herpes simplex virus 2 (HSV-2) is linked to an increased risk of infection with human immunodeficiency virus (HIV) in areas such as Sub-Saharan Africa. Thus, an effective genital herpes vaccine would be an important weapon in the fight against HIV/AIDS.
To test whether a current vaccine candidate can protect against HSV-2 from Sub-Saharan Africa, we examined the ability of an HSV-2 vaccine strain, dl5-29, and other HSV-2 replication-defective mutant strains to protect against genital challenge with US or South African strains in a murine model.
Immunization with dl5-29 reduces infection by both viruses but is significantly more efficacious against the US virus than against the African virus. Furthermore, another US vaccine strain was more efficacious against US than against African viruses, and the converse was observed for the parallel African vaccine strain. Nevertheless, protection against the African viruses was significantly less with all vaccines used in this study.
We conclude that there may be differences in protective epitopes and pathogenesis between the US and African strains that raise the need for increased doses of the existing vaccine candidate or an HSV-2 vaccine strain based on viruses from that region.
The Journal of Infectious Diseases 05/2011; 203(10):1434-41. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: In addition to K65R, the other mutation observed at HIV-1 RT codon 65 is K65N. While K65N appears to have a phenotypic effect similar to K65R, it is less frequent during clinical trials. We compared the relative impact of K→N with respect to K→R change on viral replication capacity (RC). Mutant viruses were created and replication kinetics assays were performed in PBM cells. Analysis of RCs revealed a significant loss in replication (p=0.004) for viruses containing K65N mutation in comparison to those with K65R mutation. RT processivity assays showed a significant decrease in the processivity of K65N RT in comparison to K65R RT. We demonstrated that the significant decrease in RC of K65N viruses is related to the impaired RT processivity of K65N RT in comparison to K65R, and that the selection of the K65R mutation may be favored in clinical use of antiretroviral drugs compared to K65N.
[show abstract][hide abstract] ABSTRACT: The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.
Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.
Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.
The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.
[show abstract][hide abstract] ABSTRACT: Human cytomegalovirus (CMV) is a human herpesvirus, and infection is widespread in the human population. Prevalence of seropositivity for human CMV increases with age. CMV establishes persistent infection in vascular arterial and venous endothelial tissue. It has been associated with atherosclerosis and graft rejection in heart transplant recipients. The antiviral drug ganciclovir prevents CMV disease in heart transplant patients, and valganciclovir and CMV immune globulin reduce rejection rates and cardiovascular disease. Human CMV infection has been associated with proinflammatory cytokine increases and nonresponsiveness to antiinfluenza vaccine in the elderly. Enhanced expression of proinflammatory cytokines has also been associated with enhanced mortality in the elderly. In this issue of the Journal, Roberts et al. (Am J Epidemiol. 2010;000(00):000-000) report that, in a large population-based cohort of elderly Sacramento area Latino subjects in California followed from 1998 to 2008, more than 95% were seropositive for human CMV. In that study, Kaplan-Meier survival curves suggested worse cardiovascular disease survival for individuals in the highest quartile of human CMV immunoglobulin G antibody titers over 9 years of follow-up. Theirs is the first study known to report a relation between high human CMV antibody levels and mortality.
American journal of epidemiology 08/2010; 172(4):372-4. · 5.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The purpose of this study was to evaluate the safety and efficacy of raltegravir vs efavirenz-based antiretroviral therapy after 96 weeks in treatment-naive patients with HIV-1 infection.
Multicenter, double-blind, randomized study of raltegravir (100, 200, 400, or 600 mg twice a day) vs efavirenz (600 mg every day), both with tenofovir/lamivudine (TDF/3TC), for 48 weeks, after which raltegravir arms were combined and all dosed at 400 mg twice a day. Eligible patients had HIV-1 RNA > or =5000 copies per milliliter and CD4 T cells > or =100 cells per microliter.
One hundred ninety-eight patients were randomized and treated; 160 received raltegravir and 38 received efavirenz. At week 96, 84% of patients in both groups achieved HIV-1 RNA <400 copies per milliliter; 83% in the raltegravir group and 84% in the efavirenz group achieved <50 copies per milliliter (noncompleter = failure). Both groups showed similar increases in CD4 T cells (221 vs 232 cells/uL, respectively). An additional 2 patients (1 in each group) met the protocol definition of virologic failure between weeks 48 and 96; no known resistance mutations were observed in the raltegravir recipient; the efavirenz recipient had nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance mutations. Investigator reported drug-related clinical adverse events (AEs) were less frequent with raltegravir (51%) than efavirenz (74%). Drug-related AEs occurring in >10% of patients in either group were nausea in both groups and dizziness and headache in the efavirenz group. Laboratory AEs remained infrequent. Raltegravir had no adverse effect on total or low-density lipoprotein cholesterol or on triglycerides. Neuropsychiatric AEs remained less frequent with raltegravir (34%) than efavirenz (58%). There were no drug-related serious AEs in patients receiving raltegravir.
In antiretroviral therapy-naive patients, raltegravir with TDF/3TC had potent antiretroviral activity, which was similar to efavirenz/TDF/3TC and was sustained to week 96. Raltegravir was generally well tolerated; drug-related AEs were less frequent in patients treated with raltegravir compared with efavirenz.
[show abstract][hide abstract] ABSTRACT: Cytomegalovirus (CMV) infection is a common infection in adults (seropositive 60-99% globally), and is associated with cardiovascular diseases, in line with risk factors such as hypertension and atherosclerosis. Several viral infections are linked to hypertension, including human herpes virus 8 (HHV-8) and HIV-1. The mechanisms of how viral infection contributes to hypertension or increased blood pressure are not defined. In this report, the role of CMV infection as a cause of increased blood pressure and in forming aortic atherosclerotic plaques is examined. Using in vivo mouse model and in vitro molecular biology analyses, we find that CMV infection alone caused a significant increase in arterial blood pressure (ABp) (p<0.01 approximately 0.05), measured by microtip catheter technique. This increase in blood pressure by mouse CMV (MCMV) was independent of atherosclerotic plaque formation in the aorta, defined by histological analyses. MCMV DNA was detected in blood vessel samples of viral infected mice but not in the control mice by nested PCR assay. MCMV significantly increased expression of pro-inflammatory cytokines IL-6, TNF-alpha, and MCP-1 in mouse serum by enzyme-linked immunosorbent assay (ELISA). Using quantitative real time reverse transcriptase PCR (Q-RT-PCR) and Western blot, we find that CMV stimulated expression of renin in mouse and human cells in an infectious dose-dependent manner. Co-staining and immunofluorescent microscopy analyses showed that MCMV infection stimulated renin expression at a single cell level. Further examination of angiotensin-II (Ang II) in mouse serum and arterial tissues with ELISA showed an increased expression of Ang II by MCMV infection. Consistent with the findings of the mouse trial, human CMV (HCMV) infection of blood vessel endothelial cells (EC) induced renin expression in a non-lytic infection manner. Viral replication kinetics and plaque formation assay showed that an active, CMV persistent infection in EC and expression of viral genes might underpin the molecular mechanism. These results show that CMV infection is a risk factor for increased arterial blood pressure, and is a co-factor in aortic atherosclerosis. Viral persistent infection of EC may underlie the mechanism. Control of CMV infection can be developed to restrict hypertension and atherosclerosis in the cardiovascular system.
[show abstract][hide abstract] ABSTRACT: To describe cytomegalovirus (CMV) end-organ disease (EOD) rate in AIDS patients with low CD4+ cell count despite HAART who were enrolled in a randomized, placebo-controlled trial of preemptive valganciclovir (VGCV) to prevent CMV EOD in those with CMV viremia.
Subjects (N = 338) were HIV-infected with CD4+ count <100 cells/mm3, plasma HIV RNA >400 copies/mL, and on stable or no HAART. All underwent plasma CMV DNA PCR testing every 8 weeks (Step 1); those with detectable CMV DNA were randomized to VGCV or placebo (Step 2).
Plasma CMV DNA was detected in 68 (20%), of whom 4 developed CMV EOD. During Step 1, 53 died. Of the 47 who entered Step 2 (24 VGCV, 23 placebo), CMV EOD was diagnosed in 10 (4 VGCV, 6 placebo) and 15 died (7 VGCV, 8 placebo). Of those randomized to placebo, 14% were diagnosed with CMV EOD at 12 months.
We observed a lower CMV EOD rate among subjects receiving HAART than predicted based on published literature. However, mortality was high in this study. Our findings suggest that preemptive anti-CMV therapy in patients with persistently low CD4+ cell counts in the current treatment era may not be warranted given the low incidence of CMV EOD and high all-cause mortality observed in this study population.
HIV Clinical Trials 01/2009; 10(3):143-52. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: The antiviral drugs against the human herpes viruses provided pioneering insights, which have led to the development of the
fi eld of antiviral therapy. The fi rst successful use of antiviral drugs to treat any life-threatening viral infection was
Vidarabine (adenosine arabnoside) in 1977 (1). This was followed by the development of Acyclovir as the fi rst specifi c antiviral
drug which required a viral enzyme (thymidine kinase, TK) for activation to a nucleoside triphosphate, which inhibited the
viral DNA polymerase and was a chain-terminator of viral DNA elongation (2, 3). When tested against clinical viral isolates,
acyclovir was most effective against those herpes viruses which established latency in neuronal tissue (HSV-1, HSV-2, VZV)
(4), with some activity against EBV, and very little against clinical isolates of CMV in a plaque reduction assay (4, 5).
With the possible exception of infl uenza A virus and amantadine, this marked the beginnings of antiviral therapy.
[show abstract][hide abstract] ABSTRACT: Raltegravir is an HIV-1 integrase strand-transfer inhibitor with potent in vitro activity. This study explored the antiretroviral activity and safety of raltegravir in treatment-naive patients with plasma HIV-1 RNA levels > or = 5000 copies/mL and CD4 T-cell counts > or = 100 cells/mm.
Multicenter, double-blind, randomized, controlled study of raltegravir at doses of 100, 200, 400, and 600 mg twice daily versus efavirenz at a dose of 600 mg/d, all in combination with tenofovir at a dose of 300 mg/d and lamivudine at a dose of 300 mg/d (clinicaltrials.gov identifier: NCT00100048).
In the 198 patients treated (160 on raltegravir and 38 on efavirenz), the mean HIV-1 RNA level ranged from 4.6 to 4.8 log10 copies/mL at baseline. At weeks 2, 4, and 8, the proportion of patients achieving an HIV-1 RNA level <50 copies/mL was greater in each of the raltegravir treatment groups than in the efavirenz group. By week 24, all treatment groups appeared similar, with plasma HIV-1 RNA levels <400 copies/mL in 85% to 98% of patients and <50 copies/mL in 85% to 95% of patients. These reductions were maintained through week 48 in 85% to 98% of patients and in 83% to 88% of patients, respectively. Five (3%) patients on raltegravir and 1 (3%) on efavirenz experienced virologic failure before week 48. Drug-related clinical adverse events were less common with raltegravir than with efavirenz. After 24 and 48 weeks of treatment, raltegravir did not result in increased serum levels of total cholesterol, low-density lipoprotein cholesterol, or triglycerides.
Raltegravir at all doses studied was generally well tolerated in combination with tenofovir and lamivudine. Raltegravir exhibited potent and durable antiretroviral activity similar to that of efavirenz at 24 and 48 weeks but achieved HIV-1 RNA levels below detection at a more rapid rate.
[show abstract][hide abstract] ABSTRACT: Hematopoietic stem cells are resistant to HIV-1 infection. Here, we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21), a known regulator of stem cell pool size, restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs, p27(Kip1) and p18(INK4C), had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase, SIVmac-251, and the other cell-intrinsic inhibitors of HIV-1, Trim5alpha, PML, Murr1, and IFN-alpha, were unaffected by p21. Therefore, p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected "sanctuary" in HIV disease.
Journal of Clinical Investigation 03/2007; 117(2):473-81. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.
[show abstract][hide abstract] ABSTRACT: Recent studies point to important interactions between proinflammatory cytokines and neurohumoral mediators in heart failure. Here we investigate the influence of the beta-adrenergic system on cytokines and neurohumoral factors and the sequelae of viral myocarditis. In an experimental model with virus-infected BALB/c mice, we studied the acute and chronic effects of epinephrine and propranolol on myocardial morphology, cytokine gene expression, and survival. BALB/c mice were inoculated with the encephalomyocarditis virus (EMCV) or sham inoculated with saline and followed for 30 days. Epinephrine increased the severity of inflammatory cell infiltration and myocardial necrosis induced by EMCV. Gene expression of TNF-alpha, IL-6, and IL-10 was markedly enhanced by epinephrine in EMCV-inoculated mice. Survival rate after 30 days was reduced to 40% in epinephrine-treated EMCV-inoculated mice compared with 70% in untreated EMCV-inoculated mice (P < 0.05). Treatment with the beta-blocker propranolol significantly decreased mortality, myocardial necrosis, and infiltration of inflammatory cells in EMCV-inoculated mice. Propranolol also suppressed gene expression of TNF-alpha, IL-6, and IL-10. A single dose of epinephrine 120 days after EMCV inoculation caused sudden death in 70% of infected mice; propranolol significantly reduced incidence of death to 33%. These results indicate that acute and chronic stages of viral myocarditis are modulated by the beta-adrenergic system and its interactions with proinflammatory cytokines.