Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors

Background

Interferon‐α2 (IFNα2) is routinely used for anti‐hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half‐life, relatively low local concentration and strong side‐effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector‐delivered IFNα1 was tested for its anti‐HBV effects.

Methods

Adeno‐associated viral vector (AAV‐IFNα1) was generated to deliver the IFNα1 gene into hepatocytes. IFNα1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme‐linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real‐time polymerase chain reaction.

Results

AAV‐IFNα1 effectively transduced HBV‐producing cells (HepAD38) and mouse hepatocytes, where IFNα1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV‐producing mice, the concentration of IFNα1 in the liver was eight‐fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten‐fold from day 1–5, and dropped to an undetectable level on day 9 in the AAV‐IFNα1 group. Concurrently, the level of viral DNA decreased over 30‐fold for several weeks.

Conclusions

A single dose administration of AAV‐IFNα1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV‐IFNα1 might be a potential alternative strategy for anti‐HBV therapy. Copyright © 2008 John Wiley & Sons, Ltd.

     

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice

Background

Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection.

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Tim‐3 blockade promotes iNKT cell function to inhibit HBV replication

Increased expression of T cell immunoglobulin and mucin domain‐3 (Tim‐3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim‐3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim‐3 was up‐regulated on hepatic iNKT cells from HBV‐transgenic (Tg) mice or iNKT cells stimulated with α‐galactosylceramide (α‐Galcer). Compared with Tim‐3^?iNKT cells, Tim‐3⁺iNKT cells expressed more IFN‐γ, IL‐4 and CD107a, indicating a strong relationship between Tim‐3 and iNKT cell activation. Constantly, treatment of Tim‐3 blocking antibodies significantly enhanced the production of IFN‐γ, TNF‐α, IL‐4 and CD107a in iNKT cells both in vivo and in vitro. This Tim‐3^? mediated suppression of iNKT cells was further confirmed in Tim‐3 knockout (KO) mice. Moreover, Tim‐3 blockade promoted α‐Galcer‐triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down‐regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim‐3 blockade in promoting iNKT cell‐mediated HBV inhibition. Therefore, combination of α‐Galcer with Tim‐3 blockade might be a promising approach in chronic hepatitis B therapy.     

Correlation between Vertical Transmission of Hepatitis B Virus and the Expression of HBsAg in Ovarian Follicles and Placenta

Background

The aim of this study was to investigate the correlation between the expression of hepatitis B surface antigen (HBsAg) in human ovary and placenta and the vertical transmission of hepatitis B virus (HBV).

Methodology/Principal Fidnings

Ovarian and placental tissue specimens of pregnant women infected with HBV were collected during cesarean section and immunostained for HBsAg. The sera of the corresponding newborns were tested for HBV markers and HBV DNA. HBsAg was detected in 15 out of 33 (45%) placental tissues and was further detected in capillary endothelial cells in 4 specimens (26%), of which 3 (75%) corresponding infants were infected with HBV in utero. Out of the 33 ovarian tissues, 7 (21%) were positive for HBsAg, of which 2 (28%) showed HBsAg in ovarian follicles and the 2 corresponding infants (100%) had intrauterine HBV infection.

Conclusions/Significance

HBsAg expression in cells of the ovarian follicle or placental capillary endothelium signal a higher risk for intrauterine HBV infection.     

Occult hepatitis B demonstrated by anti-HBc and HBV DNA in HIV-positive patients

Background:

In patients who are hepatitis B virus (HBV) DNA-positive, but HBV surface antigen (HBsAg) -negative, the infection is referred to as occult hepatitis B infection (OBI). Occult HBV infection is harmful when other liver diseases are present, and can aggravate liver damage in in patients with chronic liver diseases. In human immunodeficiency virus (HIV) infection the suppression of viral replication by the immune system might be inactivated, and classical HBV infection in OBI patients may occur. Health care professionals should be aware of OBI in HIV patients. The routine test for HBV infection in Iran is the enzyme-linked immunosorbent assay for the HBV surface antigen (ELISA HBsAg); therefore, the aim of this study was to evaluate the prevalence of OBI in Iranian HIV patients.

Methods:

This cross-sectional study was conducted in 2012 on sera from all the known and accessible HIV patients in Jahrom and Fassa, two cities in southern Iran. All samples were tested for the HBsAg, HBV core antibody (HBcAb). All the results were analyzed using SPSS.

Results:

Of the 91 patients, seven (7.7%) were HBsAg-positive and forty-five (49.5%) were HBcAb-positive. In patients with negative HBsAg (84 patients), 39 (46.4%) were HBcAb positive and 53 (63%) were positive for HBV DNA.

Conclusion:

The prevalence of HBV infection is relatively high in HIV patients, and more accurate tests than those presently in use should be used for diagnosis.Key Words: Hepatitis B, HIV infection, Occult hepatitis     

Effectiveness of HBV Vaccination in Infants and Prediction of HBV Prevalence Trend under New Vaccination Plan: Findings of a Large-Scale Investigation

Background

Hepatitis B virus (HBV) infection remains a severe public health problem. Investigating its prevalence and trends is essential to prevention.

Methods

To evaluate the effectiveness of HBV vaccination under the 1992 Intervention Program for infants and predicted HBV prevalence trends under the 2011 Program for all ages. We conducted a community-based investigation of 761,544 residents of 12 counties in Zhejiang Province selected according to their location, population density, and economic development. The HBV prevalence trends were predicted by a time-shifting approach. HBV surface antigen (HBsAg) and alanine amino transferase (ALT) were determined.

Results

Of the 761,544 persons screened for HBsAg, 54,132 were positive (adjusted carrier rate 6.13%); 9,455 had both elevated ALT and a positive HBsAg test (standardized rate 1.18%). The standardized HBsAg carrier rate for persons aged ≤20 years was 1.51%. Key factors influencing HBV infection were sex, age, family history, drinking, smoking, employment as a migrant worker, and occupation. With the vaccination program implemented in 2011, we predict that by 2020, the HBsAg carrier rate will be 5.27% and that for individuals aged ≤34 years will reach the 2% upper limit of low prevalence according to the WHO criteria, with a standardized rate of 1.86%.

Conclusions

The national HBV vaccination program for infants implemented in 1992 has greatly reduced the prevalence of HBV infection. The 2011 program is likely to reduce HBV infection in Zhejiang Province to a low moderate prevalence, and perinatal transmission is expected to be controlled by 2020.     

Serum adiponectin levels are associated with hepatitis B viral load in overweight to obese hepatitis B virus carriers

Objective:

This study aimed to investigate the association between serum adiponectin and chronic hepatitis B virus (HBV) infection.

Design and Methods:

We conducted a campus‐based cross‐sectional study in Northern Taiwan, an HBV‐endemic country. A total of 506 participants, including 147 chronic HBV‐infected individuals and 359 healthy controls, were assessed for anthropometric indices, serum adiponectin levels, serum HBV viral load and markers, serum alanine aminotransferase levels and metabolic factors.

Results:

Older age, male gender, higher alanine aminotransferase, higher body mass index, greater waist circumference, lower fasting glucose, higher triglycerides, and higher adiponectin were associated with chronic HBV infection in univariate analyses. In multivariate analysis, the presence of chronic HBV infection was positively associated with serum adiponectin levels (P < 0.0001) and high adiponectin levels over the 75th percentile (odds ratio, 4.25; 95% confidence interval, 2.36‐7.66; P < 0.0001) after adjusting for age, gender, body mass index, and insulin resistance index. Furthermore, serum adiponectin levels were positively associated with HBV viral load in overweight to obese HBV‐infected subjects (P = 0.018).

Conclusion:

Although chronic HBV‐infected individuals were heavier than healthy controls, they had significantly higher serum adiponectin levels than healthy counterparts. Additionally, adiponectin levels were positively associated with HBV viral load in overweight to obese HBV‐infected subjects. Future research should focus on elucidating adiponectin pathways, which may contribute to the development of adjuvant treatments for chronic HBV infection.     

Association of T-Cell Immunoglobulin and Mucin Domain-Containing Molecule 3 (Tim-3) Polymorphisms with Susceptibility and Disease Progression of HBV Infection

Purpose

T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) plays an important role in regulating T cells in hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, few researches have reported the association of Tim-3 genetic variants with susceptibility and progression of HBV infection. In this study, we focused on the association of Tim-3 polymorphisms with HBV infection, HBsAg seroclearance and hepatocellular carcinoma.

Methods

A total of 800 subjects were involved in this study. Four groups were studied here, including HBV, HBsAg seroclearance, HBV-associated HCC and healthy controls. Three single-nucleotide polymorphisms (SNPs) of Tim-3, rs246871, rs25855 and rs31223 were genotyped to analyze the association of Tim-3 polymorphisms with susceptibility and disease progression of HBV infection.

Results

Our study found that rs31223 and rs246871 were associated with disease progression of HBV infection, while none of the three SNPs was relevant to HBV susceptibility. The minor allele “C” of rs31223 was found to be associated with an increased probability of HBsAg seroclearance (P = 0.033) and genotype “CC” of rs246871 to be associated with an increased probability of HBV-associated HCC (P = 0.007). In accordance, haplotypic analysis of the three polymorphisms also showed that the haplotype block CGC* and TGC* were significantly associated with HBsAg seroclearance (P<0.05) while haplotype block CAT*, CGT*, TAC* and TGT* were significantly associated with HBV-associated HCC (all P<0.05).

Conclusions

Genetic variants of Tim-3 have an important impact on disease progression of HBV infection. With specific Tim-3 polymorphisms, patients infected with HBV could be potential candidates of HCC and HBsAg seroclearance.     

Hepatitis B Virus Infection in HIV-Positive Individuals in the UK Collaborative HIV Cohort (UK CHIC) Study

Background

Hepatitis B virus (HBV) infection is an increasingly important cause of morbidity and mortality in HIV-infected adults. This study aimed to determine the prevalence and incidence of HBV in the UK CHIC Study, a multicentre observational cohort.

Methods and Findings

12 HIV treatment centres were included. Of 37,331 patients, 27,450 had at least one test (HBsAg, anti-HBs or anti-HBc) result post-1996 available. 16,043 were white, 8,130 black and 3,277 other ethnicity. Route of exposure was homosexual sex 15,223 males, heterosexual sex 3,258 males and 5,384 females, injecting drug use 862 and other 2,723. The main outcome measures used were the cumulative prevalence and the incidence of HBV coinfection. HBV susceptible patients were followed up until HBsAg and/or anti-HBc seroconversion incident infection, evidence of vaccination or last visit. Poisson regression was used to determine associated factors. 25,973 had at least one HBsAg test result. Participants with HBsAg results were typically MSM (57%) and white (59%) (similar to the cohort as a whole). The cumulative prevalence of detectable HBsAg was 6.9% (6.6 to 7.2%). Among the 3,379 initially HBV-susceptible patients, the incidence of HBV infection was 1.7 (1.5 to 1.9)/100 person-years. Factors associated with incident infection were older age and IDU. The main limitation of the study was that 30% of participants did not have any HBsAg results available. However baseline characteristics of those with results did not differ from those of the whole cohort. Efforts are on-going to improve data collection.

Conclusions

The prevalence of HBV in UK CHIC is in line with estimates from other studies and low by international standards. Incident infection continued to occur even after entry to the cohort, emphasising the need to ensure early vaccination.     

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Background

CD8⁺ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/Principal Findings

Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8⁺ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8⁺ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8⁺ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/Significance

Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8⁺ T cell-based immunotherapy that breaks tolerance to HBsAg.     

A Bayesian Meta-Analysis on Prevalence of Hepatitis B Virus Infection among Chinese Volunteer Blood Donors

Background

Although transfusion-transmitted infection of hepatitis B virus (HBV) threatens the blood safety of China, the nationwide circumstance of HBV infection among blood donors is still unclear.

Objectives

To comprehensively estimate the prevalence of HBsAg positive and HBV occult infection (OBI) among Chinese volunteer blood donors through bayesian meta-analysis.

Methods

We performed an electronic search in Pub-Med, Web of Knowledge, Medline, Wanfang Data and CNKI, complemented by a hand search of relevant reference lists. Two authors independently extracted data from the eligible studies. Then two bayesian random-effect meta-analyses were performed, followed by bayesian meta-regressions.

Results

5957412 and 571227 donors were identified in HBsAg group and OBI group, respectively. The pooled prevalence of HBsAg group and OBI group among donors is 1.085% (95% credible interval [CI] 0.859%∼1.398%) and 0.094% (95% CI 0.0578%∼0.1655%). For HBsAg group, subgroup analysis shows the more developed area has a lower prevalence than the less developed area; meta-regression indicates there is a significant decreasing trend in HBsAg positive prevalence with sampling year (beta = −0.1202, 95% −0.2081∼−0.0312).

Conclusion

Blood safety against HBV infection in China is suffering serious threats and the government should take effective measures to improve this situation.     

A common HLA-DPA1 variant is associated with hepatitis B virus infection but fails to distinguish active from inactive Caucasian carriers

Background and Aims

Chronic infection with the hepatitis B virus (HBV) is a major health issue worldwide. Recently, single nucleotide polymorphisms (SNPs) within the human leukocyte antigen (HLA)-DP locus were identified to be associated with HBV infection in Asian populations. Most significant associations were observed for the A alleles of HLA-DPA1 rs3077 and HLA-DPB1 rs9277535, which conferred a decreased risk for HBV infection. We assessed the implications of these variants for HBV infection in Caucasians.

Methods

Two HLA-DP gene variants (rs3077 and rs9277535) were analyzed for associations with persistent HBV infection and with different clinical outcomes, i.e., inactive HBsAg carrier status versus progressive chronic HBV (CHB) infection in Caucasian patients (n = 201) and HBsAg negative controls (n = 235).

Results

The HLA-DPA1 rs3077 C allele was significantly associated with HBV infection (odds ratio, OR = 5.1, 95% confidence interval, CI: 1.9–13.7; p = 0.00093). However, no significant association was seen for rs3077 with progressive CHB infection versus inactive HBsAg carrier status (OR = 2.7, 95% CI: 0.6–11.1; p = 0.31). In contrast, HLA-DPB1 rs9277535 was not associated with HBV infection in Caucasians (OR = 0.8, 95% CI: 0.4–1.9; p = 1).

Conclusions

A highly significant association of HLA-DPA1 rs3077 with HBV infection was observed in Caucasians. However, as a differentiation between different clinical courses of HBV infection was not possible, knowledge of the HLA-DPA1 genotype cannot be translated into personalized anti-HBV therapy approaches.     

Characterization of Acute and Chronic Hepatitis B Virus Genotypes in Canada

Objective

The prevalence and distribution of hepatitis B virus (HBV) genotypes in Canada is not known. Genotypic analysis may contribute to a better understanding of HBV strain distribution and transmission risk.

Methods

HBV surface antigen (HBsAg) positive samples of acute (n = 152) and chronic (n = 1533) HBV submitted for strain analysis or reference genotype testing between 2006 and 2012 were analyzed. The HBsAg coding region was amplified to determine the HBV genotype by INNO-LiPA assay or sequence analysis. Single and multivariate analyses were used to describe genotypes’ associations with known demographic and behavioral risk factors for 126 linked cases of acute HBV.

Results

Nine genotypes were detected (A to I), including mixed infections. Genotype C (HBV/C) dominated within chronic infections while HBV/D and A prevailed among acute HBV cases. History of incarceration and residing with a chronic HBV carrier or injection drug user were the most frequently reported risks for acute HBV infection. Over time, HBV/A increased among both acute and chronic infections, and HBV/C and HBV/D decreased among chronic infections.

Conclusion

Chronic and acute HBV genotypes in Canada differ in the relative distribution and their associations with known risk factors, suggesting different routes of transmission and clinical progression of infection.     

Association of PNPLA3 rs738409 polymorphism with liver steatosis but not with cirrhosis in patients with HBV infection: Systematic review with meta‐analysis

Background

Hepatitis B virus (HBV) infection is a worldwide health issue and is well known for being the main cause of developing secondary liver complications such as cirrhosis and hepatocellular carcinoma (HCC). The PNPLA3 rs738409 polymorphism has been investigated conclusively with occurrence risk of steatosis and cirrhosis. Therefore, performing a meta‐analysis of the available studies with the aim of clarifying the association between rs738409 and occurrence risk of steatosis and cirrhosis among HBV‐infected patients would be helpful.

Methods

Chronic HBV infection was defined as the persistence of HBsAg for more than 6 months. To gather sufficient data for this meta‐analysis, reliable databases were conclusively searched using appropriate keywords. Only studies that satisfied the inclusion criteria were enrolled in the present study.

Results

This meta‐analysis pooled four studies with 1135 cases of chronic hepatitis B (CHB) to evaluate the impact of PNPLA3 SNP on liver steatosis and also pooled five studies with 3713 cases of CHB to evaluate the impact of PNPLA3 SNP on cirrhosis. The association of rs738409 with each complication was investigated. The rs738409 was found to be associated with steatosis in recessive [p = 4.57 × 10^–6, odds ratio (OR) = 2.85], dominant (p = 4.35 × 10^‐6, OR = 1.84), co‐dominant (p = 6.18 × 10^‐8; OR = 3.74) and allelic (p = 9.79 × 10^‐9; OR = 1.78) models. No association was found between rs738409 and cirrhosis development in recessive (p = 0.99, OR = 1.00), dominant (p = 0.30, OR = 0.92), co‐dominant (p = 0.74; OR = 0.96) and allelic (p = 0.45; OR = 0.96) models.

Conclusions

Although the PNPLA3 rs738409 G allele has been associated with the risk of steatosis in CHB patients, no association between this polymorphism and the risk of cirrhosis was seen.     

Virus-Specific Immune Response in HBeAg-Negative Chronic Hepatitis B: Relationship with Clinical Profile and HBsAg Serum Levels

Background & Aims

The immune impairment characterizing chronic hepatitis B (cHBV) infection is thought to be the consequence of persistent exposure to viral antigens. However, the immune correlates of different clinical stages of cHBV and their relation with different levels of HBsAg have not been investigated. The aim of the present study was to evaluate the relationship between HBV-specific T cells response and the degree of in vivo HBV control and HBsAg serum levels in HBeAg-HBeAb+ cHBV.

Methods

Peripheral blood mononuclear cells from 42 patients with different clinical profiles (treatment-suppressed, inactive carriers and active hepatitis) of cHBV, 6 patients with resolved HBV infection and 10 HBV-uninfected individuals were tested with overlapping peptides spanning the entire HBV proteome. The frequency and magnitude of HBV-specific T cell responses was assessed by IFNγ ELISPOT assay. Serum HBsAg was quantified with a chemiluminescent immunoassay.

Results

The total breadth and magnitude of HBV-specific T cell responses did not differ significantly between the four groups. However, inactive carriers targeted preferentially the core region. In untreated patients, the breadth of the anti-core specific T cell response was inversely correlated with serum HBsAg concentrations as well as HBV-DNA and ALT levels and was significantly different in patients with HBsAg levels either above or below 1000 IU/mL. The same inverse association between anti-core T cell response and HBsAg levels was found in treated patients.

Conclusions

Different clinical outcomes of cHBV infection are associated with the magnitude, breadth and specificity of the HBV-specific T cell response. Especially, robust anti-core T cell responses were found in the presence of reduced HBsAg serum levels, suggesting that core-specific T cell responses can mediate a protective effect on HBV control.     

Inhibition of HIV‐1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

Background

We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c‐c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T‐lymphocytes.

Methods

In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL‐CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL‐CD34+ cells and in the terminally differentiated macrophages using macrophage colony‐stimulating factor, granulocyte macrophage colony‐stimulating factor, interleukin‐3 and stem cell factor.

Results

Quantitative real‐time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA‐transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV‐1 NFN‐SX strain. We also developed a novel approach utilizing a mCherry‐CCR5 chimeric reporter to assess the effectiveness of CCR5 target down‐regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.

Conclusions

The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV‐1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs. Copyright © 2010 John Wiley & Sons, Ltd.