Approaches to pandemic prevention - the chromatin vaccine.

Developing effective vaccines against viral infections have significant impacts on development, prosperity and well-being of human populations. Thus, successful vaccines such as smallpox and polio vaccines, have promoted global societal well-being. In contrast, ineffective vaccines may fuel arguments that retard scientific progress. We aim to stimulate a multilevel discussion on how to develop effective vaccines against recent and future pandemics by focusing on acquired immunodeficiency syndrome (AIDS), coronavirus disease (COVID) and other viral infections. We appeal to harnessing recent achievements in this field specifically towards a cure for current pandemics and prevention of the next pandemics. Among these, we propose to apply the HIV DNA in chromatin format - an end product of aborted HIV integration in episomal forms, i.e., the chromatin vaccines (cVacc), to elicit the epigenetic silencing and memory that prevent viral replication and infection.

Systems Vaccinology in HIV Vaccine Development.

Themes of discussions in the Special Issue of T Cell Immunity and HIV-1 Pathogenicity are outlined here [...].

Toward a Cure: Does Host Immunity Play a Role?

Three decades of research on human immunodeficiency virus (HIV) and AIDS reveal that the human body has developed through evolution a genome immune system embodying epigenetic regulation against pathogenic nucleic acid invasion. In HIV infection, this epigenetic regulation plays a cardinal role in HIV RNA production that silences HIV transcription at a molecular (RNA) level, controls viral load at a cellular (biological) level, and governs the viremic stage of AIDS at the clinical (patient) level. Even though the human genome is largely similar among humans and HIV is a single viral species, human hosts show significant differences in viral RNA levels, ranging from cell to organ to individual and expressed as elite controllers, posttreatment controllers, and patients with AIDS. These are signature biomarkers of typical epigenetic regulation whose importance has been shunted aside by interpreting all of AIDS pathogenesis by the known properties of innate and adaptive immunity. We propose that harnessing the host genome immune system, defined as epigenetic immunity, against HIV infection will lead toward a cure.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus.

BACKGROUND: 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 ß3-ß4 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Invited commentary: human cytomegalovirus, inflammation, cardiovascular disease, and mortality.

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.

Sustained antiretroviral effect of raltegravir after 96 weeks of combination therapy in treatment-naive patients with HIV-1 infection.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cytomegalovirus infection causes an increase of arterial blood pressure.

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.

Rapid and durable antiretroviral effect of the HIV-1 Integrase inhibitor raltegravir as part of combination therapy in treatment-naive patients with HIV-1 infection: results of a 48-week controlled study.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Primitive hematopoietic cells resist HIV-1 infection via p21.

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.

Propranolol ameliorates and epinephrine exacerbates progression of acute and chronic viral myocarditis.

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.

A passive immunotherapy, (PE)HRG214, in patients infected with human immunodeficiency virus: a phase I study.

(PE)HRG214 (HRG) is a polyclonal antibody preparation produced by immunization of goats with purified human immunodeficiency virus (HIV) antigens. In this phase I study, HRG was administered intravenously as a single dose (1, 2, 4, 8, or 16 mg/kg) to 18 HIV-1-infected patients with CD4 cell counts >/=50 cells/microL and virus loads >/=500 copies/mL. The most frequent adverse event was a transient rash, which appeared to be both dose- and CD4 cell count-dependent. At the 16 mg/kg level, median half-life was 68.4 h, and median C(max) was 392 microg/mL, a level well above that which inhibits HIV in vitro. At that dose level, median and maximum decreases in HIV-1 RNA levels at day 8 were 0.24 log(10) and 0.58 log(10), respectively, and, at day 29, were 0.24 log(10 ) and 2.2 log(10), respectively. HRG, administered as a single dose, is reasonably well tolerated and achieves adequate plasma concentrations.

Human immunodeficiency virus type 1 nucleocapsid protein nuclear localization mediates early viral mRNA expression.

An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 x 10(9) cells/day and 10.3 x 10(9) virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.

Cocaine enhances myocarditis induced by encephalomyocarditis virus in murine model.

This study was designed to investigate whether cocaine can exacerbate viral myocarditis and increase its incidence. Recent clinical evidence suggests that cocaine abuse increases the incidence of myocarditis. However, it has not been directly demonstrated that cocaine exposure enhances murine myocarditis. BALB/c mice were divided into eight groups: saline control, encephalomyocarditis virus (EMCV), 10 mg/kg cocaine (Coc-10), 30 mg/kg cocaine (Coc-30), 50 mg/kg cocaine (Coc-50), EMCV+Coc-10, EMCV+Coc-30, EMCV+Coc-50. After inoculation with EMCV, the mice were treated daily with either cocaine or saline for 90 days. Mice were euthanized at different days after EMCV inoculation. Mortality was recorded and myocarditis severity was evaluated. The mortality of the myocarditis mice treated with cocaine increased significantly, from 22% (EMCV) to 25.7% (Coc-10+EMCV), 41.4% (Coc-30+EMCV), and 51.4% (Coc-50+EMCV) (P < 0.05), respectively. The incidence and severity of inflammatory cell infiltration and myocardial lesions was higher in infected mice exposed to cocaine. Cocaine administered only before infection did not exacerbate myocarditis. Norepinephrine (NE) assay showed that cocaine exposure significantly increased myocardial NE concentration but this increase was partially inhibited in infected animals. Adrenalectomy abolished the effect of cocaine on mortality. Furthermore, propranolol, a beta-blocker, significantly decreased the enhancing effects of cocaine on myocarditis mice. In conclusion, cocaine increases the severity and mortality of viral myocarditis in mice. Increased catecholamines may be a major factor responsible for this effect.